Some specific examples of drug-excipient impurity interactions performed internally. Benzyl alcohol, glycerin, hydrogen peroxide and reference standards were procured from sigma Aldrich, USA. HPLC grade Acetonitrile, methanol, and ortho phosphoric acid, Ammonium acetate, acetic acid were purchased from Merck, Darmstadt, Germany. Water used was obtained by using Millipore MilliQ Plus water purification system. Novel Laboratories Inc., Somerset, New Jersey, supplied drug product/Drug substance samples.

Water HPLC with 2695 separation module equipped with 2996 PDA detector used for the HPLC method development. The output signal was monitored and processed using Empower-3 software. LC-MS/MS system (Acquity UPLC coupled with TQD mass spectrometer with empower software, Waters Corporation, Milford, USA) was used for the identification of unknown compounds formed during forced degradation and stability testing studies.

Flavors used in the formulation process are the most possible origins to form impurities due to the trace level presence of aldehydes and ketones that apparently used for its aroma. Famotidine oral solution forms unknown impurity due to Benzaldehyde, which is present in the excipient of cherry flavor used in the formulation process. Cherry flavor used in the famotidine oral solution contains very small amount of benzaldehyde that formed imine reaction with the terminal nitrogen of the famotidine (Figure 1). Hotha et al. described the identification and characterization of this unknown impurity by UPLC-MS/MS and NMR (Figure 1 and Figure 2). Molecular ions of 187 and 236 confirm the formation of sulfynyl amine attached to the benzaldehyde moiety. Aldehyde free cherry flavor recommended for the preparation of oral solutions for amine containing compounds [18] .

Benzyl alcohol used as a bacteriostatic preservative at low concentration in intravenous medications, cosmetics and topical drugs. The use of benzyl alcohol as a 5% solution has been approved by the US FDA in the treatment of head lice in children older than 6 months and in adults. Majority of the parenteral solutions use Benzyl alcohol as a preservative. There were several possible degradants associated with the oxidation of benzyl alcohol (Figure 3). Benzaldehyde and benzoic acid are the possible degradants observed in the parenteral solutions, which contains benzyl alcohol (Figure 4) [19] -[21] . These oxidation products of benzyl alcohol react in a solution, which will form benzaldehyde dibenzyl acetal (Figure 5).

Flavor containing Vanillin reacts with the Active Pharmaceutical Ingredient (API), having primary amine to form imines. The finished product is a ready to use liquid and oil based formulation [10] [17] . The aldehyde functionality of vanillin leads to form cis/trans-imines and to inversion of the chiral center (Figure 6).

Munnir N. Nasser et al. reported a degradation product in the BMS-204352 clinical formulation identified as the formaldehyde adduct of BMS-204352 (hydroxymethyl derivative, BMS-215842) (Figure 7). The formaldehyde spiking study supports the hypothesis that formaldehyde in the Polysorbate 80 and PEG 300 excipients used in the formulation result in formation of the formaldehyde adduct degradant [22] [23] . Limits may need to be established for reactive impurities in formulation excipients, to ensure acceptable levels of degradants and adequate product shelf life [24] .

Michal Dousa et al. [25] studied different pharmaceutical preparations against the common cold containing phenylephrine and saccharose. The formulations containing both phenylephrine and saccharose found to be susceptible

to loss of phenylephrine activity. There is evidence that the degradation of PHE resulted from condensation reactions of phenylephrine with aldehydes (such as formaldehyde or 5-HMF) (Figure 8 and Figure 9). The results indicate that phenylephrine is molecule, which should handle with special care during pharmaceutical processing to avoid decomposition and interactions. The new identified degradation products of phenylephrine found in pharmaceutical preparations against the common cold containing phenylephrine and saccharose.

David C. Dubost et al. reported degradation of a cyclic peptide drug in a lyophilized formulation occurs in the

solid state to give an aldehyde as an oxidation product [26] . The oxidative pathway does not require oxygen but does depend on the presence of impurities in the pharmaceutical excipient used in the formulation. A mechanism involving Schiff base formation, double bond isomerization, and subsequent hydrolysis has been proposed to account for the formation of the degradant. The extent of the formation of the impurity is influenced by the amount of mannitol used as an excipient in the formulation (Figure 10).

Sugar alcohols and parabens are commonly used ingredient in oral suspension formulations. Parabens are commonly used antimicrobial agents in pharmaceuticals, food and cosmetics. However, their possible incompatibility because of transesterification reaction is a concern during formulation development. During the stability, study of an experimental batch of an oralsuspension formulation that contained sorbitol and paraben preservatives shows approximately 1% degradation for the samples stored at 30˚C for one year (Figure 11). 3, 4, 5 carbon sugar alcohols reacts with methyl paraben to form 6 isomer products were identified by HPLC and NMR by Minhui Ma et al. [28] -[30] .

The reaction between 5-aminosalicylic acid (5-ASA) and the excipient citric acid (Figure 12) during storage of an experimental enema preparation observed three isobaric reaction products of an ester and an amide with non-symmetrically substituted citric acid moieties and a symmetrical amide. Larsen et al identified and characterized these products [31] . Approximately 5% of the 5-Aminosalicylic acid present in the formulation was transformed into these impurities. Because of the relatively rapid and extensive formation of reaction, production between 5-Aminosalicylic acid and citric acid the use of citric acid in liquid 5-Aminosalicylic acid formulations not recommended. Thus based on these findings further development of a formulation 5-Aminosalicylic acid contains citric acid was abandoned and replaced with another formulation.

Drug excipient interactions between 6-aminocaproic acid and the excipients citric acid were found in highly concentrated solutions at pH 4.0 and pH 5.0 [32] . In addition to that, several degradation products of the 6-aminocaproic acid reacting with itself, e.g., dimers trimers and cyclized forms observed (Figure 13). The utilization of citric acid as buffering species in liquid formulations requires careful evaluation on a case-by-case basis.

Cetirizine with the sorbitol and glycerol to form monoesters (Figure 14 and Figure 15). At a temperature as low as 40˚C more than 1% of the cetirizine content was transformed into a monoester within 1 week using concentrations similar to those used in marketed preparations [33] .

Transesterification reaction of methylphenidate with glycerin forms different structural isomeric products at the specified RRT of 0.75 and 0.77 in a HPLC method. The formation of these impurities monitored during forced degradation studies, excipient compatibility studies and in the stability conditions. Quantification of the stability samples were analyzed and observed that about 0.6% of the Methylphenidate content was transformed into methylphenidate-glycerin isomers within 3 Months at 40˚C/75% RH and 18 Months at 25˚C/60% RH conditions. Identificaiton and characterization of these impurities (Figure 16, Figure 17) by LC-MS/MS was performed [34] -[36] . The LC-MS/MS method was developed using Acquity UPLC BEH C18 1.7 µm, 2.1 × 150 mm column as stationary phase. The mobile phase A is a 0.1% of acetic acid in water. The mobile phase B is acetonitrile. The UPLC gradient program was set as: Time (min) % solution B: 00/15, 5.58/15, 26.92/40, 26.98/15, 35.00/15. The column temperature maintained at 40˚C and the injection volume was 15 μL Milli-Q water used as Diluent. The mobile phase pumped at 0.64 mL/min⁻¹. The eluted compounds monitored at 210 nm. The run time was 35 min. Mass spectrometric conditions optimized as cone gas 10 V, Collision flow 70 L/Hr., Ion energy 0.7 Entrance and Exit Potentials 1 V, source temperature 150˚C, Desolovation gas 800 L/hr., Desolvation

temperature 400˚C. Molecular ion [M + H]⁺ shows intense ion of 294 confirms the addition of glycerin moiety with Methylphenidate (Figure 18).

The solid state reaction between citric acid and carvedilol forms three citric acid esters and three citric acid amides with the two diastereomeric pairs RR and RS (Figure 19). When citric acid monohydrate replaced with

anhydrous citric acid, the amount of esters was reduced approximately eight folds. Presence of small amounts of water significantly speeds up the solid-state reaction. Jesper Larsen et al. [37] reported all the degradants and elucidated the structures by HPLC and NMR.

Buprenorphine shows unknown impurity in the Buprenorphine and Naloxone sublingual tablets due to the presence of citric acid in formulation. This was confirmed by the excipient compatibility studies. Characterization performed by LC-MS/MS (Figure 20 and Figure 21). The unknown impurity found in Buprenorphine Naloxone sublingual tablets is Impurity I according to EP monograph [38] . This impurity is formed due to the Dehydrative cyclization of Buprenorphine to form Furanyl ring (Figure 22). The molecular weight of the impurity is 435 whereas the molecular weight of the Buprenorphine is 467. Loss of methoxy group (32 amu) in the cyclization process for Buprenorphine to form the Impurity-I.

Tacrolimus is macrolide drug that is widely used as a potent immune suppressant. Tenja Rozman Peterka et al. [41] reported the formation of alpha-hydroxy acid from the parent tacrolimus through Benzilic acid type rearrangement reaction in the presence of magnesium stearate (Figure 27). This reaction occurred in the presence of divalent metallic cations at higher pH values.

Norfloxacin reacts with magnesium stearate forms stearamide adduct (Figure 28) through Stearoyl rearrangement [17] [42] [43] . Magnesium stearate is an excipient used in the tablet formulation. This adduct formation was observed after prolonged storage at 60˚C. Since Magnesium stearate derived from multiple sources, the presence of other fatty acids like palmitic acid, arachidic acid and behenic acids can lead to form more than one adducts.

Serajuddin et al. reported [44] that Fosinopril sodium forms three degradation products when tablet formulations lubricated with magnesium stearate (Figure 29). There were two distinct pathways of degradation where magnesium ion mediated through hydrolysis. There should be a limited amount of water in the formulation, which will reduce the drug excipient compatibility because it does not depend on the hygroscopicity of the individual components.

Patric et al. reported Duloxetine to react with polymer degradation products or residual free acids present in the enteric polymers hydroxypropyl methylcellulose acetate succinate (HPMCAS) and hydroxypropyl methylcellulose phthalate (HPMCP) in dosage formulations to form succinamide and phthalamide impurities, respectively (Figure 30) [45] . The rate of formation of the impurities is accelerated by heat and humidity. The structures were deduced using molecular weights obtained from LC comparison of UV spectra, HPLC retention times, and electrospray mass spectra to independently synthesized material. It is proposed that polymer-bound succinic and phthalic substituents can be cleaved from the polymer, resulting in the formation of either the free acids or the anhydrides. It is postulated that the reaction is enabled by migration of either the free acid or anhydride or the parent drug through the formulation. The formation of these impurities was minimized by increasing the thickness of the physical barrier separating the enteric coating from the drug.

Fluoxetine contains a secondary amine forms the Maillard reaction with lactose to form formyl fluoxetine identified as a major product of this Maillard reaction (Figure 31). Water content, lubricant concentration and temperature founds to influence the degradation. David D. Wirth et al. [46] [52] reported this degradation pattern which and formation of N-formyl fluoxetine through malliard reaction.

Cetirizine contains carboxylic acid group and forms lactose esters when reacts with the free hydroxy groups of the lactose present in Hydroxy Propyl Cellulose (HPC), Low Hydroxy Propyl Cellulose (Hyprolose or LHPC-31) and Micro Crystalline Cellulose (MCC) (Figure 32) [53] . LHPC-31 is low-substituted Hydroxypropyl ether of cellulose. It is non-ionic, less reactive to active ingredients, insoluble in water and alcohol but swells in water by holding water molecules around hydroxy propyl groups that are distributed on the cellulose backbone (Figure 33). Based on the Low Molar substitution and in solubility of LHPC-31 in water, the formation of Cetirizine ester with LHPC will be very non-significant levels when compared to HPC. The molar substitution value and number of glucose units attached to the LHPC vs. HPC makes less reactive towards Cetirizine.

Serajuddin et al. [44] reported that Ceronapril is an ACE inhibitor with a primary amine group interaction with mannitol and with dibasic calcium phosphate form the degradation products (Figure 34). Based on the drug excipient compatibility studies lactose and mannitol were excluded from the formulation.

Natasa et al. reported the impact of excipients on the stability of olanzapine and confirmed the formation of 2- methyl-5,10-dihydro-4H-thieno[2,3-b][1,5]benzodiazepine-4-one and 2-methyl-4-(4-methyl-4-oxidopiperazin1- yl)-10H-thieno [2,3-b][1,5]benzodiazepine, olanzapine N-oxide (Figure 35). Olanzapine is sensitive to temperature and moisture. In samples protected from moisture, the increase in concentration of impurity was shown to be highly temperature dependent and the degradation followed zero-order kinetics. The physical state of water in drug-excipient mixtures determines its role in drug?excipient interactions. Mannitol, LM and LA anhydrous are crystalline excipients. Upon exposure to humidity, water is adsorbed onto their surfaces and then attached to the polar groups located on them. It could be postulated that degradation of olanzapine in the presence of moisture is excipient mediated. In olanzapine-mannitol mixtures, the molecular mobility is small. In addition, differences found between LM and LA anhydrous could be explained by the fact that water activity in the anhydrous form is higher because it tends to form a hydrate that is thermodynamically more stable. LM contains about 5% (w/w)

of water which is incorporated in its crystal structure and therefore, not available for chemical reactions. On the other hand, the LA anhydrous contains only 1% (w/w). It could strongly adsorb water owing to the fact that formation of Lactose Monohydrate is a favored process. Therefore, anhydrous LA uses absorbed water to form the monohydrate thereby decreasing the amount of active water that can contribute towards the degradation of olanzapine. In the course of this study, it was discovered that the choice of tablet filler excipient plays an important role in the formulation of olanzapine film-coated tablets. Degradation of olanzapine film coated tablets was governed by temperature and moisture [54] [55] .

Paul A. Harmon et al. reported the trace levels of condensation products between lactose and the amine containing Hydrochlorothiazide (HCTZ) formed when a mixture of the two solids containing 30% weight water is heated at 60˚C for 2 weeks [56] [57] . The two most abundant condensation products were characterized (Figure 36) by LCMS. Under the relatively mild conditions of formation, the amine-lactose reaction products are limited to those involving the elimination of only a single molecule or water rather than the multiple water eliminations associated with later stages of Malliard reaction.

Michael J. Lovadahl et al. reported that Pregabalin in the presence of lactose undergoes a Maillard reaction over time to form conjugates with lactose in formulated product. Seven of these conjugates, which contain the lactam form of the pregabalin moiety, were generated in milligram to gram quantities by heating pregabalin in the presence of lactose in solution. There were four degradation products (Figure 37) observed with glucose and through B lactose with Amadori rearrangement [58] .

Faran et al. reported that low levels of the Acyclovir-lactose condensation product was confirmed by liquid chromatography-tandem mass spectrometry, indicating the formation of a covalent link between Acyclovir and

lactose with the loss of a single water molecule (Figure 38). Maillard reaction of acyclovir and lactose in a solid-state formulation is less possible than in the aqueous phase. Thus, there may be some other important factors such as restricted mobility affecting the Maillard reaction in real solid dosage forms. It is also advisable to avoid wet conditions in the formulation process and/or storage of acyclovir solid-state dosage forms containing lactose [59] .

Xin Yu et al. reported that Desloratidine reacts with the open chain of maltose to form the maltose adduct through Amadori rearrangement (Figure 39, Figure 40). In one of the formulation, Desloratadine reacted with the isomer of acetylformoin, which is present in one of the starch excipient as an impurity. Xin Yu et al. performed significant efforts in identifying in characterizing this impurity by LCMS and NMR [60] .

Omari et al. found that Amlodipine besylate is unstable in presence of lactose, magnesium stearate, and water in the solid-state formulations. The major degradant observed is amlodipine besylate glycosyl (Figure 41). This is also a conformity with the Maillard reaction between primary amine and lactose [61] .

Kerry et al. reported that N oxide derivative of Raloxefine is formed due to the residual peroxides present in the excipient of povidone and crospovidone in the finished product. An oxidative degradation product in a tablet formulation of Raloxefine hydrochloride was identified as a N-oxide derivative (Figure 42). On the basis of applying the ICH guideline for acceptable levels of this degradation product, the data generated in this study can be used to set internal specifications on the level of peroxides for purchased lots of povidone and crospovidone. However, it is also known that in the presence of atmospheric oxygen, the peroxide content of povidone and crospovidone can increase The results in this study suggest that the peroxides in this tablet formulation are not continuing to form appreciably upon aging (from autoxidation), but rather are present in the excipients as process impurities that are consumed by reaction with R-HCl and also possibly by decomposition [62] [63] .

Fluphenazine Deaconate forms several N-oxides due to the Oxidation of the phenothiazine group. This could be

due to the peroxide present in the excipients or oxidation of the active drug after accelerated conditions. UPLC method developed and peroxide degradation of the impurities were identified by LC-MS/MS (Figures 43-45). The N- and S-oxides produced may be formed by ageing process, which is often the result of photo oxidation. Oxidation of the phenothiazine group of drugs is a well-documented feature of these compounds. The N- and S-oxides produced may be formed by ageing process, which is often the result of photo oxidation [64] . The

fluphenazine molecule contains four potential oxidation sites, thus fifteen o-tidesare theoretically possible (Table 3). Peroxide content and oxidation due to exposure shall be avoided while manufacturing this product. UPLC-MS/MS with positive ion mode with ammonium acetate buffer with Acquity UPLC column was employed for the identification of these impurities.