Separation Of Degradation Products Research Articles

Development and Validation of a Stability-indicating Method for Neostigmine Bromide: Separation, Identification and Characterization of Degradation Product using LC-MS and ESI-Q-TOF-MS/MS

Abstract: Objectives: Forced degradation study of the drug product and drug substance is very much important in drug development and drug discovery to establish the intrinsic stability and understand its behaviors towards different stress conditions. In the present work, stress testing of neostigmine bromide (NBr) was carried out as per ICH guidelines to identify and characterize the degradation product (DP) formed. Materials and Methods: According to ICH guidelines, drug substance was subjected to various degradation conditions. Waters BEH (Ethylene Bridge Hybrid) C-18 column (1.7 m, 100 mm 2.1 mm) having the composition of mobile phase Eluent A: 0.01M KH2PO4 in water pH-2.5 with orthophosphoric acid and Eluent B: Acetonitrile utilizing 220 nm wavelengths provided the best separation of DP and drug component. Throughout the analysis, the injection volume (5 μL) and flow rate (0.3 mLmin -1) were kept constant. Results: The limits of detection (LOD) and quantitation (LOQ) were set at 25 ngmL-1 and 50 ngmL-1, respectively. The method showed excellent linearity over a concentration range of 25-250 ngmL-1 with a regression coefficient (R2) value of 0.9999. The results showed that significant degradation was observed in base and oxidative degradation conditions whereas found in neutral, acidic, photolytic and thermal degradation conditions. Conclusion: The DP was identified and characterized using a sophisticated HRMS/MS/TOF approach for accurate mass measurement using the ESI positive mode of ionization. In the present study, the establishment of the degradation pathway of drug substance and fragmentation pathway of DP-I were explained which was never reported in any literature. Keywords: Neostigmine bromide, RP-UPLC, Stability-indicating, Stress study, Validation.

Prediction of Ocular Toxicity of Potential Degradation Products of Proparacaine Hydrochloride Subjected under ICH Recommended Stress Conditions.

Proparacaine hydrochloric ophthalmic solution (0.5%) is widely used as ophthalmic anesthetic for short conjunctival and corneal procedures. In connection to this, present research work was conducted to investigate ocular toxicity of its probable degradation products (DPs), which were generated after treating drug with stressors mentioned under International Conference on Harmonization (ICH) guideline Q1A (R2). The probable DPs were generated by placing drug under various stress conditions to generate similarity of the effect of stressor which drug supposed to face during its life cycle. The hydrolytic stress condition under acidic, basic and neutral environment formed two major DPs, while the drug was stable and did not generate any DP when subjected to oxidative, dry heat and photolytic stress. The separation of DPs was carried out using high performance liquid chromatographic system. The structural identification of DPs was carried out by subjecting degradation samples to liquid chromatography coupled with mass spectrometry. These DPs were screened using in-silico ocular toxicity prediction models Pub-SVM to predict their potential to cause irritation/corrosion to eye. The DPs identified were DP 1 (2-(diethyl amino) ethanol) and DP 2 (3-amino-4-propoxybenzoic acid). The in-silico ocular toxicity study predicted that DP 1 has potential of eye corrosion (EC) and eye irritation (EI), whereas DP 2 has a potential EI. The drug was found safe and devoid of any EC and EI potential.

Solubility determination and thermodynamic modeling of bis-2-hydroxyethyl terephthalate (BHET) in different solvents

Studies on the degradation process of waste polyethylene terephthalate (PET) have become increasingly mature, but there are relatively few studies on the separation of degradation products. The products contain many components and the separation of which is difficult. Therefore, the study on phase equilibrium thermodynamics of bis-2-hydroxyethyl terephthalate (BHET) is of great theoretical significance and practical value to provide basic data for the BHET crystallization separation. In this work, the degraded products were purified and characterized. The solubility of BHET in methanol, ethanol, ethylene glycol, water and the mixture of ethylene glycol + water were determined by static method. The experimental results were correlated with different models, such as ideal solution (IS) model, λh equation, Apelblat equation and NRTL model. Based on the van't Hoff equation, the mixing Gibbs energy, enthalpy and entropy were calculated. From this work, the basic data which can be used to guide the crystallization process of BHET were obtained, including solubility data, correlation model and thermodynamic properties.

Separation, Identification, Isolation and Characterization of Degradation Product of Osimertinib Tablets by UPLC-QTOF-MS/MS and NMR: Evaluation of <i>In-Silico</i> Safety Assessment for Osimertinib (OSM) and Degradation Product (DP)

The present work encompasses identification and characterization of major degradation product (DP) of OSM observed in base hydrolytic stress study. The separation of DP was carried out on a non-polar stationary phase by using high-performance liquid chromatography system (HPLC). Using waters X-bridge (250 mm × 4.6 mm, 5 μm) C18 column with gradient elution program. For the characterization study, stress samples were subjected to HPLC and UPLC-QTOF-MS/MS and based on mass fragmentation pattern, plausible structure was deduced. Further, the DP was isolated using semi-prepara- tive liquid chromatography and concentrated the fractions using lyophilization. The isolated DP was subjected to extensive 1D (1H, 13C, and DEPT-135) and 2D (COSY, HSQC and HMBC) nuclear magnetic resonance (NMR) studies to authenticate the structure. The impurity was unambiguously named as N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-4-metho-xy-5-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)phenyl)-3-methoxypropanamide. Add- itionally, the In-Silico structure activity relation (QSAR) assessed through statistical based software’s DEREK NexusTM, and MultiCASE, Case UltraTM widely accepted and respected software’s for DP and OSM.

Development, Validation of RP-HPLC Method and GC MS Analysis of Desloratadine HCL and IT"s Degradation Products

Introduction: Efficacy and safety of drug therapy is directly related to the stability of active pharmaceutical ingredient (API) and drug product used. Forced degradation studies (also called stress testing) are performed for better understanding of API and drug product stability. Thus, stress testing is a prognostic research tool used to ascertain stability of drug molecules and provide support for developing a stability-indicating method. Aim: The research aim is Development, Validation of RP-HPLC Method and GC MS Analysis of Desloratadine HCL and Its Degradation Products. Objective: The objective of the current study was to develop a validated specific stability-indicating reversed-phase liquid chromatographic method for the quantitative determination of Desloratadine HCl in bulk samples in the presence of degradation products. Method: Desloratadine HCL was subjected to variable pH, oxidative, dry heat and photolytic stress condition as per ICH guideline for the stability study. Stressed samples were further studied by the validated RP-HPLC method and also studied by GC-MS to characterize degradation products (Fig 15). Result: At oxidative stress, degradation products were generated and detected by GCMS. Slight degradation was observed in acidic and alkaline stress while no degradation was observed in other stress conditions. Separation of degradation products from pure drug was achieved on C18 column 5µ (4.6 X 250 mm) using the mobile phase consists a mixture of Orthophosphoric acid (0.1%V/V), Acetonitrile and Methanol (50:35:15 V/V/V). The detection was carried out at 242 nm. The proposed validated LC method was used to quantify the stressed test solutions in order to ascertain stability-indicating potential of the method. Conclusion: The established LC approach has been shown to be suitable for determining the quality of Desloratadine HCl from its dosage form and assessing its stability when required.

Development and Validation of a Stability-indicating Method for Ibrutinib: Identification and Separation of Degradation Products, Known and Genotoxic Impurities Using RP-HPLC/PDA and QDa Mass Detectors

The present work describes a novel stability-indicating method development and validation for separation and identification of degradation products, known and genotoxic impurities in recently FDA approved (Ibrutinib) an anti-cancer drug using RP-HPLC/PDA and QDa mass detectors. Separation of all impurities could be achieved using stationary phase column (X-Select CSH, C18, 150 mm × 4.6 mm × 3.5 μm) with a flow rate of 1 ml/minute and total run time 85 minutes. Degradation was carried out; Ibrutinib was sensitive to acid, base and peroxide conditions. In the base degradation sample, a major degradation impurity was found, which was identified their mass value and structure information using QDa mass detector. Ibrutinib was found to be stable under heat, UV, humidity and water conditions. No interference was found at identified impurities, degradation products and ibrutinib peak retention time. The proposed method was validated as per ICH Q2 (R1) guidelines. It was observed that the percentage recovery was found to be 95 % to 105 % and linearity over the range of LOQ level, 50 %, 100 % 150 % and 300 % level of concentrations with R2: 0.999 to 1.000. According to validation and degradation results, the method was found to be good stability indicating nature as well as user-friendly.

Isolation and Characterization of Novel Degradation Products of Eliglustat Tartrate Using 2D-NMR and HRMS: Development and Validation of Stability Indicating RP-UPLC Method for Quantification of Assay and Characterized Impurities

Eliglustat tartrate is an oral inhibitor of glucosylceramide synthase used in the therapy of Gaucher’s disease. The drug was subjected to forced degradation study using various stress parameters like acidic, basic, oxidation, photolysis and thermal conditions. The drug showed significant degradation under acidic and peroxide stress conditions, however the drug remained stable under basic, neutral, thermal and photolytic stress conditions. The present research reports the characterization of degradation products of Eliglustat by liquid chromatography-tandem mass spectrometry(HRMS) and 2D-NMR techniques along with the development and validation of stability indicating ultra performance liquid chromatographic method for determination and quantification of Eliglustat drug in presence of characterized impurities. Two novel degradation products (DP1 & DP-2) were found under stress conditions. These degradation products were isolated using preparative high performance liquid chromatography. The structure of the DP-1 was identified as the cis- diastereoisomer of Eliglustat and DP-2 as the N-Oxide impurity of Eliglustat which were not reported in any of the literature. The chromatographic method was developed on UPLC to achieve separation of degradation products and Eliglustat using Acquity; BEH; C-18; 100 x 2.1 mm; 1.7 μm column within a short run time of 8.0 minutes. The method was validated for specificity, precision, linearity and accuracy. The quantitation limits obtained were in the range of 0.3-3.0 μg ml-1 for the characterized impurities. This method is compatible to LCMS analysis which enables to identify the unknown impurities formed in the process.

Stress Testing of Quetiapine Fumarate by a Chemometric Assisted Multi-Component Ion-Interaction System

Classical experimental designs have been popularly employed in establishing robust analytical methods while achieving other advantages, viz., reduction in the number of experiments and hence lower reagent consumption and less laboratory work. To achieve optimum chromatographic condition, a computer-aided Box-Behnken Design (BBD) in ion-pairing stability-indicating RP-HPLC assay of quetiapinefumarate along with its stress related substances has been investigated here, proving to be an invaluable tool in ascertaining a reliable method. The study includes forced degradation of quetiapinefumarateunder acidic, alkaline, photo, oxidative and peroxide stress conditions followed by separation of degradation products. Critical factors including buffer pH, % organic phase (acetonitrile) and concentration of hexane sulphonate (ion-pairing reagent) susceptible to influence the separation (critical resolutions) and total analysis time were investigated by response surface methodology. The best optimal separation condition as obtained was observed on an enable C-18 column (250mm x 4.6mm i.d, 5μm particle size) using mobile phase composed of Phosphate buffer (pH 2.0) containing 0.002mM hexane sulphonate and acetonitrile (74.4:25.6 v/v) at a flow rate of 1.00ml/min. The eluents were observed at 220nm using a PDA detector. Further, the method was validated to ensure its reliability and other regulatory criteria are met.

Degradation Efficiency and Characterization of Lincomycin by Electron Beam Irradiation

Lincomycin is one of the major species among the Pharmaceuticals and Personal Care Products (PPCPs) detectedfrom the four major rivers in Korea. The structure characterization was performed of six degradation products of lincomycinformed under the irradiation of electron beam, and the degradation efficiency as a function of the various irradiation dose andsample concentration was investigated. Electron beam (10 MeV, 0.5 mA and 5 kW) experiments for the structural characterizationof degradation products that are fortified with lincomycin, were performed at the dose of 10 kGy. The separation of degradationproducts and lincomycin was carried out using a C18 column (2.1×100 mm, 3.5 μm), using gradient elution with 20 mMammonium acetate and acetonitrile. The structures of six degradation products of lincomycin were proposed by interpretation ofmass spectra and chromatograms by LC-MS/MS. The mass fragmentation pathways of mass spectra in tandem mass spectrometrywere also proposed. Experiments were performed of the degradation efficiency as a function of the irradiation dose intensityand the initial concentration of lincomycin in an aqueous environment. In addition, increased degradation efficiency wasobserved with a higher dose of electron beam and lower concentration.

A Simple Stability Indicating CZE Method for the Analysis of Octreotide Acetate

A simple stability indicating capillary zone electrophoretic method was developed and validated for the analysis of octreotide acetate (OCT-Ac). The best separation was achieved by bare fused silica capillaries (50 μm i.d.; 65.5 cm total and 57.0 cm effective length), phosphate buffer (pH = 3.25; 50 mM), at 32.5 °C. The samples were injected using 50 mbar for 5 s and subjected to the applied voltage of 27.5 kV for separation. The detection was carried out using a PAD at a wavelength of 195 nm. For improving the repeatability of the method, l-histidine was applied as an internal standard. According to the validation results, the method was linear in the concentration range of 3.30–400 μg mL−1 (correlation coefficient of 0.9996) with a limit of detection of 1.08 μg mL−1 and a limit of quantification of 3.30 μg mL−1; accuracy of the method was between 100.4 ± 0.2 and 101.1 ± 0.2 %; intra-assay precision was 0.5–2.6 % and intermediate precision was 1.3–3.2 %. The proposed method was successfully applied for the quantification of OCT-Ac in both a pharmaceutical formulation and force-degraded samples and for the detection and separation of degradation products; besides, the obtained results were used for the evaluation of the degradation kinetics of OCT-Ac under different stress conditions. So, it is concluded that the developed method could be employed as a simple, accurate and precise stability-indicating method in quality control laboratories to assess the quantity and stability of OCT-Ac pharmaceutical products.

Mechanisms of Degradation of the Natural High-Potency Sweetener (2R,4R)-Monatin in Mock Beverage Solutions

The sodium, potassium, or mixed sodium/potassium salt of the naturally occurring high-potency sweetener (2R,4R)-monatin, also known by the common name arruva, degrades over time in model beverage solutions in the presence of light. By use of UHPLC, LC-MS/MS, and peroxide assays, it has been demonstrated that degradation is accelerated by UV/visible light and the presence of trace metal ions. Data are presented that are consistent with a role for singlet oxygen (¹O₂), free radicals, and peroxides (both H₂O₂ and organic peroxides) in monatin oxidation. Separation of degradation products by UHPLC/HPLC or LC-MS/MS provided evidence for the formation of hydroxylated and peroxide species formed on the indole ring (mass increases 16 and 32, respectively) as well as multiple ring and side-chain oxidation and scission products. Model oxidation systems using the photosensitizer Rose Bengal as a source of ¹O₂ support the proposed photodegradation pathways.

STABILITY-INDICATING CHROMATOGRAPHIC METHOD FOR THE DETERMINATION OF BENZONATATE, DIPHENHYDRAMINE, GUAIFENESIN, AND PHENYLEPHRINE

A high-performance liquid chromatographic method has been developed for the simultaneous analysis of benzonatate (BNZ), diphenhydramine hydrochloride (DPH), guaifenesin (GFN), and phenylephrine hydrochloride (PEP). The separation was achieved using a Thermo C18 reversed-phase column (250 mm × 4.6 mm ID, particle size 5 µ). Dual mode gradient elution was used for the separation and UV detection was carried out at 222 nm. The method was specific and stability indicating as chromatographic conditions provided adequate separation of degradation products. The method showed good linearity in the range of 5–400, 1–120, 3–300, and 2–100 µg/mL for BNZ, DPH, GFN, and PEP, respectively. All the square of the correlation coefficients are 0.999. The degradation products were isolated and identified by NMR, IR, and mass spectroscopy. The proposed method proved to be accurate, precise, selective, and robust. The applicability of the method was evaluated in commercial dosage form analysis as well as in stability studies.

Development and Validation of a Novel Stability Indicating RP-UPLC Method for Simultaneous Estimation of Methylparaben, Mometasone Furoate and Eberconazole Nitrate in Combined Topical Pharmaceutical Dosage Form

A novel, rapid, simple, stability indicating RP-UPLC method development and validation for simultaneous estimation of methylparaben (MP), mometasone furoate (MF) and eberconazole nitrate (EN) in topical pharmaceutical dosage form. Isocratic chromatographic separation was achieved on Acquity BEH C18, column using mobile phase consist of buffer (0.1%v/v triethylamine in water pH 6.0 by Glacial acetic acid), acetonitrile in the ratio (40:60, v/v) at flow rate 0.4 mL/min and detection was monitored at 235 nm by using photo-diode array detector. The method shows excellent linearity over the range of 2.75-152.7 µg/mL, 4.45-60.7 µg/mL and 9.10-606.6 µg/mL for methylparaben, mometasone furoate and eberconazole nitrate respectively. Recovery was found for all the components to be in the range of 98.4-101.7%. Stability indicating capability of developed method was established by analysing forced degradation samples in which spectral purity of methylparaben, mometasone furoate and eberconazole nitrate along with separation of degradation products from analytes peak. The proposed method was successfully applied for the quantitative estimation of MP, MF and EN in cream sample.

Stability-Indicating Validated Novel RP-HPLC Method for Simultaneous Estimation of Methylparaben, Ketoconazole, and Mometasone Furoate in Topical Pharmaceutical Dosage Formulation

A simple, specific, precise, and accurate RP-HPLC method has been developed and validated for simultaneous estimation of Methylparaben (MP), Ketoconazole (KT), and Mometasone Furoate (MF) topical pharmaceutical dosage formulation. The separation was achieved by Waters X Terra C18 column using mobile phase consisting of buffer (triethyl amine in water, pH adjusted to 6.5 with glacial acetic acid)-acetonitrile (40 : 60, v/v) at a flow rate of 1.5 mL/min and detection at 250 nm. The method showed linearity with correlation coefficient <0.9999 over the range of 0.12–15.2 μg/mL, 0.67–149.4 μg/mL, and 0.42–7.6 μg/mL for MP, KT, and MF, respectively. The mean recoveries were found to be in the range of 99.9–101.1% for all the components. The method was validated as per the ICH guidelines for linearity, limit of detection, limit of quantification, accuracy, precision, robustness and solution stability. Stability indicating capability of the developed method was established by analyzing forced degradation of samples in which spectral purity of MP, KT, and MF along with separation of degradation products from analytes peak was achieved. The method can be successfully applied for routine analysis of quantitative determination of MP, KT, and MF in pharmaceutical dosage form.

Development and validation of a stability‐indicating assay including the isolation and characterization of degradation products of metaxalone by LC‐MS

A stability-indicating reverse-phase high-performance liquid chromatography-mass spectrometric method was developed and validated for the assay of metaxalone through forced degradation under acidic, alkaline, photo, oxidative and peroxide stress conditions. Separation of degradation products was accomplished on a reverse-phase Phenomenex C18 (250 × 4.6 mm, 5 µm) column thermostated at 25 °C using 10 mM aqueous ammonium acetate: methanol (35:65 v/v) as mobile phase in an isocratic mode of elution. The eluents were detected at 275 nm by photo diode array detector and mass detectors connected in series. Two unknown base hydrolysis products of metaxalone were identified and characterized as (a) methyl 3-(3,5-dimethylphenoxy)-2-hydroxypropylcarbamate and (b) 1-(3,5-dimethylphenoxy)-3-aminopropan-2-ol by MS, (1)H NMR and FTIR spectroscopy. The method was validated as per International Conference on Harmonization guidelines and metaxalone was selectively determined in presence of its degradation impurities, demonstrating its stability-indicating nature.

Development and validation of stability-indicating UPLC method for the determination of lafutidine and its impurities in bulk and pharmaceutical dosage form

Abstract Background A simple and rapid stability-indicating, reversed phase ultra-performance liquid chromatography (UPLC) method was developed for the quantitative determination of lafutidine and its four potential impurities. Results Separation was achieved on Acquity BEH-shield RP18 UPLC column (3.0 mm × 100 mm, 1.7 μm) under the gradient mode of elution by using mobile phase A (0.02M diammonium hydrogen phosphate/acetonitrile, 80:20 v/v) and mobile phase B (0.02M diammonium hydrogen phosphate/acetonitrile, 30:70 v/v). The flow rate was maintained at 0.5 mL min−1. UV detection was carried out at 276 nm. Conclusions Stability-indicating capability of the developed method is established by analyzing forced degradation samples in which the spectral purity of lafutidine is ascertained along with the separation of degradation products from analyte peak. The developed UPLC method is validated as per International Conference on Harmonization guidelines with respect to system suitability, specificity, precision, sensitivity, accuracy, linearity, and robustness.

Development and Validation of a Stability-Indicating RP-HPLC Method for Simultaneous Determination of Phenoxyethanol, Methylparaben, Propylparaben, Mometasone Furoate, and Tazarotene in Topical Pharmaceutical Dosage Formulation

A stability-indicating RP-HPLC method has been developed and validated for the simultaneous determination of phenoxyethanol (PE), methylparaben (MP), propylparaben (PP), mometasone furoate (MF), and tazarotene (TA) in topical pharmaceutical dosage formulation. The desired chromatographic separation was achieved on the Waters X-Bridge™ C18 (50×4.6mm, 3.5μ) column using gradient elution at 256 nm detection wavelength. The optimized mobile phase consisted of 0.1%v/v orthophosphoric acid in water as solvent-A and acetonitrile as solvent-B. The method showed linearity over the range of 5.88–61.76 μg/mL, 0.18–62.36 μg/mL, 0.17–6.26 μg/mL, 0.47–31.22 μg/mL, and 0.44–30.45 μg/mL for PE, MP, PP, MF, and TA, respectively. The recovery for all of the components was in the range of 98–102%. The stability-indicating capability of the developed method was established by analysing the forced degradation samples, in which the spectral purity of PE, MP, PP, MF, and TA along with the separation of degradation products from the analyte peaks was achieved. The proposed method was successfully applied for the quantitative determination of PE, MP, PP, MF, and TA in a cream sample.

Validation of the Stability-Indicating HPLC Method for the Major Flavonoids in Spray- Dryed Leaf Extract of Aleurites moluccana L. Willd

The HPLC-UV method previously developed for the analysis of the flavonoid 2”-O-rhamnosylswertisin (I), an active compound of dried leaf extracts of Aleurites moluccana L. Willd, was fully validated with the inclusion of another flavonoid, swertisin (II). The stability-indicating capability of the method was established by analyzing forced degradation extract samples (acid, alkali, neutral, oxidative and photolytic condition) in which the spectral purity of the markers was ascertained together with the separation of degradation products from the markers. The method proved be linear over a range of 5.89-117.8 and 1.38-27.68 μg.mL-1 for I and II, respectively. The recovery for I and II was 100.3 and 102.8%, respectively, at a level of 100%. A relative standard deviation (RSD%) < 1.0% (intra-day) and < 3.5% (inter-days) for area, and less than 0.5% for retention time indicated that the precision of the method is acceptable. Validation parameters such as selectivity and robustness were also determined. In addition, during the course of LC-MSn analysis three other flavonoids were discovered in the A. moluccana extract and their tentative structures identified.

HPLC Stability Indicating Assay Method for Metformin Hydrochloride in Bulk Drug and Tablets and Cytotoxicity of Degradation Products

The present work reports a stability-indicating reversed phase ion pair liquid chromatography method for quantitative determination of metformin hydrochloride (MTF) (drug and tablets) and assessment of the cytotoxicity of degraded MTF. Chromatographic separation was performed on a C18 (Phenomenex® Luna), 5.0 μm (250 mm x 4.6 mm) column using isocratic elution. The optimized mobile phase consisted of 10 mmol L-1 heptane sulphonic acid (pH 3.0), acetonitrile and methanol (75:8:17 v/v), flow 1.0 mL.min-1, at 30 ºC. The eluted compounds were monitored at 210 nm wavelength using a DAD detector. The stability indicating capability of the developed method was established by analyzing forced degradation samples (acid, alkali, neutral, oxidative and photolytic condition) in which the spectral purity of MTF was ascertained, along with the separation of degradation products from the analyte peak. The cytotoxicity of MTF and the degraded drug was analyzed in the L929 cell test by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The 500 mg of commercial MTF tablets (Reference, Generic and Similar drugs) showed a similar chromatographic profile, but lower intensity degradation in the same conditions, in relation to the bulk drug in almost forced conditions. Neither the degraded drug samples, nor the tablet samples degraded in acid conditions, showed any cytotoxicity. The developed stability-indicating ion-pair HPLC method was fully validated according to the International Conference on Harmonization (ICH) guidelines. The degraded sample of MTF showed no affect on cell viability, compared with the non-degraded drug. Keywords: Bulk Drug, Cytotoxicity, Degradation Products, HPLC-UV, Ion-pair liquid chromatography, Metformin hydrochloride, Method validation, MTT assay, Stability-indicating assay method, Tablets

STRATEGIES FOR THE OPTIMUM SEPARATION OF GAMMA RADIOLYTIC DEGRADATION PRODUCTS OF METHOXYCHLOR USING RP-HPLC

Different types of reversed phase (RP) columns have been incorporated for the separation of gamma radiolytic degradation products (DPs) of methoxychlor. The effect of carbon chain length, carbon content, surface area, pore size, pore volume, and column dimensions of RP columns on DPs separation has also been investigated. The separation of DPs has been optimized employing methanol-water mobile phase and UV-DAD. In methanol media two major and five minor DPs were detected in gamma irradiated MXC by RP-HPLC. The peaks eluting before the parent peak on reversed phase column are expected to be relatively polar compounds while peaks after MXC are assumed to be non-polar in nature corresponding to general RP behavior. From HPLC analysis it is inferred that at gamma radiation dose of 5 kGy ≥95% MXC has been degraded for a dose rate of 200 Gyh−1. The chemical radiation yield (G-value) along with effect of MXC concentration on degradation has also been explored. The physico-chemical parameters of RP columns have been compared under the optimum separation conditions. It may be concluded that HPLC is an excellent tool to investigate radiolytic degradation of persistent organic pollutants.