Different Spectrophotometric Methods for Simultaneous Determination of Trelagliptin and Its Acid Degradation Product

New spectrophotometric and chemometric methods were carried out for the simultaneous assay of trelagliptin (TRG) and its acid degradation product (TAD) and applied successfully as a stability indicating assay to recently approved Zafatek® tablets. TAD was monitored using TLC to ensure complete degradation. Furthermore, HPLC was used to confirm dealing with one major acid degradation product. The proposed methods were developed by manipulating zero-order, first-derivative, and ratio spectra of TRG and TAD using simultaneous equation, first-derivative, and mean-centering methods, respectively. Using Spectra Manager II and Minitab v.14 software, the absorbance at 274 nm–260.4 nm, amplitudes at 260.4 nm–274.0 nm, and mean-centered values at 287.6 nm–257.2 nm were measured against methanol as a blank for TRG and TAD, respectively. Linearity and the other validation parameters were acceptable at concentration ranges of 5–50 μg/mL and 2.5–25 μg/mL for TRG and TAD, respectively. Using one-way analysis of variance (ANOVA), the optimized methods were compared and proved to be accurate for the simultaneous assay of TRG and TAD.


Introduction
Trelagliptin (TRG) (Figure 1) is a dipeptidyl peptidase-4 (DPP-4) inhibitor for treating type 2 diabetes mellitus as a onceweekly monotherapy. Its mechanism of action includes inhibiting the DPP-4 enzyme that leads to increment of glucagon-like peptide-1 (GLP-1) and other hormone levels [1]. TRG administration once weekly showed a high e cacy and good safety pro le [2,3]. TRG kinetic analysis revealed reversible and slow-binding inhibition of DPP-4, while X-ray di raction data indicated a noncovalent interaction between DPP-4 and TRG [4,5]. DPP-4 inhibitors are weight neutral and well tolerated and provide better glycaemic control for a longer period compared to conventional therapies. Despite the fact that various drugs are available, glycaemic control remains suboptimal in approximately half of patients with type 2 diabetes mellitus, one of the major reasons for low medication adherence [6][7][8]. A novel DPP-4 inhibitor, TRG, was approved in Japan in March 2015 and is the rst once-weekly oral antidiabetic agent in the world. Medication adherence for the treatment of diabetes was reviewed in the recent phases I, II, and III clinical studies. TRG has demonstrated superiority to placebo and noninferiority to alogliptin, indicating its efcacy and tolerance in Japanese patients. TRG is expected to improve adherence and prevent complications. Due to the convenient dosing regimen, it is expected to be widely used in the clinical setting [6][7][8][9].
Although some LC methods were reported for quantication of TRG [10][11][12], no methods were found in the literature dealing with the direct UV assay of TRG in its pharmaceutical dosage form. In addition, no methods were used for the simultaneous assay of TRG and its acid degradation product (TAD) using UV spectrophotometric techniques. TAD (Figure 1) was found in the literature to be the main acid degradation product [12,13], which is 2-[(3-methyl-2, 4, 6-oxo-tetrahydro-pyrimidin-1(2H) yl)methyl]-4-uorobenzonitrile. Literature results were ascertained by separating TRG from its acid degradation product on the TLC that results in two separated spots before the complete degradation and showed only one spot afternalizing the stress conditions as reported [12,13]. Moreover, in this presented work, HPLC-UV was used to con rm that we have one major degradation product where chromatographic separation of TRG from TAD was obtained with retention times for TRG and its acid degradation product equal to 1.86 and 2.43, respectively ( Figure 1). e present work is considered as the rst spectrophotometric methods for the analysis of TRG in Zafatek ® tablets and for simultaneous determination of TRG in the presence of TAD that are considered as simple and inexpensive techniques. is study presents di erent methods resolving the overlapped spectra of TRG and TAD by manipulating their zero-order, rst-order, and ratio spectra using simultaneous equation, rst-derivative, and mean-centering methods, respectively.

Reagents, Reference Samples, and Stock and Working
Solutions. TRG was certi ed to contain more than 99.0%, and Zafatek tablets (each tablet contains 50 mg of TRG) were provided by Takeda Pharmaceuticals Ltd. (Japan). Stock solutions (1 mg/mL) and their appropriate dilution to working solutions (100 µg/mL) of TRG were prepared separately in analytical grade methanol purchased from Sigma-Aldrich (Germany).

Sample Preparation.
e coats of twenty Zafatek tablets were carefully removed; then, the tablets were powdered and mixed. An accurately weighed amount equivalent to 10 mg of TRG was transferred to a 100 mL volumetric ask, completed to volume with methanol, sonicated to dissolve, and ltered. Two, three, and four milliliters of the previously prepared extract were added separately to a series of 10 mL volumetric asks and completed to volume with methanol. e nal TRG concentrations were equivalent to 20, 30, and 40 µg/mL.

Preparation of the Trelagliptin Acid Degradation Product (TAD).
Using TRG stock solution (1 mg/mL), acidic hydrolysis of TRG was carried out in a Fischer brand disposable tube by mixing 2.5 mL of TRG stock solution with 2.5 mL of 1 N HCl and was heated for 2 hours at 90°C. At the speci ed time (after cooling the tube contents), a precalculated amount of 1 N NaOH was added to neutralize the tube contents. en, the tube contents were transferred into a 50 mL volumetric ask and completed to volume with methanol.
us, the concentration of TAD was assumed to be 50 μg/mL. Complete acidic hydrolysis of TRG was con rmed by injecting the sample into the HPLC-UV system that results in only one peak with a retention time of 2.43 (instead of TRG retention time that equals 1.86). Also, the sample was separated on TLC that results in only one spot that is corresponding to TAD. e sample was stored under 4°C until analysis.

Preliminary Investigation.
e zero-order absorption spectra of TRG (30 µg/mL) and TAD (15 µg/mL) were recorded separately against methanol as a blank. Overlay of both TRG and TAD spectra (Figure 2(a)) showed the maximum absorption (λ max ) at 274 nm and 260.4 nm, respectively. Overlay of the rst-order spectra of TRG (30 µg/mL) and TAD (15 µg/mL) showed the TAD zero crossing point at 260.4 nm and the TRG zero crossing point at 274.0 nm, as shown in Figure 2(b).

Linearity.
Aliquots of working solutions corresponding to 50-500 µg/mL and 25-250 µg/mL of TRG and TAD, respectively, were added separately into a series of 10 mL volumetric asks and completed to volume with methanol. e corresponding absorbance was measured at 274.0 nm and 260.4 nm for TRG and TAD, respectively, using methanol as a blank. Calibration curves (absorbance against concentration) were constructed for both TRG and TAD.  e simultaneous equation spectrophotometric method was successfully applied to TRG and TAD by manipulating their zero-order absorption spectra. TRG and TAD showed the maximum absorption (λ max ) at 274 nm (λ max 1) and 260.4 nm (λ max 2), respectively. Absorbance at λ max 1 and λ max 2 was plotted against the corresponding concentrations of TRG and TAD, respectively, and calibration curves were constructed. Mixtures of TRG and TAD with di erent lambdas and concentrations of TRG and TAD (C TRG and C TAD ) can be calculated by a simultaneous equation. rough absorbance of the sample at λ1 and λ2 (A1 and A2), absorptivities of TRG at λ max 1 and λ max 2 (a TRG 1 and a TRG 2) and absorptivities of TAD at λ max 1 and λ max 2 (a TAD 1 and a TAD 2) are as follows: (1) e total absorbance is the sum of TRG and TAD absorbance, as their λ max are relatively dissimilar and they do not interact chemically.

First-Derivative Method.
e rst-derivative spectrophotometric method was successfully applied to TRG and TAD by manipulating their rst-order spectra. e amplitudes of the rst-order spectra were measured for TRG and TAD at 260.4 nm and 274 nm, respectively. en, the amplitudes were plotted against corresponding concentrations of TRG and TAD to construct calibration curves.

Mean-Centering Method.
e scanned zero-order spectra of TRG and TAD were separately divided by 25 µg/mL of TAD and 50 µg/mL of TRG, respectively. Using the Minitab v.14 program, the obtained ratio spectra were mean centered and then, the mean-centered values of TRG and TAD were measured at 287.6 nm and 257.2 nm, respectively. Calibration curves were constructed by plotting the mean-centered values against corresponding concentrations.

Accuracy and Precision.
ree di erent ratios of the drug and its degradation product (TAD equals to 10%, 15%, and 20% of TRG) as 3 laboratory-prepared mixtures were applied using concentrations corresponding to 35, 40, and 45 µg/mL and 3.5, 6, and 9 µg/mL of TRG and TAD, respectively. e zero-order absorption spectra of the mixtures were recorded using methanol as a blank. Finally, the obtained spectra were manipulated by di erent methods to calculate the corresponding concentration of each drug. Furthermore, they were analyzed using the proposed methods three times within the same day (n � 3) and on three successive days (n � 3). e percent recoveries (% R) and the percent relative standard deviation (% RSD) were calculated for each method.

Assay of Zafatek Tablets.
e absorbance spectrum of the tablet extract prepared under Sample Preparation was recorded.
en, the percent recoveries and standard deviation were calculated.

Results and Discussion
Calibration curves for the simultaneous equation method were constructed by plotting absorbance of TRG and TAD (Figures 3(a) and 3(b)) against the corresponding concentrations. A simultaneous equation at the speci ed lambdas (274.0 nm and 260.4 nm) was used to get the corresponding concentration of TRG and TAD in their mixtures. en, the percent recoveries of both TRG and TAD were calculated for the three laboratory-prepared mixtures (Table 1).
Calibration curves for the rst-derivative method were constructed by plotting amplitudes of TRG and TAD (Figures 4(a) and 4(b)) against the corresponding concentrations. e concentrations of TRG and TAD were Journal of Analytical Methods in Chemistry 3 calculated by applying the corresponding regression equations, as shown in Table 2.
For the mean-centering method, the absorption spectra of TRG and TAD were separately divided by the zero-order spectra of 50 μg/mL TRG and 25 μg/mL TAD to get the ratio spectra that were mean centered using the Minitab program. In uences of di erent variables were studied, including divisor concentration and smoothing factor. Di erent concentrations were tried as divisors (30-60 μg/mL of TRG and 15-35 μg/mL of TAD). e selected divisors (50 μg/mL TRG and 25 μg/mL TAD) showed minimum noise, maximum sensitivity, and smoother ratio spectra, as shown in Figures 5(a) and 5(b).
Mean centering was applicable, as TRG and TAD are noninteractive to each other, and each of them obeys Beer's law according to the following equation (V a � A TRG C TRG + A TAD C TAD ), where V a is the vector of absorbance, A TRG -A TAD are the molar absorptivities, and C TRG -C TAD are the concentrations of TRG and TAD, respectively. After division over A TAD , the resultant ratio spectra  were mean centered; C TAD will be zero value enabling the determination of C TRG without interference from TAD and the same concept regarding A TRG , as shown in Table 3.

Validation according to ICH Guidelines
3.1.1. Linearity. e linearity of the calibration curves was con rmed by LOD-LOQ parameters, STEYX, S b , and S a as shown in Tables 1-3, where LOD is the limit of detection, LOQ is the limit of quanti cation, STEYX is the standard error of estimation, S b is the standard deviation of the slope, and S a is the standard deviation of the intercept. Also, the methods were adopted successfully for the assay of TRG and TAD in their mixtures. Acceptable results of the regression parameters were achieved as shown in Tables 1-3 [14].

Accuracy and Precision.
Accuracy was checked by calculating the percent recoveries of TRG and TAD in their laboratory-prepared mixtures, while precision values were checked using their intraday and interday records, n � 3, as shown in Tables 1-3.

Speci city and Application on the Pharmaceutical
Dosage Form. TRG was determined in its laboratoryprepared mixtures with TAD and in Zafatek tablets without interference from the excipients of the  e mean of the percent recoveries and standard deviation were calculated, as shown in Tables 1-3.

Statistical
Comparison. ANOVA comparison of the proposed methods at p � 0.05 showed no signi cant difference, as shown in Table 4.  Figure 5: e mean-centered values of the ratio spectra representing 5-50 μg/mL TRG divided by 25 μg/mL of TAD (a) and the ratio spectra of 5-25 μg/mL TAD divided by 50 μg/mL of TRG (b), using methanol as a blank.

Conclusion
e optimized analytical methods were con rmed to be precise and accurate for determination of TRG and TAD based on the simple economic assay. e methods were applied successfully on the pharmaceutical dosage form, with acceptable validation results. Simultaneous determination for laboratory-prepared mixtures of TRG and TAD was achieved through manipulating their zero-order, rst-order, and ratio spectra. e developed methods should be of interest to the analysts in the area of drug control and can be used by QC laboratories.

Conflicts of Interest
e authors declare that they have no con icts of interest.