Stability‐indicating chromatographic methods for determination of flecainide acetate in the presence of its degradation products; isolation and identification of two of its impurities

In this work, two stability‐indicating chromatographic methods have been developed and validated for determination of flecainide acetate (an antiarrhythmic drug) in the presence of its degradation products (flecainide impurities; B and D). Flecainide acetate was subjected to a stress stability study including acid, alkali, oxidative, photolytic and thermal degradation. The suggested chromatographic methods included the use of thin layer chromatography (TLC‐densitometry) and high‐performance liquid chromatography (HPLC). The TLC method employed aluminum TLC plates precoated with silica gel G.F₂₅₄ as the stationary phase and methanol–ethyl acetate–33% ammonia (3:7:0.3, by volume) as the mobile phase. The chromatograms were scanned at 290 nm and visualized in daylight by the aid of iodine vapor. The developed HPLC method used a RP‐C₁₈ column with isocratic elution. Separation was achieved using a mobile phase composed of phosphate buffer pH 3.3–acetonitrile–triethylamine (53:47:0.03, by volume) at a flow rate of 1.0 mL/min and UV detection at 292 nm. Factors affecting the efficiency of HPLC method have been studied carefully to reach the optimum conditions for separation. The developed methods were validated according to the International Conference on Harmonization guidelines and were applied for bulk powder and dosage form. Copyright © 2016 John Wiley & Sons, Ltd.     

Different stability‐indicating chromatographic methods for specific determination of paracetamol,dantrolene sodium,their toxic impurities and degradation products

A well‐known analgesic (paracetamol, PAR) and skeletal muscle relaxant [dantrolene sodium (DNS)] have been analyzed without interference from their toxic impurities and degradation products. The studied PAR impurities are the genotoxic and nephrotoxic p‐amino phenol (PAP) and the hepatotoxic and nephrotoxic chloroacetanilide, while 5‐(4‐nitrophenyl)‐2‐furaldehyde is reported to be a mutagenic and carcinogenic degradation product of DNS. The five studied components were determined and quantified by TLC–densitometric and RP‐HPLC methods. TLC–densitometry (method 1) used TLC silica gel and chloroform–ethyl acetate–acetic acid–triethylamine (7:3:0.5:0.05, by volume) as the mobile phase with UV scanning at 230 nm, while RP‐HPLC (method 2) was based on separation on a C₁₈ column using methanol–water (55:45, v/v pH 3 with aqueous formic acid) as mobile phase at 1 mL/min and detection at 230 nm. The developed methods were used for determination and quantification of the five studied components in different laboratory‐prepared mixtures. The were also applied for analysis of Dantrelax^® compound capsules where no interference among the studied components with each other or from excipients was observed. The methods were validated as per International Conference on Harmonization guidelines, and they compared favorably with the reported ones.     

Different chromatographic methods for simultaneous determination of diloxanide furoate,metronidazole and its toxic impurity

TLC densitometric and RP-HPLC methods are innovative chromatographic methods used for determination of diloxanide furoate, metronidazole and its impurity, 4-nitroimidazole. In the developed TLC densitometric method, appropriate separation was achieved using silica gel 60 F₂₅₄ TLC plates and ethyl acetate/acetone/hexane/ammonia solution (9.5:0.5:0.3:0.3, by volume), as a developing system and the separated bands were UV-scanned at 276 nm. While the developed RP-HPLC method depended on separation of components on C8 column using deionized water containing 0.05 % TEA: methanol (40:60, v/v) as a mobile phase at constant flow rate of 1 mL/min with UV detection at 276 nm. Variables affecting performance of the developed methods were studied and optimized. Regression analysis showed acceptable correlation coefficients in the selected ranges with excellent percentage recoveries. The methods showed no significant interferences from dosage form excipients, and the validity of the proposed methods was further assessed by applying standard addition technique. In addition, results obtained by applying the proposed methods were statistically compared to those obtained by applying the reported method and no significant difference was found between them. The suggested methods were successfully applied for the determination of the cited drugs in bulk powder, laboratory prepared mixtures and commercial tablets.     

Synthesis,characterization and control of eight process-related and two genotoxic fingolimod impurities by a validated RP-UPLC method

An HPLC method has been described in the European Pharmacopoeia and United States Pharmacopeia for the determination of nine organic impurities (imp A–I) in fingolimod hydrochloride, a synthetic sphingosine-1-phosphate receptor modulator. The manufacturing process of fingolimod hydrochloride consists of multistep chemical synthesis wherein controls of precursors, intermediates and process steps should be performed to assure the final quality of the drug substance. We synthesized and isolated eight process-related impurities (FINI imp A–H) of fingolimod, which were different from the pharmacopoeial impurities. One unknown process-related impurity was found as a key intermediate (FINI) and was identified by LC–MS. Characterization of all of the impurities were done using spectroscopic techniques (¹H and ¹³C NMR, FTIR, MS), and the mechanistic pathways to the formation of these impurities were also discussed. Two of these impurities were evaluated as potential genotoxic impurities owing to their alerting structures and alkylating properties (alkyl sulfonates and alkyl halides, class 3, ICH M7). We also developed and validated an RP-UPLC method in line with ICH Q2 guidelines for control these impurities (FINI imp A–H) and to assure the pharmacopoeial quality drug substance.     

Determination of Sumatriptan and Zolmitriptan in Presence of Their Corresponding Degradation Products by HPTLC Methods

Accurate, sensitive, and precise high performance thin layer chromatographic (HPTLC) methods were developed and validated for the determination of sumatriptan and zolmitriptan in presence of their degradation products. Sumatriptan was separated from its degradation products and analyzed on TLC silica gel 60 F₂₅₄ plates using chloroform–ethyl acetate–methanol–ammonia (4:3:3:0.1, v/v) as a developing system followed by densitometric measurement of the bands at 228 nm. Zolmitriptan was determined using chloroform–ethyl acetate–methanol–ammonia (3:3:3:1, v/v) as a developing system followed by densitometric measurement at 222 nm. The methods were validated over a range of 0.5–4 μg/spot for sumatriptan and 0.5–3 μg/spot for zolmitriptan. The proposed methods were successfully applied for the determination of the studied drugs in bulk powder and in their pharmaceutical formulations.     

Stability-indicating liquid chromatography method development and validation for impurity profiling of montelukast sodium in bulk drug and tablet dosage form

Montelukast sodium (MLS) is a leukotriene receptor antagonist drug used in the treatment of asthma, bronchospasm, allergic rhinitis and urticaria. A reversed-phase high performance liquid chromatography method was developed to separate, identify and quantitative determination of MLS and its eight known organic impurities in tablet dosage form using a C₁₈ column and mobile phases consisting of a gradient mixture of pH 2.5 phosphate buffer and acetonitrile. The stability-indicating character of the developed method was proven using stress testing (1 m HCl at 80°C/30 min, 1 m NaOH at 80°C/30 min, H₂O at 80°C/30 min, 3% H₂O₂ at 25°C/1 min, dry heat at 105°C/10 h and UV–vis light/4 days) and was validated for specificity, quantitation limit, linearity, precision, accuracy and robustness. For MLS and its eight known impurities, the quantitation limits, linearity and recoveries were 0.015–0.03 μg/ml, correlation coefficient > 0.997 (R² > 0.995) and 85.5–107.0%, respectively. The developed chromatographic method is suitable for impurity profiling and also for assay determination of MLS in bulk drugs and pharmaceutical formulations. The mass values (m/z) of newly formed degradation products (DP1 and DP2) of montelukast sodium were identified using liquid chromatography–mass spectrometry.     

Simultaneous HPTLC and RP-HPLC methods for determination of bumadizone in the presence of its alkaline-induced degradation product

Accurate, selective, sensitive and precise HPTLC‐densitometric and RP‐HPLC methods were developed and validated for determination of bumadizone calcium semi‐hydrate in the presence of its alkaline‐induced degradation product and in pharmaceutical formulation. Method A uses HPTLC‐densitometry, depending on separation and quantitation of bumadizone and its alkaline‐induced degradation product on TLC silica gel 60 F₂₅₄ plates, using hexane–ethyl acetate–glacial acetic acid (8:2:0.2, v/v/v) as a mobile phase followed by densitometric measurement of the bands at 240 nm. Method B comprises RP‐HPLC separation of bumadizone and its alkaline‐induced degradation product using a mobile phase consisting of methanol–water–acetonitrile (20:30:50, v/v/v) on a Phenomenex C₁₈ column at a flow‐rate of 2 mL/min and UV detection at 235 nm. The proposed methods were successfully applied to the analysis of bumadizone either in bulk powder or in pharmaceutical formulation without interference from other dosage form additives, and the results were statistically compared with the established method. Copyright © 2011 John Wiley & Sons, Ltd.     

Development and Validation of HPLC,TLC and Derivative Spectrophotometric Methods for the Analysis of Ezetimibe in the Presence of Alkaline Induced Degradation Products

Reversed phase‐high performance liquid chromatography (RP‐HPLC), thin layer chromatography (TLC) densitometry and first derivative spectrophotometry (1D) techniques are developed and validated as a stability‐indicating assay of ezetimibe in the presence of alkaline induced degradation products. RP‐HPLC method involves an isocratic elution on a Phenomenex Luna 5μ C₁₈ column using acetonitrile: water: glacial acetic acid (50:50:0.1 v/v/v) as a mobile phase at a flow rate of 1.5 mL/min. and a UV detector at 235 nm. TLC densitometric method is based on the difference in Rf‐values between the intact drug and its degradation products on aluminum‐packed silica gel 60 F₂₅₄ TLC plates as stationary phase with isopropanol: ammonia 33% (9:1 v/v) as a developing mobile phase. On the fluorescent plates, the spots were located by fluorescence quenching and the densitometric analysis was carried out at 250 nm. Derivative spectrophotometry, the zero‐crossing method, ezetimibe was determined using first derivative at 261 nm in the presence of its degradation products. Calibration graphs of the three suggested methods are linear in the concentration ranges 1–10 mcg/mL, 0.1–1 mg/mL and 1–16 mcg/mL with a mean percentage accuracy of 99.05 ± 0.54%, 99.46 ± 0.63% and 99.24 ± 0.82% of bulk powder, respectively. The three proposed methods were successfully applied for the determination of ezetimibe in raw material and pharmaceutical dosage form; the results were statistically analyzed and compared with those obtained by the reported method. Validation parameters were determined for linearity, accuracy and precision; selectivity and robustness and were assessed by applying the standard addition technique.     

Determination of Ranolazine in Tablet Formulations by High-Performance Thin-Layer Chromatography—Mass Spectrometry Using Reflectance Scanning Densitometry

JPC – Journal of Planar Chromatography – Modern TLC - A new quantitative densitometric high-performance thin-layer chromatographic (HPTLC) method was developed and validated for the...     

Two validated chromatographic determinations of an antifungal drug,its toxic impurities and degradation product: A comparative study

Tolnaftate, a thionoester anti‐fungal drug, was subjected to alkaline hydrolysis to produce methyl(m‐tolyl)carbamic acid and β ‐naphthol (tolnaftate impurity A). N‐Methyl‐m‐toluidine, tolnaftate impurity D, was synthesized and structurally elucidated along with tolnaftate alkaline degradation products using IR, H¹NMR and MS. Two stability‐indicating HPTLC and RP‐HPLC methods were developed and validated, for the first time, for determination of tolnaftate, its alkaline degradation products and toxic impurities in the presence of methyl paraben, as a preservative in Tinea Cure^® cream. The proposed HPTLC method depended on separation of the studied components on TLC silica gel F₂₅₄ plates using hexane–glacial acetic acid (8:2, v/v) as a developing system and scanning wavelength of 230 nm. The proposed RP‐HPLC method was based on separation of the five components on an Eclipse plus C₁₈ column. The mobile phase used was acetonitrile–water containing 1% ammonium formate (40:60, v/v), with a flow rate of 1 mL/min and detection wavelength of 230 nm. The proposed methods allowed the assay of tolnaftate toxic impurities, β ‐naphthol and N‐methyl‐m‐toluidine, down to 2%, allowing tracing of β ‐naphthol that could be absorbed by the skin causing systemic toxic effects, unlike tolnaftate, indicating the high significance of such determination. International Conference on Harmonization guidelines were followed for validation.     

Validated chromatographic methods for determination of hydrochlorothiazide and spironolactone in pharmaceutical formulation in presence of impurities and degradants

Two specific, sensitive, and precise stability indicating chromatographic methods have been developed, optimized, and validated for Hydrochlorothiazide (HCT) and Spironolactone (SPR) determination in their mixtures and in presence of their impurities and degradation products. The first method was based on thin layer chromatographic (TLC) combined with densitometric determination of the separated spots. The separation was achieved using silica gel 60 F(254) TLC plates and ethyl acetate-chloroform-formic acid-triethyl amine (7:3:0.1:0.1, by volume) as a developing system. Good correlations were obtained between the integrated peak area of the studied drugs and their corresponding concentrations in different ranges. The second method was based on the high-performance liquid chromatography with ultraviolet detection, by which the proposed components were separated on a reversed phase C(18) analytical column using gradient elution system with deionized water-acetonitrile (97:3, v/v) for 8 min. Then acetonitrile was successively increased to 35% in the next 2 min, and kept constant in the following 10 min, finally 3% acetonitrile was regained again to stabilize the chromatographic system. The flow rate was maintained at 2 mL/min and the detection wavelength was at 230 nm. Linear regressions were obtained in the range of 4.0-50 μg/mL and 5.0-50 μg/mL for both HCT and SPR, respectively. Different parameters affecting the suggested methods were optimized for maximum separation of the cited components. System suitability parameters of the two developed methods were also tested. The suggested methods were validated in compliance with the ICH guidelines and were successfully applied for determination of HCT and SPR in their commercial tablets. Both methods were also statistically compared to each other and to the reported method with no significant difference in performance.     

Rapid TLC Method for Estimation of Mevalonic Acid in the Leaves of Medicinal Plants

A simple, rapid, specific and sensitive thin layer chromatographic (TLC) method has been developed for the quantitative estimation of mevalonic acid (MVA) in leaves of medicinal plants; Artemisia annua, Psorelia corylifolia, Vinca rosea, Withania somnifera and Barleria proinites. The assay procedure involved conversion of MVA to its lactone, mevalonolactone (MVAL). Mevalonic acid was extracted from the leaf tissues of plants. Separation of MVAL was carried out on silica gel 60 F₂₅₄ TLC plates using benzene:acetone (3:2) as the mobile phase. The densitometric determination of MVAL was performed at 600 nm after derivatization with anisaldehyde reagent in absorption–reflectance mode. The method was validated over the linearity range of 100–500 ng spot^?1 and correlation coefficient for the calibration curve was >0.99. The average recovery of MVAL, used as internal standard, was higher than 98%. The lower limit of detection was found to be 50 ng spot^?1.     

液相色谱-串联质谱法测定吉非替尼中4种基因毒性杂质

建立了一种同时测定吉非替尼中4种基因毒性杂质3-氯-4-氟苯胺、3,4-二氟苯胺、3-氟-4-氯苯胺和3,4-二氯苯胺的高效液相色谱-串联质谱（HPLC-MS/MS）方法。用Inertsil ODS-3柱（100 mm×3.0 mm,3μm）为色谱柱,以0.1%（体积分数,下同）甲酸水溶液-0.1%甲酸乙腈溶液为流动相,在电喷雾正离子模式下进行测定。该方法在特异性、线性、精密度、准确性、稳定性和耐用性方面得到了验证。4种基因毒性杂质在0.6~96.0μg/L范围内与峰面积呈良好线性关系。检测限和定量限分别为0.2~2.0μg/L和0.6~6.0μg/L。所有杂质的回收率为91.0%~98.5%。检测后,在批号16052301和R16052501-1样品中仅检测到3-氯-4-氟苯胺,但低于杂质限度（6 mg/L）。该方法简便可靠,可用于吉非替尼中4种基因毒性杂质的测定,并为质量控制提供参考。     

Simultaneous quantification of diterpenoids in Premna integrifolia using a validated HPTLC method

A sensitive, selective and robust densitometric high‐performance thin layer chromatographic method was developed and validated for the determination of diterpenoids in the root bark of Premna integrifolia. Diterpenoids 1β,3α,8β‐trihydroxy‐pimara‐15‐ene ( A ), 6α,11,12,16‐tetrahydroxy‐7‐oxo‐abieta‐8,11,13‐triene ( B ) and 2α,19‐dihydroxy‐pimara‐7,15‐diene ( C ) were used as chemical markers for the standardization of P. integrifolia plant extracts. The separation was performed on silica gel 60F₂₅₄ high‐performance thin layer chromatography plates using hexane/acetone/ethylacetate (60:20:20 v/v) as mobile phase. The quantitation of diterpenoids was carried out using densitometric reflection/absorption mode at 475 nm after post‐chromatographic derivatization using vanillin–sulfuric acid reagent. A precise and accurate quantification can be performed for compounds A , B and C in the linear working concentration range of 1–10 μg/spot with good correlations (r²=0.9985, 0.9996 and 0.9992, respectively). The method was validated for peak purity, precision, robustness, limit of detection (LOD) and quantitation (LOQ) etc., as per the International Conference on Harmonization (ICH) guidelines. Specificity of quantitation was confirmed using retention factor (R_f) and spectra correlation of markers in standard and sample tracks. The method reported here is simple and reproducible which may be applied for quantitative analysis of above diterpenoids in the root bark of P. integrifolia.     

Rapid Validated Thin-Layer Chromatography—Densitometry for the Simultaneous Determination of Three Co-formulated Drugs Used for Common Cold Treatment

JPC – Journal of Planar Chromatography – Modern TLC - A rapid validated thin-layer chromatography (TLC)—densitometric method has been developed for the simultaneous determination...     

Quality assessment for tramadol in pharmaceutical preparations with thin layer chromatography and densitometry

Research studies have been carried out to develop a chromatographic and densitometric method suitable for identification and determination of tramadol and impurities. In addition, the stability of tramadol in solutions was investigated, including an effect of solution pH, temperature and incubation time. In the first instance the conditions for identification and quantitative determination of tramadol and impurities in pharmaceutical preparations were established. The separation was performed on silica gel-coated chromatographic plates (HPTLC) using two mobile phases: (I) chloroform-methanol-glacial acetic acid (9:2:0.1, v/v/v); (II) chloroform-toluene-ethanol (9:8:1, v/v/v). The UV densitometry was carried out at lambda = 270 nm. The developed method is of high sensitivity and low detection and determination limits ranging from 0.044 to 0.35 microg. For individual constituents the recovery ranges from 93.23 to 99.66%. The next step was to evaluate the stability of tramadol and determine a method of decomposition under various experimental conditions. It was found that tramadol decomposes in various ways in acidic and basic environments producing (1RS)-[2-(3-methoxyphenyl)cyclohex-2-enyl]-N,N-dimethylmethanamine (imp. B) and (1RS, 2RS)-2-[(dimethylamino)methyl]-1-(3-methoxyphenyl)cyclohexanol (imp. cis-T) or imp. cis-T, respectively.     

Development and validation of a TLC-densitometry method for quantitative analysis of nefopam hydrochloride beside its degradation products

A quantitative densitometric thin-layer chromatographic method for determination of nefopam hydrochloride in pharmaceutical preparations has been established and validated. Nefopam from the formulations was separated and identified on silica gel 60 F₂₅₄ TLC plates with chloroform-methanol-glacial acetic acid (9: 2: 0.1, v/v/v) as mobile phase. Densitometric quantification was performed at absorbance maximum 266 nm. The method was validated for linearity, sensitivity, precision and recovery in accordance with ICH guidelines. The presented method is selective and specific with potential application in pharmaceutical analysis. Nefopam hydrochloride was subjected to acidic and alkaline hydrolysis at different temperatures. As the method could effectively separate the drug from its degradation products, it can be employed as a stability indicating one.     

Resolution and isolation of enantiomers of (±)‐isoxsuprine using thin silica gel layers impregnated with l‐glutamic acid,comparison of separation of its diastereomers prepared with chiral derivatizing reagents having l‐amino acids as chiral auxiliaries

Thin silica gel layers impregnated with optically pure l ‐glutamic acid were used for direct resolution of enantiomers of (±)‐isoxsuprine in their native form. Three chiral derivatizing reagents, based on DFDNB moiety, were synthesized having l ‐alanine, l ‐valine and S‐benzyl‐l ‐cysteine as chiral auxiliaries. These were used to prepare diastereomers under microwave irradiation and conventional heating. The diastereomers were separated by reversed‐phase high‐performance liquid chromatography on a C₁₈ column with detection at 340 nm using gradient elution with mobile phase containing aqueous trifluoroacetic acid and acetonitrile in different compositions and by thin‐layer chromatography (TLC) on reversed phase (RP) C₁₈ plates. Diastereomers prepared with enantiomerically pure (+)‐isoxsuprine were used as standards for the determination of the elution order of diastereomers of (±)‐isoxsuprine. The elution order in the experimental study of RP‐TLC and RP‐HPLC supported the developed optimized structures of diastereomers based on density functional theory. The limit of detection was 0.1–0.09 µg/mL in TLC while it was in the range of 22–23 pg/mL in HPLC and 11–13 ng/mL in RP‐TLC for each enantiomer. The conditions of derivatization and chromatographic separation were optimized. The method was validated for accuracy, precision, limit of detection and limit of quantification. Copyright © 2014 John Wiley & Sons, Ltd.     

TLC-Densitometric Method for Quantification of Oxaprozin and its Degradation Products in Pharmaceutical Preparations

JPC – Journal of Planar Chromatography – Modern TLC - A new, simple and rapid thin-layer chromatographic method with densitometric detection has been developed and validated for...     

Impurity profile of macitentan in tablet dosage form using a stability-indicating high performance liquid chromatography method and forced degradation study

Macitentan (MAC) is a pulmonary arterial hypertension (PAH) drug marketed as a tablet and often has stability issues in the final dosage form. Quantitative determination of MAC and its associated impurities in tablet dosage form has not been previously reported. This study quantified impurities present in Macitentan tablets using a binary solvent-based gradient elution method using reversed phase-high performance liquid chromatography. The developed method was validated per International Conference on Harmonization (ICH) guidelines and the drug product was subjected to forced degradation studies to evaluate stability. The developed method efficiently separated the drug and impurities (48 min) without interference from solvents, excipients, or other impurities. The developed method met all guidelines in all characteristics with recoveries ranging from 85%-115%, linearity with r² ≥ 0.9966, and substantial robustness. The stability-indicating nature of the method was evaluated using stressed conditions (hydrolysis:1 N HCl at 80℃/15 min; 1 N NaOH at 25℃/45 min; humidity stress (90% relative humidity) at 25℃ for 24 h, oxidation:at 6% (v/v) H₂O₂, 80℃/15 min, thermolysis:at 105℃/16 h and photolysis:UV light at 200 Wh/m²; Fluorescent light at 1.2 million luxh). Forced degradation experiments showed that the developed method was effective for impurity profiling. All stressed samples were assayed and mass balance was>96%. Forced degradation results indicated that MAC tablets were sensitive to hydrolysis (acid and alkali) and thermal conditions. The developed method is suitable for both assay and impurity determination, which is applicable to the pharmaceutical industry.