Development and validation of a new analytical method for determination of linagliptin in bulk by visible spectrophotometer

Mar 06, 2023

Scientific Reports volume 13, Article number: 4083 (2023) Cite this article

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A simple, economical, and specific analytical method has been developed for determining and validating linagliptin (LNG) in bulk. This method is based on a condensation reaction between a primary amine in LNG and an aldehyde group in P-dimethylaminobenzaldehyde (PDAB) to form the yellow Schiff base with a wavelength of 407 nm. The optimum experimental conditions for the formulation of the colored complex have been studied. The optimum conditions were 1 mL of 5% w/v reagent solution with methanol and distilled water as a solvent for both PDAB, LNG respectively, also adding 2 mL of HCl as an acidic medium, heating to 70–75 °C on a water bath for 35 min. Furthermore, the stoichiometry of the reaction has been studied according to Job's and Molar ratio method which was expressing 1:1 for LNG and PDAB. The researcher modified the method. The results show that the linearity in the concentration range (5–45 µg/mL) with correlation coefficient R2 = 0.9989 with percent recovery (99.46–100.8%) and RSD was less than 2%, LOD and LOQ 1.5815 − 4.7924 μg/mL respectively. This method can show high quality and there is no significant interference with excipients and in pharmaceutical forms. None of the studies showed the development of this method before.

Linagliptin is a new dipeptidyl peptidase-4 (DPP-4) inhibitor, this enzyme is responsible for the downgrade of the incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). So this action increase the insulin level in the blood and the level of glucagon will be decreased1. It is used in combination with diet and exercise in the therapy of type 2 diabetes, either alone or in combination with other oral hypoglycemic agents (Empagliflozin, Metformin)2. The drug received FDA approval in May 20111. As an oral antidiabetic agent, it has a xanthine-based structure, that may be a significant factor in the drug's elimination half-life (more than 100 h). The long half-life of LNG may be more beneficial for patients who occasionally miss their doses of medication1. The chemical structure of LNG is 8-[(3R)-3-aminopiperidin-1-yl]-7-(but-2-ynyl-3-methyl)-1-[(4-methylquinazolin-2-yl)methyl]purine-2,6-dione.Molecular weight 472.5 g/mol (Fig. 1)2.

Chemical structure of linagliptin.

Chemical and Physical Properties of LNG: color and form: slightly hygroscopic, white to yellow solid. Melting Point: 190–195 °C. Solubility: In water, 3.33 mg/L at 25 °C; soluble in methanol; sparingly soluble in ethanol. Stability: It is stable if stored as directed; avoid strong oxidizing agents2.

Linagliptin isn't available as a specific method for analyzing in British pharmacopeia (BP) or the United States Pharmacopeia (USP). The Review of Research Literature has shown several articles for the determination of LNG in pharmaceutical forms, including the spectrophotometer in UV3,4,5, also in VIS with chemical derivation using NQS (1,2-naphtho quinine 4-sulphonic acid sodium salt), vanillin6 and picric acid7 as a chromogenic reagents. Capillary Electrophoresis (CE)8, and with high-performance liquid chromatography (HPLC)9,10,11,12,13. Those methods are specificity and selectivity but they need more time, a lot of amount an expensive solvent and equipments.

While Vis Spectrophotometer is a simple, economical analytical method that it is used in multiple fields (Clinical biochemistry, chemistry…etc.). Also, it has high speed and doesn't need extraction for detection of a small amount of material concentration compared to another method HPLC or CE. Due to LNG does not have a lot of chromophores, one chemical derivative method was used to develop a new spectrophotometer method for determination LNG in tablet products. This method needs less time-assuming, and has few solvents. Also, it has high precision and accuracy by using the derivation agent as PDAB that produces with the primary amine of LNG a yellow color of the complex having a maximum absorption at 407 nm.

The research method which is adopted for this paper was an experimental design that used an analytical approach to explore the research objectives.

UV–Visible Spectrophotometer T80 + PG Instruments Ltd—England.

Sartorius-Germany analytical balance.

water bath.

Calibrated glass pipettes.

Analytical grade Linagliptin, its purity was 99.25%, (Simson Pharma Limited—China),

Methanol (99.9%, ACROS).

Para-Dimethylaminobenzaldehyde (PDAB) 5% (w/v) (Scharlau-)

Hydrochloride acid (HCl) 37% (SCP science)

A stock solution (1000 µg/mL): was weighed 50mg of LNG in 50 mL of distilled water.

A working solution (100 µg/mL): 10 mL of stock solution is diluted to 100 mL with distilled water.

ρDAB 5% (w/v): 1.25 g was dissolved in 25 mL of methanol with good shaking, and was freshly prepared.

Aliquots volume of working solution LNG were moved into series of 10 mL volumetric flasks to perform final concentrations of 5–45 ppm. To each flask was added 1 mL of ρDAB 5% (w/v) and 2 mL of HCl 37%, then to the water bath at 70–75° for 35 min after closed and shaking very well, after those flasks were cooled and diluted to 10 mL by distilled water. The maximum absorption of the yellow color was 407 nm upon the blank. The amount of linagliptin was calculated from the calibration curve.

This research was approved by the Damascus University Faculty of Pharmacy deanship.

Our study protocol was reviewed and approved by the Damascus University Faculty of Pharmacy, Damascus, Syria.

The analysis of the results that were gathered in the course of the research paper was sustained on four levels: the absorption spectra, Optimization of Reaction conditions, Stoichiometry of reaction and Validation of the developed method.

The absorption spectra of LNG (20 µg/mL) were recorded on a vis spectrophotometer in the wavelength region of 350–700 nm. which shows the absorption maxima curve at 407 nm in Fig. 2.

Absorption spectrum of the derivative product against a reagent blank (20 µg/mL LNG).

Many solvents such as distilled water, methanol, ethanol, and acetonitrile were chosen for solved LNG and PDAB as solvents for both LNG, and PDAB respectively, and as potential diluting media. Distilled water and methanol were found to be the optimum solvent for both LNG and PDAB respectively, and the highest absorbance values and the stability of the Schiff base formulated were obtained.

The reaction between LNG and the increase of different PDAB concentrations (1–7% w/v) was studied. It was found that absorbance increases with increasing PDAB concentration and reaches its maximum value by using 5% w/v of reagent shown in Fig. 3a.

Optimization of Reaction conditions. (a) Effect of PDAB concentration (dashed line) and volume (dotted line) on the reaction of LNG with PDAB. (b) Effect of temperature (dotted line) and time (dashed line) on the reaction of LNG with PDAB. (c) Effect of HCl volume on the reaction between LNG (20 µg/mL) and PDAB 5%. (d) Stability of the reaction product.

The researcher studied the suitable of PDAB volume in the range of 0.5–3 mL, to find the suitable volume of PDAB reagent (5% w/v). So, the result had shown that the highest absorption intensity was achieved at a PDAB volume of 1 mL then it decreased (Fig. 3b).

The researcher tested different volumes of HCI 37% (1–3 mL), to select the appropriate acidic medium volume for LNG and PDAB reaction. The best results were obtained with 2 mL of HCl 37% (Fig. 3c).

To choose the best temperature which can achieve the objectives of the research, many different temperatures range (25, 60–65, 70–75, 80–85 °C) in the water bath was checked. Thus, it is possible to determine the best temperature range was 70–75 °C (Fig. 3b).

The effect of prolonged heating time on this reaction was controlled by monitoring the color development at different time intervals (10–60 min) at 70–75 °C. Maximum absorbance values were gained at 35 min (Fig. 3b).

For stability of the derivative product, it was tested by time intervals (5–130 min). The results showed that the derivative compound needs 15 min to reach the great absorption value and then the values remain constant for an hour (Fig. 3d).

The quantitative reaction rate was recorded by Job's Method of Continuous Variations and the Molar ratio method14.

Preparation of Linagliptin Standard Solution (2 × 10–3 mol/l) by weighted 94.5 mg of LNG in 100 mL of distilled water.

Preparation of ρDAB (2 × 10–3 mol/l) solution by dissolving 29.8 mg in 100 mL of methanol with good shaking, was freshly prepared.

The continuous variation plot in Fig. 4a indicated that a molar fraction of 0.5 meant the ratio 1:1 of LNG: PDAB complex with 2 mL of 37% HCl for all flasks. While the Molar ratio plot in Fig. 4b, the highest absorbance was with LNG: PDAB complex ratio = 1. So, 1 mol of LNG interacts with 1 mol of PDAB.

Stoichiometry of reaction LNG and PDAB. (a) Job's method of continuous variations between LNG and PDAB. [LNG]: 2 × 10−3 M; [PDAB]: 2 × 10−3 M; [LNG] + [PDAB]: 4 mL + [HCl]: 2 mL. (b) Molar ratio method of stichometry of the reaction between LNG and PDAB. (c): Proposed reaction pathway between LNG and PDAB.

The scheme of reaction between LNG and the reagent is shown in Fig. 4c.

The method was validated according to the procedures described in ICH guidelines, which included linearity, precision, accuracy quantitation limit, and detection limit15.

Linearity was evaluated after determining the optimum conditions, with Beer's law used over the concentration, ranges 5–45 μg/mL, The calibration curve was formed by concentration versus absorbance, using linear regression analysis with an R2 value 0.9989 and regression equation was y = 0.0265x + 0.0602.

The limit of detection (LOD) and limit of quantitation (LOQ) were calculated according to the following formula:

where: SD is the standard deviation of the blank, and b is the slope. The LOD and LOQ were found to be 1.5815 and 4.7924 μg/mL, respectively (Table 1A)

The repeatability of the method was studied by measuring six replicate specimens of one concentration (20 µg/mL) of LNG within—day, with relative standard deviation RSD \(\le \hspace{0.17em}2\%\) .

Furthermore, the intermediate precision was evaluated by analyzing three replicate solutions of LNG at three different concentrations (10–20–30 µg/mL) during the same day (intra-day) and over three successive days (inter-day), where no significant difference between intra- and inter-day precision values was observed and RSD % values were less than 2 (Table 1B).

About the accuracy that was checked by the percent mean recovery and RSD % between three concentrations of LNG (10–20–30 µg/mL). Table 1B.

The researcher had checked the interference of excipients that might be formed the pharmaceutical dosage by using this method, in order to determine the specificity of proposed method.

Sample was prepared by mixing mannitol 20 mg, maize starch 30 mg, pregelatinized starch 30 mg, copovidone 5 mg and magnesium stearate 4 mg16. These excipients were analyzed by proposed method. The results were referred good recovery 101.157% and RSD = 0.8387%. These means no interference between excipients and determination of LNG in pharmaceutical dosage by this derivative method.

The robustness of proposed method was examined, the results indicated that there were no bias in the experimental conditions (variation concentration of PDAB ∓ 0.2% (w/v) also volume of PDAB and HCl ∓ 0.2 mL, the heating time ∓ 2 min, measurement wavelength (nm) ∓ 2 nm, while others parameter were constant so RSD was less than 2%.

Linagliptin is a new oral antidiabetic agent. LNG isn't available a specific analytical method in pharmacopeia. LNG has many articles that aim to assay LNG by HPLC, this method needs expensive solvents and specific equipments. So, this paper was described a new, simple analytical method depending in condensation reaction between LNG with PDAB as a chemical reagent for determination LNG in bulk and pharmaceutical dosage. The conditions of optimal conditions of this analytical research were studied and it was found that distilled water and methanol were the best solvents for both LNG and PDAB, 1 mL of PDAB 5% as a derivative reagent with 2 mL of HCl 37% as an acidic medium with heating to 70–75 °C on a water bath for 35 min to form the yellow Schiff base with a wavelength at 407 nm and the stability of Schiff base formed for one hour. Validation of the proposed method was showed that this reaction has linearity 5–45 μg/mL, according to the correlation coefficient R2 = 0.9989 with percent recovery (99.46–100.8%) within an accepted criteria and RSD was less than 2%, so this proposed method has good accuracy and precision and high specificity.

Furthermore, the molar ratio of this reaction was selected between LNG and PDAB it was (1:1) complex depending on two methods the Job of continuous variations and molar ratio.

The proposed derivation method was characterized by the use of a PDAB reagent that is easy to apply and does not consume expensive materials compared to other analytical methods that require many devices and materials.

The data can be made available upon reasonable request from the corresponding author.

ρ-Dimethylaminobenzaldehyde

Linagliptin

Incretin hormones glucagon-like peptide-1

Glucose-dependent insulinotropic polypeptide

Food and Drug Administration

British pharmacopeia

United States Pharmacopeia

Ultra-violet

Visible

Capillary electrophoresis

High-performance liquid chromatography

Hydrochloride acid

Weigh/volume percent

International conference of harmonization

Relative standard deviation

Standard deviation

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Department of Analytical and Food Chemistry, Faculty of Pharmacy, Damascus University, Damascus, Syria

Lujain Sahloul & Maisam Salami

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L.S.: Data curation; Investigation; Resources; Validation; original draft; Writing—review & editing; Methodology; Project administration. M.S.: Investigation; Resources; Validation; original draft; Writing—review & editing; Methodology; Supervision; Project administration. All authors have read and approved the manuscript.

Correspondence to Lujain Sahloul.

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Sahloul, L., Salami, M. Development and validation of a new analytical method for determination of linagliptin in bulk by visible spectrophotometer. Sci Rep 13, 4083 (2023). https://doi.org/10.1038/s41598-023-31202-w

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Received: 21 July 2022

Accepted: 08 March 2023

Published: 11 March 2023

DOI: https://doi.org/10.1038/s41598-023-31202-w

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