Validated HPLC Method for the Determination of Linagliptin in Rat Plasma. Application to a Pharmacokinetic Study

Journal of Chromatographic Science, 2016, Vol. 54, No. 9, 1573–1577 doi: 10.1093/chromsci/bmw106 Advance Access Publication Date: 26 June 2016 Article Article A Validated HPLC Method for the Determination of Linagliptin in Rat Plasma. Application to a Pharmacokinetic Study Abeer Hanafy1,2 and Hoda Mahgoub3,4,* 1 Department of Pharmacology and Toxicology, Faculty of Pharmacy, King Abdulaziz University, Jeddah 80260, Saudi Arabia, 2Department of Pharmacology, Faculty of Veterinary Medicine, Kafrelsheikh University, Kafrelsheikh 33516, Egypt, 3Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdulaziz University, Jeddah 80260, Saudi Arabia, and 4Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, University of Alexandria, Alexandria 21521, Egypt *Author to whom correspondence should be addressed. Email: Received 15 May 2016 Abstract A sensitive and reproducible HPLC method for the determination of linagliptin (LNG) in rat plasma was developed and validated using pindolol (PIN) as the internal standard. Both LNG and PIN were separated on a Zorbax Eclipse XDB C18 column kept at ambient temperature using as mobile phase a combination of 75% methanol: 25% formic acid 0.1% pH 4.1 at a flow rate of 1.0 mL min−1. UV detection was performed at 254 nm. The method was validated in compliance with ICH guidelines and found to be linear in the range of 5–1,000 ng mL−1. The limit of quantification (LOQ) was found to be 5 ng mL−1 based on 100 µL of plasma. The variations for intra- and inter-assay precision were <10%, and the accuracy values were ranged between 93.3 and 102.5%. The extraction recovery (R%) was >83%. The assay was successfully applied to an in vivo pharmacokinetic study of LNG in rats that were administered a single oral dose of 10 mg kg−1 LNG. The maximum concentration (Cmax) and the area under the plasma concentration–time curve (AUC0–72) were 927.5 ± 23.9 and 18,285.02 ± 605.76 ng mL−1, respectively. Introduction Diabetes mellitus (Type 2 ( presents ∼95% of all diabetes cases that shows increasing incidence. The complications of this disease will significantly reduce the life expectancy (1). In May 2011, the US Food and Drug Administration has approved Tradjenta (linagliptin) tablets, used with diet and exercise, to improve blood glucose control in adults with Type 2 diabetes (http://www.hhs.gov/morgan. ). Linagliptin (LNG), 8-[(3R)-3-aminopiperidin1-yl]-7-(but-2-yn-1-yl)-3-methyl-1-[(4-methylquinazolin-2-yl) ethyl]-3, 7-dihydro-1H-purine-2,6-dione] (Figure 1) belongs to dipeptidylpeptidase-4 inhibitor class (2, 3). DPP-4 inhibitors represent a new therapeutic approach for the treatment of Type 2 diabetes that functions to stimulate glucose-dependent insulin release and reduce glucagon levels. This is done through inhibition of the inactivation of incretins, particularly glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP), thereby improving glycemic control (4). Linagliptin is not extensively metabolized, 90% of dose is excreted unchanged. The small portion of drug that is metabolized, the main metabolite is CD1790 and is pharmacologically inactive with respect to DPP-4 inhibition (5). High performance liquid chromatography (HPLC) is considered a valuable separation technique for the quantitative determination of analytes in complicated biological matrices. The development of an analytical method for the elimination of matrix interferences is very essential for a successful HPLC analysis of drugs in biological fluids. The literature revealed few methods developed for the determination of LNG by HPLC technique (6–11). Some of these methods were only applied for LNG assay in pharmaceutical tablets with a LOQ not <0.03 µg mL−1 (6–8). Other methods are using HPLC coupled to tandem mass spectrometric technique, (HPLC–MS/MS) which are © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: 1573 1574 Hanafy and Mahgoub Standard and working solutions Stock standard solutions of LNG (1 mg mL−1) and PIN (1 mg mL−1, IS) were prepared by dissolving the appropriate amount of pure substance in methanol. Working standard solutions were obtained by diluting the stock standard solutions with the mobile phase. All standard solutions were stored at 4°C. Matrix calibration curves were daily prepared in drug-free rat plasma and appropriate volumes of the working standard solutions and the drug-free rat plasma were added to test tubes in order to achieve concentrations ranging from 5 to 1,000 ng mL−1. Quality control (QC) samples were independently prepared in the same way to achieve final concentrations of 10, 100 and 1,000 ng mL−1. QC samples were prepared from a stock solution that was different from the one used to generate matrix calibration curve samples. These QC samples were run in each assay to investigate intra- and inter-run variations. Sample preparation Figure 1. Chemical structure of linagliptin and pindolol. sophisticated and much more sensitive and selective analytical methods (5, 9–11). However, these methods are not affordable for most routine laboratories due to their special requirements and high equipment cost. In addition, these methods are mainly designed for human biological samples in a relatively large volume of plasma. Rats could be considered ideal for the preclinical pharmacokinetic studies of drugs because of their small size, low cost, and ease in handling. Under the scope of this view, the aim of this work was to develop and validate an accurate and reproducible HPLC method for the determination of LNG with high sensitivity in rat plasma. The method will be validated in compliance with ICH guidelines (12) and involves a single extraction step of a very small plasma volume (100 µL). It has been successfully applied in a pharmacokinetic study of LNG in rats. Experimental Materials and reagents HPLC grade reagents (methanol, ethylacetate, formic acid, sodium hydroxide) were obtained from BDH Chemicals Ltd, Poole, Dorset, England. LNG analytical standard (99.6%) was obtained from Eli Lilly and Company, Indianapolis, IN, USA and PIN was purchased from SIGMA, Steinheim, Germany. Equipment An HPLC system Agilent 1100 series (Agilent Technologies, Santa Clara, USA) was used and was consisted of a G1314A variable wavelength UV detector, G1321A fluorescence detector, G1311A quaternary pump, G1379A microvaccum degasser and G1313A autosampler. The HPLC system control and data processing was performed by Chemstation (Agilent Technologies). Liquid chromatographic conditions The analytes of interest were separated on a Zorbax Eclipse XDB- C18, 250 × 4.6 mm, 5 µm column (Agilent Technologies), kept at ambient temperature and protected by a precolumn (Zorbax Extend-C18). The mobile phase was consisted of methanol:formic acid 0.1% pH 4.1 (75:25 v/v%) and delivered at a flow rate of 1.0 mL min−1. The UV detector was set at 254 nm. Plasma sample (100 (...truncated)