Degradation Impurities Research Articles

Chromatographic analysis of ponatinib and its impurities: method development, validation, and identification of new degradation product.

Ponatinib, a third-generation tyrosine kinase inhibitor, is employed in the management of adult chronic myeloid leukemia. Nevertheless, the presence of process impurities and degradation impurities linked to ponatinib may potentially influence its effectiveness and safety. Therefore, the objective of this research was to establish a robust liquid chromatography method and systematically validate it for the detection of substances related to ponatinib. The separation of ponatinib and its impurities was conducted using an Agilent 5HC-C18 chromatographic column (4.6mm × 250mm, 5μm). The mobile phase A comprised a mixture of water and acetonitrile in a 9:1 ratio, with an aqueous solution of pH 2.4 containing 2mM potassium dihydrogen phosphate and 0.4% triethylamine. Mobile phase B, consisting of acetonitrile, was eluted in a gradient fashion. The flow rate was set at 1.0mL/min, detection wavelength at 250nm, column temperature at 40°C, and injection volume at 10μL. The method demonstrated high specificity, sensitivity, solution stability, linearity, precision, accuracy, and robustness. Additionally, this research unveiled a novel compound, imp-B, generated via the oxidative degradation of ponatinib. The molecular structure of the newly discovered product was elucidated through the utilization of nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS). In conclusion, the chromatographic method developed in this study has the potential to be utilized for the detection of ponatinib and its impurities, thereby offering significant insights for quality assessment in ponatinib research.

Case Study on Regulatory Approaches for New Degradation Impurity Exceeding ICH Thresholds in Solubilized Ibuprofen Capsules During Stability Testing

The article discusses the identification, isolation, characterization, and toxicology study of a new degradation impurity found in stability samples of Ibuprofen soft gelatin capsules. An unknown impurity was detected during stability analysis, reaching 0.28% level, which prompted further investigation. A preparative method was developed to isolate the impurity quickly, followed by its identification and characterization using spectroscopic techniques. The impurity, named 2,3-dihydroxxypropyl 2-(4-isobutylphenyl)propanoate, was confirmed through various analyses, including HPLC, LC-MS, NMR, and FT-IR. Forced degradation studies suggested that the impurity formation might be due to the reaction of Ibuprofen with Glycerin present in the product. Toxicology studies were conducted to evaluate the safety of Ibuprofen with the impurity, concluding that it posed no significant toxicity concerns. Based on the findings, a specification limit of not more than 0.3% for the impurity was proposed, supported by justification from various studies and guidelines. To the best of our knowledge, this impurity has not been reported in pharmaceuticals elsewhere; it can be helpful in the pharmaceutical industry for regulatory considerations. This would enhance the pharmaceutical industry's perspectives on unknown impurities and knowledge of degradation impurities, which would positively impact pharmaceutical companies' approaches.

Analytical Method Development and Validation to Determine Oxidized Follitropin Recombinant Follicle Stimulating Hormone (rFSH) Injection by RP-HPLC

Purpose: A simple, rapid and precise reverse phase–High performance liquid chromatography (RP-HPLC) method was developed for the determination of degradation impurity i.e. Oxidised follitropin in recombinant Follicle Stimulating Hormone (rFSH) Injection. Methods: Chromatographic separation was performed using a C4 column of size: 250 × 4.6 mm, 5 μm along with C3 Guard Column (SB-C3 size: 125 mm x 4.6 mm, 5 μm) and using gradient elution. The flow rate was kept at 1.0 ml per minute and the detection wavelength was at 210 nm. The retention time of oxidised follitropin was ~14 to 15 minutes. The detector showed a linear response between the range of 2.46–63.325 μg/ml (5% - 125%) with a correlation coefficient value of 0.9987. After establishing the procedure, it was ensured for its intended usage by validation of the analytical parameters like specificity, linearity, accuracy, repeatability, and robustness as per the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) [Q2 (R1) Validation of Analytical Procedures: Text and Methodology]. Results: All of the parameters performed using the current method yielded results that met the acceptance requirements. The detector showed a linear response between the range of 2.46– 63.325 μg/ml (5% - 125%) with a correlation coefficient value of 0.9987. Conclusions: As a result, a newly designed RP-HPLC method was capable of effectively separating impurities while maintaining acceptable limits.

Identification and characterization of two new oxidation degradation impurities in cinnarizine through LC‐HRMS/MS and 1H NMR, along with in silico toxicity predictions of its degradation products

AbstractCinnarizine (CIN) drug substance is a US FDA and EMA approved antihistaminic drug, There is no report available on CIN for the identification of degradation products and their degradation pathway. Herein, we report a stability‐indicating assay method for CIN, the formation and characterization of its major degradation products using LC‐HRMS/MS and 1H‐NMR techniques. CIN was subjected to oxidation, acid, base, thermal and photolytic degradation conditions. Two unknown degradation products (DP‐1 and DP‐2) of CIN were formed under oxidative conditions. We successfully separated these degradants using gradient elution on an Inertsil ODS 3 V column (150 × 4.6 mm, 5 μm) using mobile phase A consisting of 0.1% formic acid and the mobile phase B consisting of 0.1% formic acid/acetonitrile (20/80, v/v). CIN was labile to oxidative conditions and stable to acidic, alkaline hydrolytic, photolytic and thermal conditions. The degradation pathways were derived from the nature of the product formed under oxidative degradation conditions and available reports for confirmation of the mechanism. Since the stability‐indicating assay method can be utilized for stability studies and routine quality control of CIN in both the pharmaceutical industry and research laboratories. This method has been validated in compliance with the guidelines set forth by the ICH.

Investigation of the Influence of Copovidone Properties and Hot-Melt Extrusion Process on Level of Impurities, In Vitro Release, and Stability of an Amorphous Solid Dispersion Product.

Hot-melt extrusion (HME) is a widely used method for creating amorphous solid dispersions (ASDs) of poorly soluble drug substances, where the drug is molecularly dispersed in a solid polymer matrix. This study examines the impact of three different copovidone excipients, their reactive impurity levels, HME barrel temperature, and the distribution of colloidal silicon dioxide (SiO2) on impurity levels, stability, and drug release of ASDs and their tablets. Initial peroxide levels were higher in Kollidon VA 64 (KVA64) and Plasdone S630 (PS630) compared to Plasdone S630 Ultra (PS630U), leading to greater oxidative degradation of the drug in fresh ASD tablets. However, stability testing (50 °C, closed container, 50 °C/30% RH, open conditions) showed lower oxidative degradation impurities in ASD tablets prepared at higher barrel temperatures, likely due to greater peroxide degradation. Plasdone S630 is suitable for ASDs with drugs prone to oxidative degradation, while standard purity grades may benefit drugs susceptible to free radical degradation, as they generate fewer free radicals post-HME. ASD tablets exhibited greater physical stability than milled extrudate samples, likely due to reduced exposure to stability conditions within the tablet matrix. Including SiO2 in the extrudate composition resulted in greater physical stability of the ASD system in the tablet; however, it negatively affected chemical stability, promoting greater oxidative degradation and hydroxylation of the drug substance. No impact of the distribution of SiO2 on drug release was observed. The study also confirmed the congruent release of copovidone, the drug substance, and Tween 80 using flow NMR coupled with in-line UV/vis. This research highlights the critical roles of peroxide levels and SiO2 in influencing the dissolution and physical and chemical stability of ASDs. The findings provide valuable insights for developing stable and effective pharmaceutical formulations, emphasizing the importance of controlling reactive impurities and excipient characteristics in ASD products prepared by using HME.

Isolation and characterization of a degradation product of mexiletine hydrochloride using FT-IR, LC-MS and NMR: method development and validation of stability indicating RP-HPLC method for quantification of characterized impurity

Mexiletine Hydrochloride is an antiarrhythmic drug used to treat acute and chronic ventricular arrhythmias. Significant degradation of unknown impurity was observed while evaluating the impurity profile of the Mexiletine Hydrochloride stability studies. The levels of the unknown impurity are more than the regulatory identification/qualification threshold level (0.08%). Mexiletine was forcefully subjected to various stress conditions, such as acidic, basic, oxidation, photolysis, and thermal. Among these stress conditions, we identified an unknown degradation impurity in the thermal stress (105°C for 15 days) of Mexiletine, the same as that of an unknown impurity observed in the stability studies. The present research reports the isolation of unknown impurity in thermal stress by preparative HPLC and the characterization of isolated unknown degradation impurity using LC-MS, FT-IR, and NMR techniques. The spectral data elucidated the structure of the degradation product, and it was identified as “(E)-2-(((1-(2,6-dimethylphenoxy)propan-2-yl)imino)methyl)-6-methylphenol”. A stability-indicating HPLC method was developed and validated to determine and quantify the degradation product in Mexiletine Hydrochloride. The levels of the unknown degradation product reduced to below the limit of detection (<0.007%) from 0.10% after applying its relative response factor “16.5” and complying with the regulatory threshold limits. It helps to extend the shelf life of Mexiletine.

Eco-Friendly Graphene-Based Electrochemical Sensor for Selective Determination of Lesinurad in Its Pharmaceutical Formulation and in the Presence of Its Degradation Products

The objective of the present study was to create solid contact ion selective electrodes (SC-ISEs) for the purpose of selectively determining lesinurad (LES) in both its pure form and pharmaceutical dosage form where potential degradation products and impurities may be present. To achieve that goal, an electrochemical sensor with graphene nanomaterial as an ion-to-electron transducer was constructed using the glassy carbon electrode (GCE) as a substrate. A number of plasticizers were tested to find the best plasticizer for creating the potentiometric sensors, where 2-nitrophenyloctyl ether (o-NPOE) revealed the optimum response and nearly Nernstian slope. Sensor was characterized and the linear range was 5.0 × 10−7 to 1.0 × 10−3 M, and the calculated LOD was found to be 4.9 × 10−7 M. The developed sensor’s performance was evaluated as per the IUPAC requirements. Lesinurad was successfully determined in its pharmaceutical tablets using the proposed sensor. Additionally, statistical comparison of the developed method with the reported HPLC results has been carried out using student’s t-test and F-value, where no significant difference was found. Using the AGREE tool, the suggested method’s greenness was assessed and contrasted with the published HPLC one.

New stability indicating RP-HPLC methods for the determination of related substances and assay of trametinib acetic acid: a mass balance approach.

New stability indicating related substances and assay methods for trametinib acetic acid by RP-HPLC have been developed and validated through forced degradation and mass balance studies for the first time. In related substances (RS) method, trametinib acetic acid was successfully separated from its six related substances namely, cyclopropanamide impurity, desiodo trametinib, desacetyl trametinib, trione acetamide intermediate, trione intermediate, and trione PTSA intermediate using YMC-Triart C18 (150 × 4.6mm, 3µm) column. Orthophosphoric acid (0.15%) in water was used as buffer. Gradient elution was programmed using mobile phase-A (buffer and acetonitrile mixture in 80:20 v/v ratio) and mobile phase-B (buffer and acetonitrile mixture in 20:80 v/v ratio). Acetonitrile and methanol mixture (1:1 v/v) was used as diluent. Flow rate, injection volume, column temperature, and wavelengths were kept as 0.8mL/min, 10µL, 55°C, and 240nm, respectively. Desacetyl trametinib and cyclopropanamide impurity were identified as potential degradation impurities in acid and base degradation conditions, respectively. Assay method specific to above six related substances was also developed. Assay of forced degradation samples was also determined, and the mass balance was established by adding total impurities formed in RS method to assay of trametinib acetic acid.

Development and validation of stability indicating assay method for mitapivat: Identification of novel hydrolytic, photolytic, and oxidative forced degradation products employing quadrupole-time of flight mass spectrometry.

Mitapivat is a novel, first-in-class orally active pyruvate kinase activator approved by the US Food and Drug Administration in 2022 for the treatment of hemolytic anemia. There is no literature available regarding the identification of degradation impurities of mitapivat. The present study deals with the degradation behavior of mitapivat under various stress conditions such as hydrolytic, photolytic, thermal, and oxidative stress. The multivariate analysis found that the independent variables, that is, molarity, temperature, and time, are interacting with each other to affect the degradation of mitapivat. A specific, accurate, and precise high-performance liquid chromatographic (HPLC) method was developed to separate mitapivat from its degradation products. The separation was achieved on the C-18 column (250mm × 4.6mm × 5µm) using the combination of 0.1% formic acid buffer and acetonitrile in gradient elution profile. The method was validated as per the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use Q2(R2) guideline. LC-electrospray ionization-Quadrupole-time of flight was employed to identify degradation products. A total of seven novel degradation products of mitapivat were identified based on tandem mass spectrometry and accurate mass measurement. In-silico toxicity of mitapivat and its degradation products was qualitatively evaluated by the DEREK toxicity prediction tool.

Study on the impurity profiles of cloxacillin and flucloxacillin based on liquid chromatography tandem ion trap/time-of-flight mass spectrometry.

Cloxacillin and flucloxacillin are prone to degradation and polymerization in humid and hot environments, and their polymers have long been recognized to trigger allergic manifestations. A series of the degradation and polymerized impurities in cloxacillin and flucloxacillin were separated and characterized to ensure safe use of these drugs by the public. By studying the chromatographic behavior of the degradation impurities and polymerized impurities in reversed-phase high-performance liquid chromatography (RP-HPLC) gradient elution, the impurities in cloxacillin and flucloxacillin were effectively separated and eluted. RP-HPLC tandem ion trap/time-of-flight mass spectrometry (MS) was applied to characterize the structures of unknown impurities eluted from the RP-HPLC methods for cloxacillin and flucloxacillin. The mechanisms of formation of the impurities in cloxacillin and flucloxacillin were also investigated. The structures of 10 unknown impurities in cloxacillin and 8 unknown impurities in flucloxacillin were elucidated based on the high-resolution MSn data at positive and negative modes, respectively. Six polymerized impurities were found and characterized, of which three were from the polymerization of cloxacillin and three were from the polymerization of flucloxacillin. The study on the impurity profiles of cloxacillin and flucloxacillin provided a scientific basis for improving their production processes and quality control.

Stability-indicating Reverse Phase-HPLC Method Development and Method Validation for Quantitative Determination of Degradation Products in Favipiravir API and Drug Product

Introduction: Favipiravir is an antiviral medication shown to be broad spectrum activity against RNA viruses, and potentially treating the COVID-19. Methodology: In this study, the HPLC method for the quantification of degradation impurities (Favipiravir Acid Impurities) were developed and validated for Favipiravir in Tablet dosage form. The specificity of the method was achieved in analytical column Agilent HPLC-C18, 5µm, (4.6 x250) mm. using a suitable mobile phase was 10 mM Phosphate buffer (pH 3.5 with orthophosphoric acid) and Acetonitrile in the Isocratic more of 70:30 v/v. The flow rate is 1.0 mL/min. the injection volume is 10 µL, detection at 320 nm in UV and total run time is 8.0 minutes. The samples were made for forced degradation under hydrolysis, oxidation, photolytic and thermal conditions. The method was validated for specific, selective, linear, robust and accurate as per the ICH guidelines. Results and Conclusion: The linearity of the method for Impurities and the analytes was found from 25 to 150 % concentration level with the correlation coefficient (r2) &gt; 0.999. The accuracy for impurity and the analytes was performed from 50 to 150% level concentration, and mean recovery was found from 98-102%. The analytical degradation and validated study results indicate its unstable nature in acidic, basic and thermal conditions. Therefore, this method is simple, selective and sensitive, this method can be used in pharmaceutical research and development and quality control departments.

A drug–excipient interaction impurity of bromhexine hydrochloride injection: Structure and formation mechanism elucidation

A long-term stability study using high performance liquid chromatography (HPLC) revealed an unidentified impurity in the bromhexine hydrochloride injection, which was employed as a mucolytic agent. Investigations into stress degradation and elemental impurities revealed one of the elemental impurities Fe3+ in this injection as the primary generator of these impurities. This impurity, named N-carboxymethyl bromhexine, was a product formed during drug–excipient interaction between bromhexine and tartaric acid with Fe3+. The structure of the impurity was identified through ultra-high-performance liquid chromatography with diode array detector (UHPLC-DAD), liquid chromatograph mass spectrometer (LC-MS). Further, the formation mechanism of the impurity was discussed. Overall, this study elucidates the cause, origin, and mechanism of an unknown impurity in bromhexine hydrochloride injection, providing a basis for quality control for bromhexine hydrochloride injections and drug products containing both amine and tartaric acid.

Isolation and characterization of major degradants in dihydroergotamine injection

AbstractIn this study, we report the systematic approach for characterization of two major degradant impurities, which are not listed in any compendia and were formed during the stability studies of Dihydroergotamine mesylate injection (DHE). An ion-pair UPLC chromatographic method was developed to quantify the related substances present in the DHE injection drug product. The same was used to monitor the impurity profiling during its stability. The two unknown impurities were observed at RRT about 0.08 (Impurity-1) and RRT about 0.80 (Impurity-5) and found to be significantly increasing on stability. Forced degradation studies revealed the nature of the impurity and conditions required for enriching them. A Mass compatible HPLC method was developed to quantify only these two impurities using 25% ammonia and formic acid in water. Their mass numbers were identified using LC MS/MS with triple quadruple mass spectrometer coupled with a HPLC. These two impurities were then isolated from enriched products using preparative HPLC. These impurities were then characterized using Mass and NMR analysis along with Q-TOF elemental analysis.

Identification and structural characterization of potential degradation products of baricitinib using liquid chromatography combined with quadrupole time-of-flight mass spectrometry

AbstractBaricitinib (BRT) is a drug substance with potent anti-inflammatory activity indicated in rheumatoid arthritis, atopic dermatitis, severe alopecia areata and recently for the treatment of Covid-19. Process impurities of the drug during its formulation are quite known, however studies regarding its degradation products (DPs) under stress conditions are limited. In this study, the drug was subjected to forced degradation under various degradation conditions, including acidic hydrolysis, alkaline hydrolysis, oxidative and thermal, to determine its inherent stability. To this purpose, a novel HPLC method was developed for the determination of degradation impurities of BRT. Alkaline hydrolysis test showed a selectivity towards breaking C–C bonds. This resulted to five DPs formed by chain scission reactions occurred at the pyrrolo-pyrimidine group between C6–C10 and C8–C9. Also, the ethylsulfonyl-azetidin-ylidene group was subjected to C–C bond cleavage at C12–C15 and C16–C18. Degradation products were further characterized with the use of liquid chromatography quadrupole time of flight tandem mass spectrometry (LC-Q-TOF-MS/MS).

Biomonitoring Equivalents for ethylene thiourea

Ethylene thiourea, or ETU, is used in the rubber industry and is a degradation product and impurity in some fungicides. The general public may be exposed to low concentrations of residues of ETU in a variety of ways, including food treated with ethylene bis-dithiocarbamate (EBDC) fungicides or migration from rubber products. Biomonitoring of ETU in urine is useful for an assessment of integrated exposures to ETU across different sources and routes of exposure. In this evaluation, we review available health-based risk assessments and toxicological reference values (TRVs) for ETU and derive Biomonitoring Equivalent (BE) values for interpretation of population biomonitoring data. BEs were derived based on existing TRVs derived by Health Canada, yielding a BE of 27 μg of total ETU/L in urine associated with the Acceptable Daily Intake (ADI) and 6.7 μg/L associated with a 1e-6 cancer risk. These BEs are based on an analytical method that involves a digestion step to liberate conjugated ETU, thus producing ‘total’ ETU in urine. The BE values derived in this manuscript can serve as a guide to help public health officials and regulators interpret population based ETU biomonitoring data in a public health risk context.

High-performance thin-layer chromatography‒spectrodensitometric determination of diltiazem hydrochloride and its commonly occurring degradation impurity

High-performance thin-layer chromatography‒spectrodensitometric determination of diltiazem hydrochloride and its commonly occurring degradation impurity

LC-HRMS and NMR studies for the characterization of degradation impurities of ubrogepant along with the in silico approaches for the prediction of degradation and toxicity

Ubrogepant is the first oral calcitonin gene-related peptide (CGRP) receptor antagonist which is used for the acute treatment of migraine in adults. The present study employs liquid chromatography-high resolution mass spectrometry (LC-HRMS) and nuclear magnetic resonance spectroscopy (NMR) techniques for the identification and characterization of degradation impurities of ubrogepant. The forced degradation study of ubrogepant was performed as per the International Council for Harmonisation (ICH) Q1A and Q1B guidelines. The in silico degradation profile of ubrogepant was predicted by Zeneth. It was observed that ubrogepant was labile to acidic hydrolysis, basic hydrolysis, and oxidative degradation conditions (H2O2), although it was stable in neutral hydrolysis and photolytic (UV light and visible light) conditions. Eight degradation impurities were formed, which were separated on reversed-phase HPLC with a gradient program on an InertSustain C8 column (4.6 × 250 mm, 5 µm) using 10 mM ammonium formate (pH unadjusted) and acetonitrile as the mobile phase. The structures of all the degradation impurities were characterized using the exact masses obtained from the HRMS/MS. Further, NMR studies were conducted on two major degradation impurities (UB-4 and UB-7). A plausible mechanism was proposed to support the structures of all the degradation impurities of UBR. In silico toxicity and mutagenicity assessment were done by DEREK Nexus, SARAH Nexus, and ProTox-II.

Tri-chromic sustainable tools of derivative/discrete Fourier transform HPLC convoluted peaks: Application on LCZ696, an antihypertensive supramolecular complex, with its in process and degradation impurity analysis

The aim of this manuscript is to discuss the use of chemometrics approaches in handling the response data of HPLC to sustainable analytical chemistry procedure development. This was performed on FDA approved LCZ696, an antihypertensive supramolecular complex of sacubitril (SAC) and valsartan (VAL), with the in process and degradation impurity (BMP). Structure similarity between SAC and its in process and degradation impurity (BMP) makes it difficult for their simultaneous determination and need too much time and solvent consumption for their separation. It was found that convolution of the resulting derivative curves using 8-points sin xi polynomials (discrete Fourier functions) after derivative treatment of the chromatographic response data was beneficial in handling one of the most common chromatographic problems which is overlapping of the chromatographic peaks without complete resolution, thus, reducing time of analysis and solvent consumption. The chemometric data handling succeeded in analyzing LCZ696, with its closely related in process impurity in less than 4.5 min. reaching impurity detection limit of 0.5 % in presence of the parent drug, SAC, while the direct chromatographic measurement showed higher LOD and LOQ of BMP with poor recovery, outside the accepted range 98–102 %, ranging from 80.56 to 104.41. Moreover, the proposed chemometric methodology will be compared with other chromatographic methods in terms of greenness, blueness and whiteness (tri-chromic assessment) using Analytical Eco Scale, AGREE, Blue Applicability Grade Index (BAGI) and the up-to-date Chlortox scale in addition to the white RGB 12 model in order to ascertain the influence of chemometric methods in minimizing the amount of time spent conducting the analysis and reducing solvent, energy and waste consumption.

Identification and Synthesis of Oxidative-Degradation and Starting Materials-Attributed Impurities in Lisdexamfetamine Dimesylate

Abstract: Impurities are an integral part of drug substances, even though they have not been studied in the pharmacological evaluation of the Benefit-Risk (BR) profiles. Hence, understanding their origin and controlling them have prime importance during drug substance development. The structures of some of the impurities in lisdexamfetamine dimesylate, a central nervous system stimulant drug, have been confirmed based on literature and analytical data. The study has been undertaken to evaluate impurities arising from oxidative degradation and impurities due to material attributes. All the listed impurities have been identified, synthesized, and characterized by spectral tools. We have, herein, reported the synthesis of two oxidative degradant impurities, 2-hydroxylisdexamfetamine dimesylate and 4-hydroxylisdexafetamine dimesylate. In addition, chiral isomeric impurities of lisdexamfetamine dimesylate as (S,R), (R,S) and (R,R)-lisdexamfetamine dimesylate have also been reported. These impurities have been identified and synthesized, and a control strategy for mitigating risk in lisdexamfetamine dimesylate is provided.

Identification, Isolation, and Characterization of a Novel Degradation Impurity of Busulfan Using Preparative Chromatography, NMR, and LC-MS.

Busulfan is the most effective medication for treating chronic myelogenous or granulocytic leukemia because it has cytotoxic properties that harm or kill hematopoietic cells. It cannot absorb light in the Ultraviolet range due to its structure. Because of this, it is very challenging to quantify using traditional HPLC coupled with UV/Photodiode Array detectors. So, using sodium diethyldithiocarbamate, a derivatization method was developed to quantify related impurities. A significant unknown impurity was identified in derivatized samples of busulfan and a noticeably high percentage level was discovered during routine drug testing. We aimed to isolate, and characterize the unknown impurity observed in the samples and to identify its root cause. Preparative HPLC was used to isolate the unidentified, derivatized impurity, and 1H NMR, 13C NMR, and MS were used to decipher its structural components. The spectral characterization data analysis showed that the unknown impurity was related to busulfan. Additionally, it was noted that the impurity developed as a result of the residual buffer used to prepare the derivatizing reagent. The isolated impurity was found to be same as comparable to that found in busulfan drug substances, according to the results of the characterization tools. An alternative method of reagent preparation was optimized and deemed satisfactory because the buffer used in reagent preparation is the only factor contributing to the formation of impurities. Using cutting-edge analytical characterization tools, it was possible to explain the structural characteristics of an unknown impurity and discover that it was a novel impurity, which undoubtedly contributes to the comprehension of drug substance reaction properties.