## 1 Introduction  
Drug safety and effectiveness are important concerns due to potential adverse effects through their degradation products on patient health and the environment [1, 2]. Investigation of possible molecular changes in a drug molecule over time without interference from excipients, impurities, and degradation products (DPs) is required [3]. Excipients used to prepare the pharmaceutical form of a drug and/or inappropriate environmental conditions can also promote the degradation of a drug substance [2]. For instance, primary and secondary amines can react with carbohydrates to form glycosylamines. Many of the reported drug-excipient reactions involve hydrolysis, oxidation, or a specific interaction of drugs with reactive impurities in excipients [2, 4]. The impurities and potential DPs may cause changes in chemistry, and the pharmacological and toxicological properties of drugs having significant impact on product quality and safety [5, 6]. Furthermore, the degradation molecules and their concentration levels have a vital importance not only for a patient's safety but also other living things exposed to the molecules via environmental factors such as soil, water, vegetables, and fruits [1]. It should be noted that even if concentrations found in the environment are at generally low levels, medicinal products are developed to be highly potent substances, and thus concentration levels on their own are not the precise indication of the associated risks [1, 7]. The risk factors are associated with the nature of a substance such as degradability, toxicity of a compound, exposure duration, toxicity of metabolites, and physicochemical properties. For these reasons, forced degradation (stress-testing) studies of pharmaceutical preparations became necessary to assure degradation mechanisms and potential DPs [8; 9; 10]. The authorities such as FDA and ICH guide stability tests to figure out the quality of a drug substance and drug product. According to the regulatory guidelines, the stress factors must include acid and base hydrolysis, oxidation, thermal degradation, photolysis, and freeze-thaw cycles and shear [11; 12]. In this study, the degradation behavior of pemetrexed (PMT), a structure shown in Figure 1, is investigated under stress conditions such as oxidative and hydrolytic stress following ICH guidelines [11]. PMT is an antifolate chemotherapy drug which is first-line treatment for patients with advanced non-squamous, non-small cell lung cancer. PMT applies its effect by disrupting folate-dependent metabolic processes essential for cell transcription [13]. It acts by inhibiting thymidylate synthase, dihydrofolate reductase, and glycinamide ribonucleotide formyl transferase enzymes used in purine and pyrimidine synthesis [14]. PMT prevents formation of DNA and RNA, which are required for the growth and survival of both normal and cancerous cells. The degradation profiles of PMT using stability-indicating methods stated by guidelines are also reported in the literature on high-performance liquid chromatography (HPLC) and HPLC coupled with tandem mass spectrometry (MS/MS) [15; 16; 17; 18; 19; 20; 21; 22; 23]. In most of these studies, the decrease in the concentration of the parent and increase in DPs are monitored under stress conditions. In a few studies, DPs were investigated after their isolation using preparative chromatography, and characterized by mass spectroscopy and NMR techniques for confirmation [18; 23]. Additionally, some of the studies of PMT and its metabolites in biological fluids have been conducted using LC/MS methods [24; 25; 26; 27]. [PERSON] et al. [18] and [PERSON] et al. [15] identified several PMT DPs through oxidation and hydrolysis using HPLC and NMR. First, the DPs were separated in a reversed-phase preparative HPLC method, then they were identified with NMR. In another study, [PERSON] et al. also identified only two potential DPs, des-glutamate and glutamic acid [19].