Simultaneous spectrophotometric determination of compounds having relatively disparate absorbance and concentration ranges; application to antidiabetic formulation of linagliptin and metformin

Accepted Manuscript Simultaneous spectrophotometric determination of compounds having relatively disparate absorbance and concentration ranges; application to antidiabetic formulation of linagliptin and metformin

Ola M. Abdalla, Ahmed M. Abdel-Megied, Ahmed S. Saad, Shymaa S. Soliman PII: DOI: Reference:

S1386-1425(18)30456-6 doi:10.1016/j.saa.2018.05.064 SAA 16098

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received date: Revised date: Accepted date:

7 September 2017 10 May 2018 15 May 2018

Please cite this article as: Ola M. Abdalla, Ahmed M. Abdel-Megied, Ahmed S. Saad, Shymaa S. Soliman , Simultaneous spectrophotometric determination of compounds having relatively disparate absorbance and concentration ranges; application to antidiabetic formulation of linagliptin and metformin. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Saa(2017), doi:10.1016/j.saa.2018.05.064

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ACCEPTED MANUSCRIPT Simultaneous spectrophotometric determination of compounds having relatively disparate absorbance and concentration ranges; application to antidiabetic formulation of Linagliptin and Metformin

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Ola M. Abdallaa.b, Ahmed M. Abdel-Megied*C, Ahmed S. Saad d, Shymaa S. Solimane

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a Analytical Chemistry Department, Faculty of Pharmacy, Al-Azhar University (Girls), Cairo, Egypt. b Pharmaceutical Chemistry Department, Faculty of Pharmacy, Badr University in Cairo (BUC), Badr City, Cairo,

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Egypt.

c Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Kafrelsheikh University, Kafrelsheikh City, Egypt.

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d Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Kaser El-Aini Street, P.O. box 11562, Cairo-Egypt.

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e Pharmaceutical Chemistry Department, October 6 University, 6th October City, Egypt.

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*Correspondence:

Dr. Ahmed M. Abdel-Megied, Pharmaceutical Analytical Chemistry

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Department, Faculty of Pharmacy and Pharmaceutical Manufacturing,

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Kafrelsheikh University, Kafrelsheikh City, Egypt.

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E-mail: [email protected], [email protected]

ACCEPTED MANUSCRIPT Abstract The limited linear range of UV-Visible spectrophotometry may be insufficient to occupy multiple components with wide variations in their concentrations or absorptivities that will hinder the simultaneous spectrophotometric determination and may require spiking or measurements in subsequent dilution steps. The current work introduces the

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absorptivity target concentration (ATC) values, a simple way for the proper choice of the

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working spectral region to execute accurate and linear spectrophotometric measurements.

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Simultaneous spectrophotometric determination of linagliptin (LNG) and metformin (MET) that are present in a ratio of 1:400 was carried out using traditional

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spectrophotometric techniques such as third derivative and derivative ratio as well as recently developed techniques such as ratio difference and factorized dual wavelength. The proposed methods were able to determine MET in the concentration range of 50 –

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1200 μg mL-1. On the other hand, LNG was successfully determined from its zero-order absorption UV-spectrum at λmax (296 nm) in the concentration range 2.5-25 μg mL-1. The

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mentioned methods were successfully applied for the determination of the LNG and MET

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in their combined dosage form. The methods were validated according to the ICH guidelines. The proposed ATC value can be employed as a novel concept for the proper

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choice of the working spectral region where spectrophotometric measurements can be

Keywords:

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deployed accurately and precisely.

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Derivative spectrophotometry, Ratio derivative, Linagliptin, Metformin hydrochloride

ACCEPTED MANUSCRIPT 1. Introduction Type II diabetes mellitus (T2D) is considered a progressive disorder caused by β-cell dysfunction and insulin resistance [1]. Currently, the prevalence of type II diabetes in Egypt is around 15.6% of all adults aged 20 to 79. For instance, patients with diabetes are two to four times more likely to have a fatal or not fatal stroke [2]. Gliptins as oral

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hypoglycemic drugs work by preventing the breakdown of a naturally occurring hormone

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called glucagon-like peptide 1 (GLP-1) which helps the body produce insulin in response to high blood glucose levels, but It is rapidly broken down [3]. Linagliptin (LNG), (8-

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[(3R)-3-amino-1-piperidinyl]-7-(2-butynyl)-3,7-dihydro-3-methyl-1-[(4-methyl-2quinazolinyl)methyl]-1H-purine-2,6-dione) is a potent, orally dihydro purine dione-based

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inhibitor of dipeptidyl peptidase 4 (DPP-4). Fig.1. Metformin (MET), N,N-Di methyl imido dicarbonimi dicdiamide hydrochloride is a biguanide hypoglycemic agent which

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stimulates glycolysis in peripheral tissues and considered to be a vital component in mixed therapies of oral hypoglycemic [4]. In 2012, the once-daily formulation approval

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was announced by FDA [5]. The literature review revealed few methods developed for the determination of LNG and MET combined dosage forms, spectrophotometry [6],

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HPLC [7-9], HPTLC [10] and LC-MS/MS [11].

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The relatively large divergence in the absorptivity and ratio of LNG and MET in the pharmaceutically available dosage forms represented a challenge for their simultaneous

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spectrophotometric determination; however, the proper choice of the spectral region to execute spectrophotometric analysis represented the core of the current work and proved

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to be a powerful tool to deal with this common challenge. The aim of the present work is to develop univariate and bivariate spectrophotometric methods with different manipulation pathways for resolving a binary mixture of the spectral interfering problem without preliminary separation. The utilized methods were very simple, accurate, and precise; did not require any sophisticated apparatus or computer programs.

ACCEPTED MANUSCRIPT 2. Experimental 2.1. Materials and reagents Pure standard LNG was purchased from Sigma Aldrich (St. Louis, MO, USA), while pure standard MET was supplied by chemical Industries Development (CID Pharmaceutical) Co. (Cairo, Egypt). Methanol (HPLC grade) was obtained from Merck

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(Darmstadt, Germany).

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2.2. Instrumentation

All the spectrophotometric measurements in solutions were carried out using Shimadzu spectrophotometer

dual

beam,

model

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UV-Visible

UV-1800

was

used

with

Probe 2.32 system software.

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2.3. Pharmaceutical formulation

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matched quartz 1cm cells and absorbance spectra were obtained by using Shimadzu UV-

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Jentadueto Tablets were manufactured by Boehringer Ingelheim Pharma GmbH & Co.KG Germany and are labeled as containing 2.5 mg LNG and 1000 mg MET.

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2.4. Standard solutions

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Stock standard solutions of LNG and MET were freshly prepared by dissolving 50 and 25 mg of LNG and MET separately in 25-mL volumetric flasks (1 mg mL-1 for LNG and 2

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mg mL-1 for MET) and completed to the volume with methanol. Series of working solutions of LNG and MET were prepared by the appropriate dilution to reach the concentration ranges of 2.5-50 and 50 – 1200 µg mL-1 for LNG and MET, respectively.

ACCEPTED MANUSCRIPT 2.5. Procedure 2.5.1. Construction of calibration curve Aliquots equivalent to 25 – 250 µg mL-1 of LNG working standard solution (50 µg mL-1) were transferred into a series of 10-mL volumetric flasks and completed to mark with

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methanol. The zero order (D0) UV- spectra were recorded against the used solvent as a

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blank. The calibration curve was constructed relating the absorbance at 296 nm to the

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corresponding LNG concentrations, and the regression equation was computed in which LNG can be determined at its λmax (296 nm) without the interference of MET.

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While aliquots equivalent to 2000 – 10000 µg mL-1 of MET stock standard solution (2000 µg mL-1) were transferred into a series of 10-mL volumetric flasks and completed

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to mark with the same solvent in which zero order UV- spectra for each solution of MET were computed versus methanol. The determination of MET was applied by using four

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different spectrophotometric methods.

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2.5.2. Third derivative method (D3)

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The obtained spectra were derivatized to obtain a third derivative spectrum then the calibration plot was constructed with respect to the absorbance at 268.7 nm to the

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corresponding concentrations and the regression equation was computed.

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2.5.3 Derivative ratio method (DD1) Zero order absorption spectra of each solution of MET were divided by the spectrum of LNG (25 µg mL-1) as a divisor. The first derivative of the ratio spectra was obtained, then a calibration curve was constructed relating the amplitude at 264 nm to the corresponding MET concentration, and finally, the regression equations were computed.

2.5.4. Ratio difference method (RD)

ACCEPTED MANUSCRIPT The scanned spectra of MET were divided by the spectrum of LNG (25 µg mL-1) as a divisor. A calibration curve was constructed relating the difference in the amplitudes of the resulting ratio spectrum at 262 and 268 nm (∆P262–268 nm) to the corresponding MET concentration, and the regression equations were computed.

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2.5.5. Factorized dual wavelength method (FDW)

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The overlain spectra of LNG and MET were observed in which the calibration curve and

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a regression equation was derived relating the difference in absorbance at 264 and 268 nm to the corresponding MET concentrations, and the regression equation was computed.

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2.6. Analysis of laboratory-prepared mixtures

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Laboratory prepared mixtures were prepared by using a different ratio of the previously prepared solutions of LNG and MET into 10-mL volumetric flask, then those mixtures

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were analyzed by using the other previously mentioned spectrophotometric methods. According to the spectrophotometric methods used for the analysis; the final

regression equation

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2.7. Assay of tablets

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concentration of each drug for each mixture was calculated from its corresponding

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Five tablets of Jentadueto® were accurately weighed and finely powdered. An accurately weighed amount of the powder equivalent to 50 mg was transferred to a 50-mL

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volumetric flask, mixed with methanol then sonicated for 15 min and centrifugated by using Falcon tube. 1 mL of the clear solution was accurately transferred into a 10-mL volumetric flask and complete to the mark with methanol to get a concentration of 1000 µg mL-1 then this application was analyzed by using the previously mentioned spectrophotometric methods. The validity of the methods was assessed by standard addition technique which performed by adding a different known concentration of pure LNG and pure MET to the previously prepared application. The recovery of the added standards was then calculated after applying the proposed methods.

ACCEPTED MANUSCRIPT 3. Results and discussion Several approaches have been developed to resolve overlapping spectral data for selective simultaneous determination of each of the overlapping components. The use of spectrophotometric methods such as derivative spectrophotometry, ratio spectra spectrophotometry and other chemometric spectral calibration techniques were found to

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be more beneficial instead of other sophisticated chromatographic analytical

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instrumentations as GC-MS/MS and LC-MS/MS. The variety of UV-spectrophotometric methods let analyst have the opportunity to select the most appropriate method for

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analysis according to the extent of the overlap which may be partially or completely. Simultaneous spectrophotometric determination of MET and LNG was extremely

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difficult since MET exhibits higher absorptivity and present in relatively higher concentration than the other component LNG which has lower absorptivity and present in

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low concentration as shown in Fig.2. Upon initial scanning of the UV-spectra of LNG and MET in methanol, it was observed an overlapped spectral bands in-between 210–250

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nm as shown in Fig. 3. This work is aimed to develop simple, accurate and low coast spectrophotometric methods for simultaneous determination of LNG and MET in bulk

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and dosage form. Thus, various methods have been applied for quantitative determination

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of LNG and MET simultaneously without any interference. For successful simultaneous spectrophotomeric measurements, careful selection of the

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working spectral region is mandatory whenever the components exhibit a wide difference in their concentration or absorptivity. The choice of the working part of the spectrum

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should be carried out such that the components must exhibit relatively similar absorptivity target concentration value (ATC value). The hereby introduced ATC value can be used as a mean for the proper selection spectral regions to execute accurate and precise spectrophotometric measurements. The value should be within the linear window of the spectrophotometric measurements, which in most cases lies between 0.2 and 2.0; however, this value may subject to variation according to the efficiency and quality of the employed instrument. Selective determination of LNG in the presence of MET was feasible at its wavelength of maximum absorbance, 296 nm, where MET showed zero absorbance and no interference the spectral region beyond 285 nm.

ACCEPTED MANUSCRIPT 3.1. Third derivative method (D3) Neither of the first and the second spectra was able to locate a wavelength for the selective determination of MET in the presence of LNG, due to the absence of a zero crossing for LNG at which MET express proper linearity as shown in Fig. 4(a, b). However, the third derivative method showed that MET could be determined by

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measuring the amplitude at 268.7 nm as shown in Fig. 4(c). with the advantage of

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3.2. Derivative ratio method (DD1)

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narrowing the spectral band and increase sensitivity.

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The advantage of this method was the elimination of entire UV-spectrum from interfering substance. In addition, it was found that the choice of the selected wavelength for

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calibration was not a crucial issue as in the derivative method. For optimization of DD1 method, the selected divisor concentrations were 5, 10, 15 and 25 μg mL-1 of the LNG, it

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was found that the best-used divisor was of 25 μg mL-1 LNG. The interference of the constant amplitude of the LNG in the binary mixtures was omitted using the

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differentiation function at the first derivative DD1. Good linearity and reproducibility

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were achieved for LNG determination at 264 nm as shown in Fig. 5.

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3.3. Ratio difference method (∆P) The ratio difference method could be applied for resolving absorption spectra of two

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components with a high degree of overlap as in the case of LNG and MET. A calibration curve was constructed relating the difference in the amplitudes of the resulting ratio spectrum at 262 and 268 nm (∆P262–268

nm)

to the corresponding MET concentration.

Regarding simplicity, the proposed ratio difference method showed minimal data manipulation; instead of applying a certain order derivative in the derivative ratio method, simply the following step will be calculating MET at ∆P262–268 nm as shown in Fig. 6.

ACCEPTED MANUSCRIPT 3.4. Factorized Dual wavelength method (FDW) The prerequisite for dual wavelength method is the selection of two such wavelengths where the interfering component shows the same absorptivity whereas the component of interest shows significant difference absorptivity values. A condition that may sometimes hinder the application of the method; however, with a simple modification by including

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the ratio of the absorptivity values at the two wavelengths as a factor will help to

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overcome that condition and extend the application of the dual wavelength method to all

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binary mixtures, provided that the component of interest still proves proper differences in absorptivity after using the mentioned factor.

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The factor is calculated using the pure spectrum of the interfering component to be the quotient of the absorptivities at any two selected wavelengths, calculated from the

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calibration curves of the interfering component at the two-selected wavelength.

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F = A1 / A2

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Where A1 is the absorptivity at λ1 and A2 is the absorptivity at λ2. The calculated factor will be used as a multiplier for the absorbance value of λ2 before

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subtraction from that of λ1 for the quantitation of the compound of interest in binary mixtures of the two components [12]. The wavelengths 264 and 268 nm were selected for

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determination of MET, Fig. 7.

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4. Methods Validation The methods were validated according to the ICH guidelines [13]: The linearity of the previously mentioned methods was evaluated at different concentrations of LNG and MET ranging from 2.5-25 µg mL-1and 50-1200 µg mL-1 respectively. Each concentration was performed by analyzing the same sample, in triplicate. The corresponding concentration ranges and other statistical parameters for the proposed methods are summarized in Table 1.

ACCEPTED MANUSCRIPT The accuracy of the results was checked by applying the proposed methods for determination of different samples of LNG and MET. While the specificity was assessed by analyzing laboratory prepared mixtures containing different concentrations of both drugs. The concentrations were obtained from the corresponding regression equations from which the percentage recoveries and standard deviations suggested good accuracy

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and specificity of the proposed methods.

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Repeatability and intermediate precision were determined using three concentrations of

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each of LNG (5, 10, 15 µg mL-1) and MET (400, 600, 800 µg mL-1) the intraday precision was assessed by analyzing three concentrations in triplicate of each sample in

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the same day while the interday precision was assessed by analyzing the same sample, in triplicate, in three successive days using the previously proposed methods as shown in

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Table 1.

The method was applied for the determination of the two drugs in their pharmaceutical

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dosage form Jentadueto® tablets, and standard addition technique was applied to ensure the validity of the proposed for the selective quantification of the two drugs within the

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tablet matrix, as shown in Table 2(a,b).

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5. Conclusion

The simultaneous determination of LNG and MET in their combined dosage within the

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limited linear range of spectrophotometric technique was hindered because of the divergent absorbance values. The selection of the region of the spectum meticulously was

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mandatory in order to carry out linear spectrophotometric measurements and execute valid spectrophotometric methods. Within their meticulously selected working spectral region, four different spectrophotometric methods were accurately and selectively applied for the determination of LNG and MET in their pure powder form, binary mixtures, and their marketed formulation.

ACCEPTED MANUSCRIPT References [1] R. Turner, C. Cull, R. Holman, United Kingdom Prospective Diabetes Study 17: A 9-year update of a randomized, controlled trial on the effect of improved metabolic control on complications in non-insulin-dependent diabetes mellitus, Ann. Int. Med. 124 (1996) 136-145.

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[2] R. Hegazi, M. El-Gamal, N. Abdel-Hady, O. Hamdy, Epidemiology of and Risk Factors for Type 2 Diabetes in Egypt, Ann. Glob. Health. 81 (2015) 814-820.

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[3] N. Bohannon, Overview of the gliptin class (dipeptidyl peptidase-4 inhibitors) in clinical practice, Postgrad. Med. 121 (2009) 40-45.

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[4] M. Kirby, D.M. Yu, S. O'Connor, M.D. Gorrell, Inhibitor selectivity in the clinical application of dipeptidyl peptidase-4 inhibition, Clin. Sci. 118 (2009) 31-41.

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[5] https://www.accessdata.fda.gov/drugsatfda_docs/label/2012/201281s000lbl.pdf.

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[6] R. El-Bagary, E. Elkady, B. Ayoub, Spectrophotometric Methods for the Determination of Linagliptin in Binary Mixture with Metformin Hydrochloride and Simultaneous Determination of Linagliptin and Metformin Hydrochloride using High Performance Liquid Chromatography, Int J Biomed Sci, 9 (2013 ) 41–47.

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[7] B. Ayoub, UPLC simultaneous determination of empagliflozin, linagliptin and metformin, RSC. Adv. 5 (2015) 95703-95709.

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[8] S. Shirisha, M.A. Haque, D. Sireesha, V. Bakshi, S. Harshini, Development and Validation of RP-HPLC Method for Simultaneous Estimation of Metformin and Linagliptin in Combined Pharmaceutical Dosage Form, Int. J. Pharm. Res. and Health. Sci. 2 (2014) 491-495.

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[9] M. Attimarad, S.H. Nagaraja, B. E. Aldhubaib, B. Nair , K.N. Venugopala, Simultaneous Determination of Metformin and Three Gliptins in Pharmaceutical Formulations Using RP HPLC, Ind. J. Pharm. Edu. and Res. 48 (2014) 45-53.

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[10] E.I. El-Kimary, D.A. Hamdy, S.S. Mourad, M.A. Barary, HPTLC Determination of Three Gliptins in Binary Mixtures with Metformin, J. Chromatogr. Sci. 54 (2016) 79-87. [11] M. Al Bratty, H. Alhazmi, S. Javed, K. Lalitha, M. Asmari, J. Wölker, S. El Deeb, Development and Validation of LC–MS/MS Method for Simultaneous Determination of Metformin and Four Gliptins in Human Plasma, Chromatographia. 80 (2017) 891–899. [12] A.S. Saad, N.F. Abo-Talib, M.R. El-Ghobashy, Novel ratio difference at coabsorptive point spectrophotometric method for determination of components with wide variation in their absorptivities, Spectrochim. Acta. A Mol. Biomol. Spectrosc. 152 (2016) 480-484. [13] ICH Harmonized Tripartie Guidline Q2B (R1) : Validation of Analytical Proceudures: Text and Methodology, (2005). http://www.ich.org.

ACCEPTED MANUSCRIPT Fig 1. Chemical structure of (A) Linagliptin and (B) Metformin Hydrochlorid Fig 2. Zero order absorption spectra of 12.00 µg mL -1 LNG (-----), 12.00 µg mL-1 MET (___) and a mixture containing 12.00 µg mL -1 LNG and 12.00 µg mL-1 MET (…..)

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measured in methanol

Fig 3. Zero order absorption spectra of 25.00 µg mL -1 LNG (-----) and 1000.00 µg mL-1

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MET (___) in methanol

Fig 4(a). First derivative absorption spectra of 25.00 µg mL-1 LNG (---), 200.00 µg mL-1

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MET (___) and a mixture containing 25.00 µg mL -1 LNG and 200.00 µg mL-1 MET

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(…..) measured in methanol

Fig 4(b). Second derivative absorption spectra of 25.00 µg mL-1 LNG (---), 200.00 µg

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mL-1 MET (___) and a mixture containing 25.00 µg mL -1 LNG and 200.00 µg mL-1

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MET (…..) measured in methanol

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Fig 4(c). Third derivative absorption spectra of 25.00 µg mL-1 LNG (---), 200.00 µg mL-1 MET (___) and a mixture containing 25.00 µg mL -1 LNG and 200.00 µg mL-1 MET (…..) measured in methanol

Fig 5. The ratio spectra (a) and the first derivative of the ratio spectra (b) for 25.00 µg mL-1 LNG (---), 200.00 µg mL-1 MET (___) and a mixture containing 25.00 µg mL -1 LNG and 200.00 µg mL-1 MET (…..) using 25.00µg mL-1 LNG as a divisor measured in methanol.

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Fig 6. The ratio spectrum of 25.00 µg mL-1 LNG (---), 200.00 µg mL-1 MET

(___) and a mixture containing 25.00 µg mL -1 LNG and 200.00 µg mL-1 MET (…..)

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using 25.00 µg mL-1 LNG as a divisor measured in methanol

(__) and a mixture containing 25.00 µg mL

-1

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Fig 7. Zero order absorption spectra of 25.00 µg mL-1 LNG (---), 200.00 µg mL-1 MET LNG and 200.00 µg mL-1 MET (…..)

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measured in methanol.

ACCEPTED MANUSCRIPT

Table 1. Assay validation sheet of the proposed methods for the determination of LNG and MET. LNG Third

Derivative

Ratio

spectrophotom

derivative

ratio

difference

etric method

spectrophotom

spectrophotom

spectrophotom

h

etric method

etric method

etric method

manipulat

(D3)

(DD1)

(RD)

ion

99.60 ± 1.68

99.52 ± 0.91

99.96 ± 0.68

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100.02 ± 0.55

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100.15 ± 1.48

90.03 ± 0.97

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lity

98.99 ± 1.42

99.33 ± 1.07 Intermedi ate

101.31 ± 0.79

100.01 ± 1.25

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Precision

Repeatabi

100.05 ±

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y

:

100.00 ± 1.05

wavelengt

0.60

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Specificit

Dual

(DW)

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Accuracy

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r

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Zero order

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Paramete

MET

100.77 ± 0.44

100.00 ± 0.47

100.00 ± 0.76

100.00 ± 1.08

94.09 ± 1.04

99.87 ± 0.40

96.48 ± 0.76

100.00 ± 0.38

ACCEPTED MANUSCRIPT precision

Linearity

0.0026

- 0.0006

0.0194

0.0017

0.9999

0.9999

0.9999

50.00 –

50.00 –

50.00 –

0.0414

- 0.0064

- 0.0061

Intercept

0.0081

0.0744

0.0416

0.9996

0.9999

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Slope

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Correlatio n

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coefficien

50.00 –

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2.50 – 25.00 µg

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t (r)

Range

th

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mL-1

Waveleng

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T

:

296.00 nm

1200.00 µg mL- 1200.00 µg mL- 1200.00 µg mL-

1200.00

1

1

1

µgmL-1

268.70 nm

264.00 nm

262.00 &

264.00 &

268.00 nm

268.00 nm

ACCEPTED MANUSCRIPT Table 2a. Standard addition results obtained by zero order spectrophotometric method for determination of LNG. Standard addition technique Claimed

%

dosage form

concentration

found

Amount

Amount

taken

added

µg ml-1

± SD

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tablets labeled to

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contain 2.50 mg LNG

µg ml-1

mg

5.00

5.26

105.36

10.00

10.44

104.43

15.00

15.54

103.64

93.46

M

1000

%

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Jentadueto

and

found

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µg ml-1

Amount Recovery

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Pharmaceutical

MET

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± 1.21

2.50

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CE

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2.50 mg

Mean

104.47

SD

0.86

RSD %

0.82

ACCEPTED MANUSCRIPT

Table 2b. Standard addition results obtained by third derivative, derivative ratio, ratio difference, dual wavelength for determination of METin presence of LNG. % found ± SD

Standard addition technique Recovery %

RD

1

me Claim

blet

ed

dos

conce

age

ntrati

for

on

W me

tho

me

tho

me

d

tho

d

tho

d

d

ts label

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92.0 3

±0.6 3

1000.00

LNG and 1000 .00

tak add en

M CE

table

mg

t

D 3

ed

D

R

D

D

D

R

D

D

D

W

3

D

D

W

1

1

µg

ml-1

ED

o

2.50

t

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duet

ain

oun oun

ml-1

Jenta

cont

µg ml-1

µg

m

ed to

Am Am

AN

Ta

D

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DD

US

D3

IP

T

Amount found

mg

91.9 7

±0.6 6

93.0 4 ±0.7 0

50.00

49.

50.

51.

53.

99

10

10

10

96

55

75

72

.9

1.1

3.5

7.4

3

1

1

5

91.1 0 ±0.8 0

1000.

100.0

00

0

98.

99.

10

10

71

90

2.3

4.2

98

99.

10

10

3

4

.7

90

2.3

4.2

3

4

1

ACCEPTED MANUSCRIPT mg ME 150.0

14

14

0

9.0

6.4

0

5

15

15

99

97.

10

10

4.0

5.0

.3

63

2.7

3.3

5

8

3

0

9

IP

T

T

CR

Mea n

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SD

AN

RSD

AC

CE

PT

ED

M

%

99

99.

10

10

.3

55

2.8

5.0

5

2

2 0.

1.7

0.6

2.1

61

6

0

4

0.

1.7

0.5

2.0

61

7

8

4

ACCEPTED MANUSCRIPT Highlights ● A challenge spectrophotometric methods for simultaneous determination of linagliptin (LNG) and metformin (MET) in their tablets.

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● Simultaneous analysis of binary mixtures with a high variety in

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absorptivities

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● Applicable on recently approved pharmaceutical dosage form.

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● The proposed method is suitable for QC laboratories

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7