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Most chemical syntheses use highly reactive molecules, which are often DNA-reactive (mutagens). They may be present in the active substance (AS) or in the drug product as genotoxic impurities (GI). The mechanism of action of most GIs is based on DNA modification by direct binding (electrophilic - alkylating agents), insertion into the DNA chain (topoisomerase inhibitors) and antimetabolites (purine/pyrimidine analogs). Structural alerts (SA) for carcinogenic activity are defined as functional groups or substructures of compounds associated with carcinogenic activity (1). SA indicates a chemical group that causes toxic effects through one or several common mechanisms of action. GIs can bind directly to DNA or after metabolic transformations (oxidation and reduction), thus the SA structure may indicate the formation of several toxic metabolites. The risk assessment for the presence of GIs is a mandatory part in the module 3 (AS/DP impurity profile) submitted in the marketing authorization procedure, also in the approval of clinical trials. Guideline ICHM7(2) provides recommendations for identification, categorization, qualification and control strategy of mutagenic impurities in order to limit the potential carcinogenic risk. According to ICHM7, the classification (class 1-5) of all impurities is performed on the basis of data on carcinogenicity and bacterial mutagenicity (BM) from databases/scientific literature. If data are not available, computational toxicological assessment (QSAR) is performed and BM is predicted with two independent models. If any of model shows SA structures, a BM assay (Ames test) is performed. The high potent mutagenic carcinogens (aflatoxins, nitrosamines, alkyl-azoxy compounds) referred to as “cohort of concern”. U većini hemijskih sinteza se koriste veoma reaktivni molekuli koji su često i DNK – reaktivni (mutageni). Oni mogu biti prisutni u aktivnoj supstanci (AS) ili gotovom proizvodu (GP) kao genotoksične nečistoće (GN). Mehanizam dejstva većine GN se zasniva na modifikaciji DNK i to direktnim vezivanjem (elektrofili - alkilujući agensi), umetanjem u lanac DNK (inhibitori topoizomeraze) i antimetaboliti (purinski/pirimidinski analozi). Upozoravajuće strukture (SA) za kancerogenu aktivnost se definišu kao funkcionalne grupe ili delovi strukture jedinjenja koje su povezane sa kancerogenom aktivnošću (1). SA ukazuje na hemijsku grupu jedinjenja koja izaziva toksične efekte kroz jedan ili nekoliko obično zajedničkih mehanizama delovanja. GN mogu direktno da se vezuju za DNK ili nakon metaboličkih transformacija (oksidacija i redukcija) pa SA struktura može da ukaže na nastanak nekoliko toksičnih metabolita. Procena rizika na prisustvo GN u delu koji se odnosi na profil nečistoća AS/GP je obavezan deo dokumentacije o kvalitetu leka koji se podnosi u postupku registracije leka, kao i odobravanja kliničkih ispitivanja. U smernici ICHM7(2) su date preporuke za identifikaciju, kategorizaciju, kvalifikaciju i kontrolnu strategiju mutagenih nečistoća kako bi se ograničio potencijalni kancerogeni rizik. Prema ICHM7 se vrši klasifikacija (klase 1-5) svih nečistoća na osnovu podataka o kancerogenosti i bakterijskoj mutagenosti iz baza i naučne literature. Ako podaci nisu dostupni sprovodi se in silico toksikološka procena (QSAR) i predviđa se bakterijska mutagenost sa dva nezavisna modela. Ako jedan od modela pokaže SA strukture, radi se test povratne mutacije na bakterijama (Ames test). Posebno se razmatraju visoko potentni mutageni kancerogeni (aflatoksini, nitrozamini, alkil-azoksi jedinjenja). VIII Kongres farmaceuta Srbije sa međunarodnim učešćem, 12-15.10.2022. Beograd

Ziprasidone, bensiothiasol piperazynylindolone derivative is second generation antipsychotic drug used for the treatment of schizophrenia and in acute maniac/mixed episodes associated with bipolar disorder. It has unique G-protein coupled receptor binding profile with relatively low propensity for weight gain (1). Recently, it became an official active pharmaceutical ingredient in European Pharmacopoeia, where there are three official chromatographic systems, one for the assay and two other for early-eluiting and late-eluting impurities. Therefore, the purpose of this investigation was to develop and validate a fast, highly sensitive UHPLC-MS/MS method for the analysis of ziprasidone and its five impurities, significantly differing in polarity and pKa. Separation was performed using Thermo ACCELA UHPLC system (Thermo Scientific, Waltham, MA, USA) equipped with triple quad Mass Spectrometer Thermo TSQ Quantum Access Max (Thermo Scientific, Waltham, MA, USA) with a heated electro-spray ionization interface. Satisfactory chromatographic separation was achieved using a gradient elution with mobile phase A (10mM ammonium formate buffer, pH 4.7) and mobile phase B (acetonitrile) on a Acquity UPLC BEH C18 (50×2.1 mm, 1.7 μm) column with mobile phase flow rate of 300 μL/min. Sample injection volume was 10 μL. The analysis runtime was 7 minutes. The method was validated according to the International Conference of Harmonization (ICH) guidelines and validation included parameters such as specificity, linearity, accuracy, precision, limit of quantification and limit of detection. The proposed rapid and sensitive method is convenient and reliable for the assay and purity control in raw materials and in dosage forms (2). Ziprasidon, derivat benzizotiazol piperazinilindolona, je antipsihotik druge generacije koji se koristi za lečenje šizofrenije, kod akutnih maničnih ili mešovitih epizoda povezanih sa bipolarnim poremećajem. Ima jedinstveni profil vezivanja za G protein-spregnute receptore (GPCR) i relativno retko neželjeno dejstvo povećanja telesne težine (1). Nedavno je monografija dve soli ziprasidona postala oficinalna u Evropskoj farmakopeji u kojoj se ispitivanje vrši pomoću tri hromatografska sistema: ispitivanje sadržaja i dva odvojena sistema za više i manje polarne nečistoće. Svrha ovog istraživanja bila da se razvije i validira brza i visoko osetljiva UHPLC-MS/MS metoda za istovremeno ispitivanje ziprasidona i njegovih pet nečistoća, koje se značajno razlikuju po polarnosti i pKa. Hromatografska analiza je vršena na Thermo ACCELA UHPLC sistemu koji je spregnut sa tripl kvadrupolskim masenim analizatorom Thermo TSQ Quantum Access Max (Thermo Scientific, Valtham, MA, USA) sa elektrosprej jonizacijom na povišenoj temperaturi (HESI) kao jonskim izvorom. Zadovoljavajuće razdvajanje dobijeno je korišć enjem gradijentog eluiranja sa mobilnom fazom A (10 mM amonijum-formijat, pH 4,7) i mobilnom fazom B (acetonitril) na Acquity UPLC BEH 50×2,1 mm, 1,7 μm (Waters) stacionarnoj fazi na temperaturi od 30ºC i pri protoku mobilne faze od 300 μL/min. Zapremina injektovanja ispitivanih rastvora bila je 10 μL. Trajanje analize je 7 minuta (2). Metoda je validirana u skladu sa ICH (International Council for Harmonisation) smernicom i pokazana je specifičnost metode, linearnost, tačnost, preciznost, limit kvantifikacije i limit detekcije. Potvrđeno je da se brza i osetljiva UHPLC-MS/MS metoda može primeniti za ispitivanje ziprasidona i njegovih nečistoća u aktivnoj supstanci i doziranim oblicima. VIII Kongres farmaceuta Srbije sa međunarodnim učešćem, 12-15.10.2022. Beograd

Guideline ICHQ2(R1) (June 1995) provides general recommendations about performing the validation of standard analytical procedures (HPLC/TLC/GC) used for testing the active substance (AS)/finished product in order to show that the method is suitable for its intended use (ensuring the quality of the medicine). The development of a new guideline ICHQ14 Analytical procedure development is underway, which consequently led to a revision of the guideline ICHQ2 in the R2 version (comments until July 2022). The aim of this paper is to present an overview of new regulatory elements that should be considered during the validation of the analytical procedure. In relation to R1, ICHQ2(R2) provides recommendations for performing/evaluating various validation tests taking into account the specificity of each analytical procedure. Validation principles covering the use of spectroscopic/spectrometric data (e.g. NIR/Raman/NMR/MS) are described, some of which often require multivariate statistical analyzes. Guideline-R1 referred to the most frequently tested specification/process parameters (identification, testing of impurities quantitatively/limit test, assay (1,2)). R2 describes the methodology for performing validation for testing: elemental impurities (ICP-OES/ICP-MS), genotoxic impurities (LC/MS), dissolution testing (HPLC), particle size determination, core tablet assay by NIR (e.g. in continuous manufacturing), quantitative 1 H-NMR for the assay of AS. The validation methodology used in specific tests for biological/biotechnological drugs for biological assay (ELISA, SPR) and impurity testing-quantitative PCR, is also described. If scientifically justified, relevant data from development studies (ICHQ14) can be used instead of validation or the validation process can be abbreviated when an already established platform analytical procedure is used for a new purpose. Smernica ICHQ2(R1) (jun 1995.) daje opšte preporuke o načinu izvođenja validacije standardnih analitičkih metoda (HPLC/TLC/GC) koje se koriste za ispitivanje aktivne supstance (AS) i gotovog proizvoda što ima za cilj da pokaže da je metoda pogodna za predviđenu svrhu (obezbeđenje kvaliteta leka). U toku je razvoj nove smernice ICHQ14 o razvoju analitičkih metoda što je posledično dovelo do revizije smernice ICHQ2 u verziju R2 (na javnoj raspravi do jula 2022.). Cilj ovog rada je da se prikaže pregled novih regulatornih elemenata koje treba razmatrati u toku validacije analitičke procedure. U odnosu na R1, ICHQ2(R2) daje preporuke za izvođenje i procenu različitih testova validacije uzimajući u obzir specifičnost svake analitičke procedure. Biće opisani postupci validacije koji pokrivaju upotrebu spektroskopskih/spektrometrijskih podataka (npr. NIR, Raman, NMR ili MS) od kojih neki često zahtevaju multivarijantne statističke analize. Smernica-R1 se odnosila na najčešće ispitivane specifikacijske/procesne parametre (identifikacija, ispitivanje nečistoća kvantitativno/limit test, sadržaj AS (1,2)). Verzija R2 opisuje metodologiju izvođenja validacije za ispitivanje elelmentalnih nečistoća (ICP-OES/ICP-MS), ispitivanje genotoksičnih nečistoća - LC/MS, ispitivanja brzine oslobađanja aktivne supstance-HPLC, određivanje veličine čestica, validacija NIR metode za ispitivanje sadržaja AS u tabletnom jezgru (npr. kod kontinuirane proizvodnje), validacija H-NMR za određivanje sadržaja AS. Opisana je i validaciona metodologija koja se primenjuje kod spefičnih testova za biološke/biotehnološke lekove za određivanje sadržaja AS (ELISA, SPR) i ispitivanje nečistoća-PCR. Ako je to naučno opravdano, odgovarajući podaci dobijeni iz razvojnih studija (ICHQ14) mogu se koristiti umesto validacije ili postupak validacije može biti skraćen kada se već utvrđena platforma analitičke procedure koristi u novu svrhu. VIII Kongres farmaceuta Srbije sa međunarodnim učešćem, 12-15.10.2022. Beograd

Ziprasidone (ZIP) is the second generation antipsychotic drug with unique G-protein-coupled (GPCR) receptor binding profile. It is a highly lipophilic and unstable compound. Our group developed and validated the single liquid chromatographic (LC) system for simultaneous determination of ZIP and its five main impurities (IMPs) and we modelled the Quantitative Structure Retention Relationship (QSRR) of the additional ten compounds including unknown detected degradant. One of two proposed structures were confirmed by UPLC-MS/MS study. The further characterisation of unknown degradant was performed with NMR studies as un versatile tool for characterisation of each compound and it is presented. Through several experiments which consists of investigation of chemical shitfs in NMR spectra of ZIP degradation products the structure of unknown degradant was proposed and confirmed as in previous experiments. 16th International Conference on Fundamental and Applied Aspects of Physical Chemistry, September 26-30, 2022 Belgrade, Serbia