Proposal Registration Code	PN-III-P1-1.1-PD-2021-0117
Project Registration Code	PD 30/2022
Project Acronym	IMP_DPP_IV
Purpose and project planning	The project aims to develop novel analytical methods for the determination of chiral and achiral impurities of dipeptidyl peptidase-4 (DPP-IV) inhibitors, using high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) separation techniques. The use of cyclodextrins as chiral selectors in CE, and polysaccharide-, macrocyclic antibiotic-, and cyclodextrin-based chiral stationary phases in HPLC along with the application of aqueous background electrolytes and reversed-phase chromatographic conditions, respectively, could contribute to the development of efficient and environmentally friendly chiral separation methods allowing the determination of enantiomeric purity in bulk and formulation samples. Simultaneous separation methods for the determination of synthesis-related impurities and degradation products of DPP-IV inhibitors will also be developed. The impurity and degradation profiling will be performed, while HPLC-mass spectrometry and other additional techniques will be employed to characterize the structures of the products formed during forced degradation. In both chiral and achiral method development, the methodologies of the Quality by Design concept will be implemented, in order to obtain reliable and robust separation methods applicable in the routine drug analysis.
Project Start Date	01.04.2022
Project End Date	31.03.2024
Project Duration	24 months
Total budget value	250000 lei
Funding	Ministry of Research and Innovation, CNCS-UEFISCDI
Main objectives	Development of separation methods for the enantiomeric purity analysis of DPP-IV inhibitors. Development of analytical methods for the analysis of organic impurities and structural characterization of the degradation products of DPP-IV inhibitors.
Team:	Name	Role
	Lajos Attila Papp	Project leader
	Gabriel Hancu	Mentor
Results	Developed and validated chiral and achiral separation methods for the impurity analysis of DPP-IV inhibitors.
Host institution	George Emil Palade University of Medicine, Pharmacy, Science, and Technology of Targu Mures

Development of enantio- and chemo-selective separation method for the simultaneous analysis of the chiral and achiral impurities of vildagliptin: A new, reversed-phase HPLC method was developed for the simultaneous determination of the dipeptidyl-peptidase-IV-inhibitor antidiabetic drug vildagliptin (VIL) enantiomeric impurity and four other achiral related impurities. Among the four related substances,  dehydroxy-VIL and VIL-dimer are two important process-related impurities, while VIL-amide and VIL-diketopiperazine can be considered to be the main degradation products of VIL An initial screening was performed on five polysaccharide-type chiral stationary phases (Lux Amylose-1, Lux Amylose-2, Lux-Cellulose-1, Lux-Cellulose-2, Lux-Cellulose-3) in polar organic mode with methanol, ethanol, 2-propanol, or acetonitrile containing 0,1% diethylamine as mobile phase to identify the best conditions for the separation of VIL enantiomers. Lux-Cellulose-2 column was found to provide the best chiral resolution for VIL enantiomers. Further experiments were conducted using different aqueous-organic mobile phases to achieve the simultaneous chiral-achiral separation of the selected compounds. Experimental design-based optimization was performed by using a face-centered central composite design. The optimal separation conditions (Lux Cellulose-2 stationary phase, 45 ◦C, mobile phase consisting of methanol/water/diethylamine 80:20:0.2 (v/v/v), and 0.45 mL/min flow rate) provided baseline separation for all 6 compounds. The optimized method was validated according to the ICH guideline and proved to be reliable, specific, linear, precise, and accurate for the determination of at least 0.1% for all impurities in VIL samples. The validated method was applied for determinations from a commercially available drug formulation and proved to be suitable for routine quality control of both enantiomeric and organic impurities of VIL.

Development of enantiomeric separation method of vildagliptin by capillary electrophoresis – study of enantiomeric complexation A systematic screening of 16 CD derivatives as chiral selectors was performed at three pH levels using phosphate (pH 2.5, pH 7.0) and acetate (pH 4.5) buffers. Native α-CD at acidic pH proved to be the most suitable separation medium for VIL enantiomers with favorable migration order (R-VIL followed by S-VIL). Method optimization was performed by experimental design approach, by examining the effect of such parameters as the concentration of buffer and CD, buffer pH, capillary temperature, and the applied voltage on the chiral resolution and analysis time. The optimized analytical conditions (75 mM acetate buffer pH 4.5, containing 50 mM α-CD, 18 kV applied voltage, and 15°C capillary temperature) provided baseline separation of VIL enantiomers within 9 min. The analytical performances of the optimized method were determined and the method was applied for the determination of enantiomeric purity of VIL from pharmaceutical products. The complexation mechanism between VIL enantiomers and α-CD responsible for chiral recognition was studied by NMR experiments and molecular docking, and the possible interactions responsible for the chiral recognition have been described.

Determination of the degradation profile of alogliptin benzoate (ALO) and the study of its chemical interactions with different pharmaceutical excipients: A new reversed-phase HPLC-UV method with gradient elution was developed for the purity analysis of ALO. The method was validated according to the ICH guidelines and proved to be applicable in forced degradation studies as well as in accelerated stability studies in the presence of different pharmaceutical excipients. During the impurity profiling of ALO the applied stress conditions were optimized in order to obtain a comprehensive view of the possible degradation pathways and mechanisms of the compound. The most important degradation products of ALO were detected and their chemical structure was elucidated using LC-MS technique. Moreover, an accelerated stability study of the drug in the presence of pharmaceutical excipients having different physicochemical properties was performed. The mixture samples were analyzed by FT-IR spectroscopy and LC-MS to identify the possible chemical interactions between the drug and the different excipients studied. An important interaction of ALO with an excipient (lactose) was identified and its mechanism has been described.

A review article was published regarding the utilization of chiral stationary phases (CSPs) in the context of single-column simultaneous chiral–achiral HPLC separation methods. While CSPs have traditionally been pivotal for enantioselective drug analysis, contemporary CSPs often exhibit notable chemoselective properties. Consequently, there is a discernible trend towards the development of methodologies that enable simultaneous enantio- and chemoselective separations utilizing a single CSP-based chromatographic column. The review provides an exhaustive overview of reported HPLC methods in this domain, with a focus on four major CSP types: cyclodextrin-, glycopeptide antibiotic-, protein-, and polysaccharide-based CSPs. This work delves into the diverse applications of CSPs, encompassing various chromatographic modes such as normal phase, reversed-phase, and polar organic mode. A critical discussion on method development is presented, emphasizing the additional chemoselective separation mechanisms of CSPs. It also explores possibilities for method optimization and development, concluding with future perspectives on this evolving field. Despite the inherent challenges in understanding the retention mechanisms involved in chemoselective separations, this review highlights promising trends and anticipates a growing number of simultaneous enantio- and chemoselective methods in pharmaceutical analyses, pharmacokinetic studies, and environmental sample determinations.

Publication in extenso in ISI-quoted journals with impact factor:

Papp LA, Hancu G, Szabó ZI. Simultaneous determination of enantiomeric and organic impurities of vildagliptin on a cellulose tris(3-chloro-4-methylphenylcarbamate) column under revered-phase conditions. J Pharm Biomed Anal. 2023, 234:115495. doi: 10.1016/j.jpba.2023.115495, IF 3,4

Papp LA, Hancu G, Szabó ZI, Székely-Szentmiklósi B, Gáti T, Fiser B. Kraszni M, Tóth G. Chiral Separation of Vildagliptin by Capillary Electrophoresis—The Study of Enantiomeric Complexation. Symmetry (Basel). 2024, 16, 17, doi:10.3390/sym16010017, IF 2,7

Papp LA, Szabó ZI, Hancu G, Farczádi L, Mircia E. Comprehensive Review on Chiral Stationary Phases in Single-Column Simultaneous Chiral–Achiral HPLC Separation Methods. Molecules. 2024, Vol. 29, Page 1346 2024, 29, 1346, doi:10.3390/MOLECULES29061346, IF 4,6

Communications at scientific conferences:

Papp LA, Hancu G, Szabó ZI, Székely-Szentmiklósi B, Tóth G, Kraszni M. Determination of enantiomeric purity of vildagliptin by capillary electrophoresis 21st International Symposium and Summer School on Bioanalysis c10-15.07.2023,  Târgu Mureș (poster).

Papp LA, Szabó ZI, Hancu G. Simultaneous determination of chiral and achiral impurities of vildagliptin by high-performance liquid chromatography 31st Scientific session of the Medical and Pharmaceutical Sciences Section of Transylvanian Museum Society, 20-22.04.2023, Miercurea Ciuc (oral presentation).