Analytical Strategies for Busulfan Quantification: A Review of HPLC Method Development and Validation

A bifunctional alkylating agent, busulfan (1,4-butaned?iol dimet?hanesulfonate) is a member o?f the alkane sulfonic acid esters therapeutic class that has anti-tumor properties.It works by covalently alkylating the N7 position of guanine bases in DNA, which ca?uses intrastran?d and interstrand cross-linking, inhibits DNA replication, and triggers apoptosis^[1].Because it can ablate bon?e marrow before grafting, busulfan has been a mainstay in h?ematopoi?etic stem cell transplantatio?n (HSCT) regi?mens, especially when combined with cy?clophosphamide or fludarabine^[2].Busulf?an's pharmacokinetics show considerable patient vari?ability despite its therapeutic significance, main?ly because of variations in absorption, hepatic metabolism via glut?athione conjugation, a?n?d renal clearance of i?ts metabolites? ^{[3,4 ].} This diversity may result in serious toxicities, such as pulmonar?y fibrosis and veno-occlu?sive disease (VOD), or t?hera?peu?tic failure (“Busulfan lung”), and seizures ^[?5].During formulation development and clinical monitoring, accurate Busulfan level quantification is essential to guarantee safety and efficacy.  Because of its excellent accuracy, repeatability, an?d capacity to ident?ify low quantities in complex matrices, high-perf?ormance liquid chromato?graphy? (HPLC) has become a reliable analytical platform for Busulfan quantification ^[6] . Busulfan concentrations i?n plasma, serum, and pharmaceutical formulations have been determined by researchers usin?g a variety of HPLC and LC-MS/MS techniques that have? been developed and confirmed throughout time ^[7–9]. However, more standardization has been required due to differences in detection wavelengths, mobile phase composition, and chromatographic parameter s between studies. Validation of? analytical procedu?res is essential to determining thei?r depend ability and repeatabili?ty. As per the Q2 (R1?) criteria of th?e Internati?onal Council for Harmoni?zation (ICH), factors including specificity, linearity, accuracy, precision, and detection limit, quantitation limit, and robu?stness must be thoroughly evaluated ^[10,11]. The present review integrates both literature findings and experimental results to propose a validated HPLC method optimized for the quantitative deter?mination of Busulfan. The developed method pr?ovides superior chromatographic resolution and sh?ort analysis time while maintaining high sens?i?tivity. Furthermore, th?e study includes forced degradat?ion assessments under various stress conditions acidic, alkaline, oxidative, thermal,? and photolytic to confirm the met?hod’s stability-in dicating na?ture ^[12–14]. This int?egrated approach not only contributes to t?he q?uality assurance of Bu?sulfan formulations but also a?ligns with regulatory expectations for analytical method valid?ation in phar?mac?eutical research and industry. 

Goal To develop and validate a novel high-performance liquid chromatography (HPLC) method for the precise, sensitive, and reliable quantification of Busulfan in pharmaceutical formulations and biological matrices, guaranteeing suitability for quality control and therapeutic drug monitoring. 

• To investigate the physicochemical and pharmacological characteristics of busulfan that are pertinent to the development of analytical methods. 
• To design and optimize an HPLC technique for selective Busufan analyses using appropriate mobile phase composition, column type, and detection wavelength.  
• To verify the developed HPLC technique in accordance with ICH Q2 (R1) requirements, including: The specificity, Accuracy, precision, robustness, sensitivity (LOD & LOQ), and linearity 
• To assess the method's stability-indicating capacity using forced degradation studies under acidic, bacterial, oxidative, and photolytic conditions.

2. DRUG PROFILE  

3. OVERVIEW OF BUSULFAN

3.1 Pharmacological Overview of Busulfan

A common cell-cycle-non?specific alkylating drug used for myeloablation prior to he?matopoietic stem cell transplantation (HSCT) is busulfan ^[1]. It is s?tructurally ?functiona?l alkane-sulfonate ester that spontaneously hydrolyzes in aqueous solution to produce derivatives o?f met?hanesulfonic acid that can alkylate DNA's nucleophilic spots ^[2]. At guanine's N7 position, the agent creates covalent connections that result in intra- and inter-strand cross-links that prevent tran?scription and repl?ication, ultimately causing ap?optosis ^[3]. Busulf?an absorption and clearance exhibit significant inter -individual var?ia?bility, which is frequently imp acted by hepatic glutathione levels, glutathione S-transf?erase (GST) polymorph?isms, and concurrent medication d?elivery, according to ph?armacokinetic stu?dies ^[4].To keep pla?sma conce?ntrations with in the idea the?rapeutic window, therapeutic drug monitoring (TD?M) is cru?cial ^[5]?.The fibrosis, hyperpigmentation, and veno- oc?c?lusive disease (VOD), highlights the necessity of accurate adverse-e?ffect profile of busulfan, which includes neurotoxicity, pulmonar?y quantitative techniques to direct dosage modifications ^[6].

3.2 Analytical Requirements for Busul?fan Quantification

Busulf?an's strong reactivity, low aqueous stability, and narrow concen?tration range in biologi?cal samples make an?alytical monitoring difficult ^[7]. Colorimetric and titrimetric assays were used in early proc?edures, but these techniques lacked sensitivity and selectivity ^[8].Chromatographi?c methods, especially high- performance liquid c?hroma?tography (HPLC), have emerged as the gold standard? for Busulfan assessment in plasma samples and pharmaceutical dosage forms throughout the last thirty years ^[9].  HPLC provides better reproducibility, faster ru?n times, and co?mpatibility with derivatization techniques that enh?ance detection when compared t?o spectrophotometr?ic and gas-chromatographic methods^[10]. Busulfan and its metabolites can now be simultaneously detected with enhanced sensitivity in the nanogr?am per millil?iter ran?ge because to the development of LC–MS/MS techniques for therapeutic monitorin?g ^{[11, 12]}. Despite? this advancement, conventional HPLC with UV detection remains popular in qualitycontrol laboratories because it requires simpler instrumentation, lower c?ost, and e?asier v?alidation ^[13].

3.3 Evolut?io?n of HPLC Methods for Busulfan

In? the late 1970s, silicon columns and non-aqueous mobile phases were used in the first H?PLC procedures for Busulfan ^[14].Due to column instability, these proces?ses offered low rep?eatability and limited resolution.Selectivity and peak symmetry were signi?ficantly enhanced by the use? of high-purity solvents and reversed-phase C18 columns ^[15]. Acetonit?rile-water c?ombinations, occasionally altered with methanol or tetrahydrofuran? to improve elution s?trength, have become the standard mo?bile phases ^[16]. Due of Busulfan's significant UV absorp?tion in th?e vicinity of 280 nm, this wavelength is frequently? used for dete ction ^[17].To account for? injection variability and derivatization ef?ficiency, internal standards like d8?-Busulfa?n or but?yl p-toluen?esulfonate are commonl?y ut?ilized^[18]. Miniaturized and ecologically friendly (green?) chromatogr?aphic systems have been the focus? of recent advancements.  Analysis periods can be reduced to less than five minutes with ultra-high-performance liquid chromatography (UHPLC) without sacrificing sufficient resolution ^[19]. For rest?ricted biologica?l samples, micro- and nano-HPLC designs improve sensitivity and? further minimize s?olvent use [20].

3.4 Derivatization and Detection Strategies

Because busulfan lacks a potent chromophore, UV detection frequently requires derivatization.  N, N-dimethylacetamide reacts wit h sodium diethyldithiocarbamate to form a pe?rsistent, highly absorbing derivative that can be seen at 280 nm ^[21].By increasing sensitivity and linearity,? this  deri?va?tization  st?age makes it possible to detect in plasma at concentrations lower than 20 ng/mL^[22].There have also been reports of? fluorescence deriva?tization employi?ng t?hiol reagents, albeit these techniques are less prevalent since t?hey require additional reaction steps ^[23].B?y tracking protonated molecule ions (m/z 264 → 151) unde?r positive-ion electrospray conditions, LC–MS/MS techniques completely avoid derivatization ^[24].Although LC?-MS/MS o?ffers bet?ter select?ivity, routine quality-control laboratories cannot widely us?e it due to its high operating costs and maintenan?ce requirements ^[25].

3.5 Validation Guidelines and Regul atory Expectations

Global standards for analytical-method validation a?re provi?ded b?y the Inte?r?national Council for Harmonization (IC H) guideline Q2 (R1), whi?ch covers specificit?y, li?nearity, a?ccuracy, precisio?n, detection lim?its, q?uantitation limits, range, and robustness^[26]. Furthermore,similar frameworks that emphasiz?e reproducibili?t?y and depend?ability are outlined by the European? Pharmacopeia (EP 2.2.46) an?d the United States Pharmacopeia (USP <12 25>) (27, 28).  Precision and a?ccuracy are crucial for Busulfan assays since even small differences m?ight result i?n overdos?e or underdosing during conditi?oning therapy. Recovery should stay between 98%? and 102%, while acceptable %RSD val?ues for retention time and area re?sponse are usual?ly less than 2% ^[29].The stability-indica ting characteristic of the analytical appro?ach is further established by forced-degradation testing in a?cidic, alkaline, oxidative, thermal, and photolyt?ic environments ^[30].Validated chromatographic procedures for a?ntineoplastic agents are expected by regulatory bodies like the European Me?dici?nes Agency (EMA) and the U.S. Food and Drug Administration (FDA) to show not only analytical soundness but also environmental safety through decreased solvent use and was?t?e ^[31].

3.6 Comparative Assessment of Analyt?ical Techniques

Table 1 summarizes representative analytical methods for Busulfan reported in the literature from 20?15 to 2024, highlighting chroma?tographic conditions and perform?ance charac?teristics.

Table 1 “Representat?i?ve HPLC and LC–MS/MS methods for Busul?fan de?termin?ation (2015– 20?24)

3.7 Gaps in Previous Research and Literature (Existing)

Despite the surge in published studies on the development of the Busulfan assay, many gaps and challenges remain.  A number of published works that emphasize sensitivity also neglect the versatility and environmental sustainability of the assay ^[32].Moreover, few studies integrate validation and forced-degradation data to ascertain true stability-indicating ability[^33].Discrepancies among laboratories arise from differences in internal standards and derivatization conditions ^[34]. Recent studies aim to identify critical method parameters and ensure lifecycle management of HPLC methods, concentrating on AQbD (Analytical Quality by Design) ^[35].The current study addresses these gaps by providing a fully validated, stabilityindicating RP-HPLC method that is appropriate for both academic research and routine qualitycontrol laboratories.

3.8 Summary

Overall, the revision of the published literature indicates that the chromatographic methods for Busulfan are in a state of transformation and development to meet the requirements of precision, robustness, and sustainability. Although the AQbD and ICH Q2 (R1) compliant validation frameworks being implemented in pharmaceutical laboratories around the world may not have kept pace with the advances in sensitivity and speed offered by new technologies in LC–MS/MS and UHPLC, chromatographic methods for Busulfan.

4. MATERIALS AND? METHODS

4.1 Chemicals and R?eagent?s

A authorized? pharmaceutical supplier in? India sup?plied the Busulfan injectable fo?rmulations a?nd reference standard (purity ≥99%). Merck Ltd. (Mumbai, India) supplied HPLC-grade aceto nitrile, methanol, water, tetrahydrofuran (THF), and N, N-dimethylacetamide. M?erck als?o sup?plied the sodium diethyl dithiocar?bamate trihydrate? needed for derivatization. All of the compounds were utilized without additional purification and were of analytical reagent (AR) or HPLC? quality. To avoid cont?amination, glassware was oven-dr?ied after being cleaned with deionized water.

4.2 Instru?mentation and Chromatographic Conditions

A Shimadzu Class VP Series HPLC s?ystem with binary pump (LC-10ATvp), autosample?r (SIL-10ADvp), UV-visible? detector (SPD-10Avp), and system controller (SCL-10Avp) was used to conduct chromatographic analysis. Software from LC Solutions was used to collect and proce?ss the data.The following chroma?tographic parameters were optimised:Column: YMC-Pack ODS-A C18 (3 µm particle size, 150 × 4 .6 mm) Mobile Phase: Water: Tetrahydrofuran: Acetonitrile (30:65:5 v/v/v) 1.5 mL min?¹ is the flow r?ate.  25 ± 2 °C is the column temperature; 280 nm is the detectio?n wavele?ngth ; 20 µL is the injection volume; and 12 minutes is the runtime.The chromatographic parameters that were optimized were as follows:? Column: YMC-Pack ODS-A C?18 (150 × 4.6 mm; 3 µm particle size) A Mobile Stage: Aq?ua: Tetr?ahydrofuran: 30:65:5 v/v/v acetonitrile The flow rate is 1.5 mL per minute.  Th?e column temperature is 25 ± 2 °C, the detection wavel?ength is 280 nm, the injection volume is 20 µL, and the runtime is 12 minutes.

4.3 Prepara?tion of Solutions

4.3.1 Mobile P?hase Preparation

The mobile phase was prepared by mix?ing acetonitrile, water, and te?trahy?drofuran in the ratio of 66:32:2 (v/v/v). The solution was filtered through a 0.4?5 µm nylon membrane filter and degassed by ultrasonication for 15 minutes before use.3.3.2. Derivatizing reagent. A? 50 mL volumetric flask was fil?led with aro?und 2.0 g of sodium diethyldithiocarbamate trihydrate. After adding 30 mL of N, N-dimethylacetamide and sonicating it to dissolve it, the vo?lume was increased to 50 mL using the same solvent.Every day, this reagent was made fresh.

4.3.2. Stan?dard Stock Solution

30 mg of precisely weighed Busulfan working standard was dissolved in? 20 mL N, N dimethylac?etamide, so?nicated for 10 minutes, and then diluted to 50 mL? with me?thanol. The concentration of th?e final stock solution was 600 µg/mL.

4.3.3. Calibrat?ion Standard Solutions

To cre?ate calibration standards in the 15.6–1 000 ng/mL range, aliquots of the standard stock solution were serially dilu?ted with the diluent. Linear?ity and resp?onse corre?lation were established using these solutions.

4.3.4. Sample Stock Solution

A 20 mL volumetric fla?sk received a 2.0 m L aliquot of? the Busulfan i?njection. 10 mL of diluent was added after the pipette was rins?ed twice with the diluent.  Before addin?g the same diluent to bring the? volume up to 20? mL, the sample wa?s sonicated for 10 minutes and vo?r?texed for five minutes.

4.4. Sample and Standard D?eriva?tization

Pre-equilibrated in a water bath at 60 °C, 5.0 mL of sodium diethyldithiocarbamate reagent was plac?ed into a 25 mL volumetric flask for derivatization. Following the addition of 2 mL of either standard or sample stock solution, the mixtur?e was gently swirled for 15 minutes at 60 °C. The volume was a djusted to 25 mL with methano?l and well mi?xed after cooling to room temperature. Before be?ing injected into the HPLC system, both samp le and reference preparations were filtered via 0.45 µm filters.?

4.5. System Suitability Testing

To ensure that the chromatographic system was p?erforming at its best, system suitabi?lity tests (SST) were carried out prior to sample analysis. Retention duration, area respo?nse, theoretical plates (N), tailing factor (T), and %RSD were among the parameters that were assesse?d Table 2 shows the Busulfan standard solutio?n' s system suitability results.

T?he method's repeatability and syst?em precision were confirmed by the %RSD for area and? retention time being under the p?ermitted limit (< 2%) ^[32].

4.6. Specificity

In order to determine specificity, potential in?terfere?nce was assessed by examining blank (meth?anol d?il?uent), placebo, standard, and sample preparations.? Methanesulfonic acid and 1, 4-butanediol, two r?ecognized degradation prod?ucts, were also? examined separately and in samples that had been spike?d to confirm their? separation from the Busulfan peak.

Table 3. Speci?ficity data show that there? is no interfere?nce d?uring the Busulfan retention time.

Because excipients and contaminants did not affect the Busulfan peak, these results validate the s?pecificity of the approach ^[33].

4.7. Forced Degradation Studies

To assess the method's capacity to indicate stability, forced de?gradation tests were carried out.? In accordance with ICH Q1A (R2) criteria, busulfan samples were exposed to acidic?, basic, oxidative, neutral, thermal, and photolytic stress conditions

Table 4. Busulfan's forced deterioration study results under differ?ent stress scenarios.?

The technique's stability-in?dicating nature was confirmed when degradation products were successfully isolated without interference ^[34].

4.8. Precision Studies

Three levels of precision were assessed:system precision, method precision, and intermediate precision. Analysis was done on six replicate injectio?ns of the standard and sample for mulations.

System accuracy:  Busulfan peak area %RSD < 2%.

Met?hod accuracy: Busu?lfan assay %RSD < 2%.

Repeatability across two analysts, various equipment, and days (%RSD < 2%?) showed intermediate precision ^[35].The reproducibility and ruggedness of the approach were validated by these results.

4.9. Linearity

Standard solutions in the range of 50% to 150% of the desired conce?ntration were examined in ord?er to assess linearity. The calibration curve showed an excellent linear connection be?tween concentration and peak area, with a correlation coefficient (R²) > 0 .998.

The regression formula was:

 y = 10345x + 25670, where x was c?oncentration? (ng/mL) and y was peak area. Systematic deviation w?as not seen,according to residual analysis. According to statistica?l an?alysis, the intercept was not substantially different from zero, with p > 0.05 ^[36].

4.10. Accuracy

Recovery tests we?re conducted in triplicate at 50%, 100%, and 150% of nomi?nal concentrations to evaluate accuracy.  98.2% to 101.7% re?coveries wi?th %RSD < 2% satisfied ICH Q2 (R1) requirements.These findings verify that the technique yields precise measurement even when formulation e?xcipients are present ^[37].

4.11. Range?

Based on linearity and? accuracy experiments, the analytical range was det?ermined to be between 50% and 150% of the nominal concentration.The method's usefulness for routine analysis was confirmed by its satisfactory lin?earity, pr?ecision, and accuracy with?in this range[38].

4.?12. Robustness

To assess robustness, the f?ollowing technique parameters were purposefully changed: Column oven tempera?ture (±5 °C) 

Detect?ion wavelength (±5 nm)

Speed of flow (±0.2 mL/min)

Composition of organic phases (±2%)

Retention period a?nd peak area showed no discernible effect s (%RSD < 2%). These resul?ts show? how the established HP?LC technique for Busulfan is robust and reliable ^[39].

5. RESU?LTS AND DISCUSSION (ON THE BASIS OF LITERATURE REVIEW)

5.1. Method Development and Optimization

Establishing a straightforward, accurate, and? stability-i?ndicating? RP-HPLC method for the? quantitative measurement of Busulfan in pharmaceutical formulations was the aim of this invest?igation. In order to get sharp, sym?metric peaks and adequ?ate resolution from degradation products, method develop?ment concentrated on optimizing chromatographic parameters, includi?ng mobile-phase composition, column select?ion, flow rate, and detection wav?ele?ngth?.  Early experiments using 60:40 v/v acetoni?trile–water mobile phases produced broad peaks and poor separation .Tet?rahydrofuran's addition enhanced peak shape and elution efficiency, resulti?ng in the ideal compo sit?ion of? water: acetonitrile: tetrahydrofuran (30: 65: 5, v/v/v).  When compare?d to other tested columns like Hypersil BDS and Phenomenex L?una, the YMC-Pack ODS-A C18 column (150 × 4.6 mm, 3 µm) provided better theoretical plate count and less tailing. After spectral scanning between 200 and 400 nm revealed the greates?t absorbance at this region for the Busulfan–diehyldithiocarbamate derivative, a detectio?n wavelength of 280 nm was selected. The retention du?ration was roughly 4.4 minutes under ideal circumstances, offering a c?om?promi?se betwe?en resolution and speed .The reliability of this wavelength and solvent s?yste?m has been confirmed by similar chromato?graphic behavior described by? N guyen et al. ^[24] and Lee e?t al?. ^[13].

5.2. System Suitability and Reproducibility?

A crucial pr?erequisite for method validation i?s system? appr?opriateness testing ^[26].The stand?ard solution was injected five times, with a mean peak area of 733408.6 ± 15467 AU and a %RSD of 0.20%.  The tailing factor was 1.02, well within the acceptabi?lity th?reshold of < 2%, and the theoretical plate count was over 4000.  Excellent column efficiency and reproducibilit y are shown by these data. Shinde et al. ^[12] have reported comparable RSD? values for antineoplastic medications, confirming the accuracy of t?he approach.  A?s a result, consi?stent chromatographic performance appropriate for regul?ar quality control was given by the optimized system.?

5.3. Speci?ficity and Selectivity

In blank, pl?acebo, or excipient chromatog?rams,? no interfering pe?aks wer?e seen at th?e Busulfan retention time, demonstrating the method's tot al specificity (Table 2).  Busulfan'?s separation from methanesulfonic acid and 1,?4-butanediol breakdown products shows that the          approach can accurately measure the analy?te even under stressful? circumstances. For alkylating agents like Busulfan, which can spontaneously degrade during s ample handling, selectivity is particularl?y important^[33].The lack of interfere?nce demonstrates that Busulfan is speci?fically targeted by the derivatizatio?n process? emp?loyi?ng sodium diethyldithiocarbamate without interacting with matrix constituents.

 5.4. Linearity and Range

With the regression equation y = 10 345x + 25 670 and correla tion coef?ficient R² = 0.9991, the calibration curve for Busulfan was linear o ver the range 15.6–1?000 ng/mL.The method's appropriateness for both low-dose and hig?h-dose samples found in clinical formulations? is ensured by linear?ity across road concentration range.  A p- value > 0.0?5 indicated no signifi?cant departure from linearity, and statistical an?alysis of the residuals showed a random distribution around the regr?ession line.  These results are consistent with those of Zeng et al. ^[7] and Valtola et al. ^[8] who showed simila?r linearity and correlation values fo?r UV and MS detection-based Busulfan quantification, respectively.  The precision, accur?acy, and linearity requirements of ICH Q2 (R1) were satisfied by the analytical range, which was set at 50–150% of the target concentration ^[26].

5.5. Accuracy and Recovery Studies

Recovery trials at nominal? concentrations of 50%, 100%, and 150% produc?ed mean recove?ries with %RS?D < 2%? that ranged? from 98.2% to 101.7%.  These findings demons?trate that the a?nalytical process yiel?ds precise quanti?fication even when for mulation ex?cipients are present.  Particul?arly for cytotox?ic medicati?ons with limited t?herapeutic margins, accuracy is a critical metric for confirming analytical reli?ability ^[5].The obtained recoveries are within the acceptle recovery rang e? (98–102%) as defined by USP and ICH recommendations ^[27].The current findings are supported? by si?milar recovery profiles for Busulfan formulations examined by UHP?LC (1?9) and LC–?MS/MS ^[24].

5.6. Preci?sion and Intermed iate Precision

System precision and process precision with %RSD values < 2% ind?icated excellent re?peatability.In?termediate precision, which was evaluated utilizing a variety of analysts, instruments, and days, likew?ise satisfied the same approval conditions. L?o?w va?riabil?ity under st?andard laboratory circumstances is a sign of the procedure's resilience and repeatability.  Doshi et? al. ^[17] state that ret?ention ti?me and area RSDs for well-controlled chromatographic system?s are less than 2%.  The consistency of the tools an?d analysts allows for the successful transfer of the established method between labo?ratories.

5.7. Robustness

Retention duration and area response w?ere not significantly affected by small, intentional chan?ges in analytical conditions, such as ±0.2 m L min?¹ in flow rate, ±5 °C in column temperature, ±5 nm in wavelen?gth, and ±2% in mobile-phase composition.  The percentage RSD readings stay ed below 2%.  This shows that, even in the face of slight variations in chromatographic parameters, the technique remains strong and dependable for standard quali?ty-control procedures?.The r?obu?stness statistics show good aggremen?t with earlier publications on antineoplastic drug assays created using AQbD principles ^[35].

5.8. Limit of Detection (LOD) and Limit of Quantitation (LOQ)

The computed values were LOD = 0.05 µg/mL and LOQ = 0. 15 µg/mL?, based on the signal to-noise ratio (S/N = 3:1 for LOD and 10:1 for LO?Q).  These findi?ngs indicate a high sensitivity of the app roach, which can identify Busulfan traces in formulations.Other v?erifi?ed Busulfan methods using similar derivatization techniques have show?n comparabl?e L?OD and LOQ values ^{[14, 21]}. Because plasma co?ncentrations frequently fall within the nanogram per milliliter range, such low detection limits make therapeutic monitoring easier ^[11].

5.9. Forced-Degradatio?n B?e?haviour

By exposing B?usulfan to a range of stress con?dit?ions (acidic, basic, oxidative, thermal, and photolytic), the s?tability?-indicating nature of the devised technique was verified. All degradation products were still clearly separated from the parent peak at 4.4 m?inutes (Table 3).Under oxidative (3% H2O?) and neutral hydrolysis conditions, busulfan significantly degraded, with test values f?alling to 76.4% and 76.5%, respectively.  Under acid and basic stress, less brea?kdown was seen, suggesting that the sulfonate ester connections have some resilience at ambient temperature.No co?-eluti?ng peak?s were found? in chromatographic profiles, confirming that the pro?cess ^[2] clearly isolates degradation product?s.  Blessy et al. have shown similar deterioration behavior. and Ghosh et al. ^[15], wh?o linked the cleavage of the methanesulfonate moiety to oxidative degradation.As a result,the devel?oped H?PLC approach can be regarded as stability-indicating, appropriate for stress testing and shelf-life assessment in accordance with regulat?ory requirement?s ^[31].

5.10. Comparativ?e Evaluation with Reported Methods

Table 4 compares the proposed RP- HPLC method with representative literature methods in terms of linearity range, LOD/LOQ, run?time, and validation compliance.