Process-related Substances Research Articles

A validated stability-indicating ultra performance liquid chromatography method for the determination of potential process-related impurities in eplerenone.

A simple, sensitive, and accurate stability-indicating analytical method has been developed and validated using ultra high performance liquid chromatography. The developed method is used to evaluate the related substances of eplerenone (EP). The degradation behavior of EP under stress conditions was determined, and the major degradants were identified by ultra high performance liquid chromatography with tandem mass spectrometry. The chromatographic conditions were optimized using an impurity-spiked solution, and the samples, generated from forced degradation studies. The resolution of EP, its potential impurities, and its degradation products was performed on a Waters UPLC BEH C18 column (50×2.1 mm, 1.7 μm) by linear gradient elution using a mobile phase consisting of 10 mmol/L ammonium acetate adjusted to pH 4.5, methanol and acetonitrile. A photo-diode array detector set at 245 nm was used for detection. The flow rate was set at 0.3 mL/min. The procedure had good specificity, linearity (0.02-3.14 μg/mL), recovery (96.1-103.9%), limit of detection (0.01-0.02 μg/mL), limit of quantitation (0.03-0.05 μg/mL), and robustness. The correction factors of the process-related substances were calculated.

Isolation and characterization of related substances in alogliptin benzoate by LC-QTOF mass spectrometric techniques

A highly specific and efficient LC-QTOF mass spectrometric method was developed for the separation and characterization of process related substances and the major degradation products in alogliptin benzoate and its tablets. The separation was performed on Phenomenex Gemini-NX C18 column (250mm×4.6mm, 5μm) using 0.2% formic acid-0.2% ammonium acetate in water as mobile phase A, acetonitrile and methanol (60:40, v/v) as mobile phase B in linear gradient elution mode. Forced degradation studies were also conducted under ICH prescribed stress conditions. Alogliptin benzoate and its tablets were tending to degrade under acid, alkaline, oxidative and thermal stresses, while relatively stable to photolytic stress. A total of seven related substances were detected and characterized through liquid chromatography-high resolution QTOF mass spectrometry techniques, including process related substances and degradation products, and two of them were further synthesized and characterized by NMR spectroscopy. Based on the related substances elucidation and the plausible formation mechanisms, efficient approaches were proposed to reduce or eliminate related substances, and in consequence the quality of alogliptin benzoate and its tablets have been promoted obviously. Therefore, the impurity profiles obtained are critical to the quality control and manufacturing processes optimization and monitoring of alogliptin benzoate and its tablets.

Identification and characterization of related substances in pomalidomide by hyphenated LC–MS techniques

The current study dealt with the separation, identification and characterization of related substances in pomalidomide by hyphenated techniques. Complete separation was obtained with an Inertsil ODS-SP column (250mm×4.6mm, 5μm) by linear gradient elution using a mobile phase consisting of 0.1% formic acid solution and acetonitrile. They were characterized by hyphenated chromatographic techniques with the accurate mass determination using high resolution LC–TOF–MS methods as well as the product MS spectra determination and elucidation. The degradation behaviors of pomalidomide under ICH prescribed stress conditions were also conducted. Pomalidomide was found to be labile to degrade under acid, alkaline, oxidative and thermal stress conditions, while it was relatively stable to photolytic stress. 13 related substances were detected and identified to be 10 degradation products and three process related substances. The hyphenated LC–MS method with high resolution accurate mass determination facilitated the qualitative analysis of the unknown compounds than that of the conventional HPLC–UV. The related compounds identified are valuable for pomalidomide manufacturing process optimization and quality control.

Characterization of process‐related impurities including forced degradation products of alogliptin benzoate and the development of the corresponding reversed‐phase high‐performance liquid chromatography method

The characterization of process-related impurities and forced degradants of alogliptin benzoate (Alb) in bulk drugs and a stability-indicating HPLC method for the separation and quantification of all the impurities were investigated. Alb was found to be unstable under acid and alkali stress conditions and two major degradation products (Imp-F and Imp-G) were observed. The optimum separation was achieved on Kromasil C18 (250 × 4.6 mm, 5 μm) using 0.1% perchloric acid (pH adjusted to 3.0 with triethylamine) and acetonitrile as a mobile phase in gradient mode. The proposed method was found to be stability indicating, precise, linear (0.10-75.0 μg/mL), accurate, sensitive, and robust for the quantitation of Alb and its process-related substances and degradation products. The structures of 11 impurities were characterized and confirmed by NMR spectroscopy, MS, and IR spectroscopy, and the most probable formation mechanisms of all impurities were proposed according to the synthesis route.

Liquid chromatographic separation, determination and ESI-MS/MS, FT-IR and NMR characterization of the forced degradation products of cinacalcet

A validated stability indicating RP-HPLC assay of cinacalcet was developed by separating its related substances and degradants on a Phenomenex C8 (250 × 4.6 mm, 5 µm) column using 10 mM aqueous ammonium acetate–acetonitrile as the mobile phase in a gradient mode of elution at a flow rate of 1.0 mL min−1 at 25 °C. The column effluents were monitored by a photo diode array detector set at 270 nm. The method was validated for accuracy, precision and linearity according to ICH guidelines. The limits of quantification of the impurities were in the range of 0.23–0.30 µg mL−1. The forced degradation of cinacalcet was carried out under acidic, basic, thermal, photo, and peroxide conditions, and the degradation products were isolated and characterized by ESI-MS/MS, FT-IR, 1H and 13C NMR spectroscopy. The method was successfully applied not only to quantify the degradation products but also to quantify process related substances of cinacalcet in bulk drugs.

RP-HPLC separation and characterization of unknown impurities of a novel HIV-protease inhibitor Darunavir by ESI-MS and 2D NMR spectroscopy

A selective RP-HPLC method for separation and determination of darunavir from its process related substances has been developed and validated. The separation was accomplished on a Hiber, LiChrospher® 60, RP-select B, C8 (250mm×4.6mm, 5μm) column connected to a photo diode array detector (PDA) using 10mM phosphate buffer with 0.1% of triethylamine (pH: 4.0 adjusted with orthophosphoric acid)/acetonitrile as a mobile phase under gradient elution. Two unknown impurities of darunavir were isolated and characterized by 1H, 13C, 2D-NMR and mass spectrometry. The method was validated in terms of accuracy, precision, linearity, robustness, LOD and LOQ.

Evaluation of polysaccharide‐based chiral stationary phases in quality control of (S)‐mirtazapine

High-performance liquid chromatographic methods were developed for separation of the enantiomers of mirtazapine and its four process-related substances. The direct separations were achieved on chiral stationary phases containing amylose tris(3,5-dimethylphenylcarbamate) (Chiralpak AD-H), cellulose tris(3,5-dimethylphenylcarbamate) (Chiralcel OD-H) and cellulose tris(4-methylbenzoate) (Chiralcel OJ-H ). The experimental data were utilized to discuss the effects of the mobile phase composition, the nature of the alcoholic modifier and the specific structural features of the analytes on retention and separation. The elution sequence was determined under the optimized separation conditions.

Development and Validation of a Reversed-Phase HPLC Method for Separation and Simultaneous Determination of Process-Related Substances of Mirtazapine in Bulk Drugs and Formulations

A simple and rapid reversed-phase high-performance liquid chromatographic method has been developed for the separation and simultaneous determination of related substances of mirtazapine in bulk drugs and pharmaceutical formulations. Six impurities, including one degradation product of mirtazapine, have been separated on a BDS Hypersil (4.6 x 250 mm; particle size 5 microm) column with a mobile phase consisting of 0.3% triethylamine (pH 3.0)-acetonitrile (78:22 v/v) eluted in an isocratic mode and monitored with a photo diode array detector at 215 nm. The chromatographic behavior of all the analytes was studied under variable compositions of different solvent systems, temperatures, buffer concentrations, and pH values. The method was validated in terms of accuracy, precision, and linearity. The inter- and intra-day assay precision was found to be < 0.98% [relative standard deviation; (RSD)] and the recoveries were in the range 95.54-102.22% with RSD < 2.21%. The correlation coefficients for calibration curves for mirtazapine as well as impurities were in the range of 0.9941-0.9999. The method was successfully applied to the analysis of commercial formulations and the recoveries of mirtazapine were in the range of 99.38-100.73% with < 0.52% RSD. The method is useful not only for rapid evaluation of the purity of mirtazapine, but also for the simultaneous determination of related substances in bulk drugs and pharmaceutical formulations.

Separation, Characterization, and Quantitation of Process-Related Substances of the Anti-Hypertensive Drug Doxazosin Mesylate by Reversed-Phase LC with PDA and ESI-MS as Detectors

A simple and rapid reversed-phase liquid chromatography (LC) method with photodiode array (PDA) and electrospray ionization (ESI)-mass spectrometry (MS) as detectors was developed and validated to separate, identify, and quantitate the related substances of Doxazosin mesylate (DXZN) for monitoring the reactions involved during process development. The high-performance liquid chromatography profiles of related-substances of DXZN are used as fingerprints to follow the procedures used in the manufacturing units. The separation is accomplished on an Inertsil ODS-3 column with acetonitrile-ammonium acetate (10 mM, pH 4.0) as the mobile phase, using a gradient elution mode and monitoring the eluents by a photodiode array detector at 265 nm at ambient temperature. LC-ESI-MS-MS is used to identify the additional impurities formed during the synthesis. The identified impurities were synthesized and characterized by UV, Fourier transform-IR, 1H NMR, and MS data. The detection limits for the impurities are 0.74 - 4.14 x 10(-9) g, and the method is found to be suitable not only for the monitoring of synthetic reactions, but also for quality assurance of DXZN in bulk drugs and formulations.

Simultaneous separation and determination of process-related substances and degradation products of venlafaxine by reversed-phase HPLC

A simple and rapid gradient RP HPLC method for simultaneous separation and determination of venlafaxine and its related substances in bulk drugs and pharmaceutical formulations has been developed. As many as four process impurities and one degradation product of venlafaxine have been separated on a Kromasil KR100-5C18 (4.6 mm x 250 mm; particle size 5 microm) column with gradient elution using 0.3% diethylamine buffer (pH 3.0) and ACN/methanol (90:10 v/v) as a mobile phase. The column was maintained at 40 degrees C and the eluents were monitored with photo diode array detection at 225 nm. The chromatographic behaviour of all the compounds was examined under variable compositions of different solvents, temperatures, buffer concentrations and pH. The method was validated in terms of accuracy, precision and linearity as per ICH guidelines. The inter- and intraday assay precision was < 4.02% (%RSD) and the recoveries were in the range of 96.19-101.14% with %RSD < 1.15%. The correlation coefficients (r2) for calibration curves of venlafaxine as well as impurities were in the range of 0.9942-0.9999. The proposed RP-LC method was successfully applied to the analysis of commercial formulations and the recoveries of venlafaxine were in the range of 99.32-100.67 with %RSD <0.58%. The method could be of use not only for rapid and routine evaluation of the quality of venlafaxine in bulk drug manufacturing units but also for the detection of its impurities in pharmaceutical formulations. Forced degradation of venlafaxine was carried out under thermal, photo, acidic, basic and peroxide conditions and the acid degradation products were characterized by ESI-MS/MS, 1H NMR and FT-IR spectral data.

Development of a scalable synthesis of a nonbasic inhibitor of the serine protease tryptase

A chromatography-free process for the synthesis of a bis(benzimidazole)difluoromethane inhibitor of the serine protease tryptase is described. This synthesis features the introduction of the gem-difluoro moiety using the electrophilic fluorinating reagent N-fluoro-bis(phenylsulfonimide) as well as the stepwise introduction of both benzimidazole rings. A protocol for the destruction of reactive, process-related substances produced in the synthesis is also presented.

Enantiomeric resolution of doxazosin mesylate and its process-related substances on polysaccharide chiral stationary phases

High-performance liquid chromatographic methods for separation of racemic doxazosin mesylate and its synthetic precursors on polysaccharide based stationary phases viz., amylose tris-(3,5-dimethylphenylcarbamate) (Chiralpak AD-H) and cellulose tris-(3,5-dimethylphenylcarbamate) (Chiralcel OD-H) were developed. The base line separation with R s > 1.50 was obtained using a mobile phase containing n-hexane–alcohol–0.1% diethylamine (ethanol, 1-propanol and 2-propanol) in various proportions. The effect of concentration of the alcoholic modifiers on the resolution was studied. A good separation was achieved on amylose based Chiralpak AD-H column when compared with cellulose based Chiralcel OD-H. The effects of structural features of the solutes and solvents on discrimination between the enantiomers were examined. The detection was carried out at 240 nm with UV detector while identification by polarimetric detector connected in series. The method was suitable not only for process development of doxazosin mesylate but also determination of enantiomeric purity of bulk drugs and pharmaceuticals.

Rapid Separation and Determination of Process-Related Substances of Paracetamol Using Reversed-Phase HPLC with Photo Diode Array as a Detector

A simple and rapid gradient reversed-phase high-performance liquid chromatographic method for simultaneous separation and determination of paracetamol and its related compounds in bulk drugs and pharmaceutical formulations has been developed. As many as nine process impurities and one degradation product of paracetamol have been separated on a Symmetry C18 column (4.6 x 250 mm i.d., particle size 5 microm) with gradient elution using 0.01 M potassium dihydrogen phosphate buffer (pH 3.0) and acetonitrile as mobile phase and photo diode array detection at 215 nm. The chromatographic behavior of all the compounds was examined under variable compositions of different solvents, temperatures, buffer concentrations and pH values. The correlation coefficients for calibration curves for paracetamol as well as impurities were in the range of 0.9951 - 0.9994. The proposed RP-LC method was successfully applied to the analysis of commercial formulations; the recoveries of paracetamol were in the range of 99-101%. The method could be of use not only for rapid and routine evaluation of the quality of paracetamol in bulk drug manufacturing units but also for detection of its impurities in pharmaceutical formulations.

LC‐PDA and LC‐ESI‐MS separation and determination of process‐related substances arising from stilbene‐type fluorescent whitening agents. Application to monitoring of their photodegradation products in industrial effluents and aqueous environmental systems

A simple and rapid gradient elution high-performance liquid chromatographic method using photodiode array and electrospray ionization mass spectrometric detectors was developed for separation and determination of the process-related substances and photodegradation products of stilbenesulfonic acids, viz. 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNSDA), 4-amino-4'-nitrostilbene-2,2'-disulfonic acid (ANSDA), and 4,4'-diaminostilbene-2,2'-disulfonic acid (DASDA) in industrial waste waters. Gradient elution was carried out using ammonium acetate and acetonitrile as mobile phase and an Inertsil-ODS 3V column for separation. The negative-ion electrospray ionization mass spectra containing [M-H]- ions of sulfonic acids allowed molecular mass determination of unknowns and the structures were proposed on the basis of the fragment ions in the MS/MS spectra.

Development and validation of a heart-cutting liquid chromatography–mass spectrometry method for the determination of process-related substances in cetirizine tablets

A heart-cutting liquid chromatography–mass spectrometry (LC–MS) method was developed for the simultaneous analysis of seven process related substances (PRS) at ng ml −1 level in tablets containing cetirizine (CTZ) as active ingredient. After dissolution and filtration, 10 μl of sample was injected onto a cyano-column coupled to an electrospray ionization–mass spectrometer (ESI–MS) equipped with a single quadrupole. A switching valve placed between the analytical column and the MS detector allowed for the transfer of only the PRS fractions into the MS. The separation of PRS and CTZ was achieved in <14 min with a binary gradient using a 50 mM ammonium acetate solution (pH 7) and acetonitrile. The ESI–MS detection was performed using single ion monitoring (SIM). The method was validated. Weighted linear regression was found appropriate in a concentration range of 2.5–250 ng ml −1 and the limit of quantification (LOQ) was estimated at 10 ng ml −1 for each analyte. Method selectivity and robustness was demonstrated. Method performance was evaluated in terms of accuracy and precision using quality control (QC) samples over the investigated range. Finally, the heart-cutting LC–MS method was successfully applied to commercially available tablets.

Dual-mode gradient HPLC procedure for the simultaneous determination of chloroquine and proguanil.

In order to assay the antipaludic capsule of the Service de Santé des Armées (SSA), that contains two antimalarial drugs, i.e. chloroquine sulfate (CQS, cp1) and proguanil hydrochloride (PGH, cp5), a HPLC procedure was developed. A reversed-phase ion-pair high-performance liquid chromatography (HPLC) method with an ultraviolet detection at 254 nm was set up and validated. Elution system includes programming of both organic concentration and flow-rate known as 'dual-mode gradient'. This method allows the simultaneous determination of both active compounds and separation of four process related substances. The method is simple, rapid, selective and accurate, and the precision is good with an inter- and intra-assay of <2%. The sensitivity is particularly suitable for pharmaceutical quality control.

Near-IR Detection of Polymorphism and Process-Related Substances

This paper reports a fast, sensitive pattern recognition method for determining the polymorphic quality of a solid drug substance, polymorph A. The pattern recognition method employed can discriminate between the desired polymorphic form of the drug substance and another undesired polymorph. In addition, it can reliably detect samples containing minor levels of the undesired polymorph. The method can also discriminate between the desired polymorph and other crystalline forms. Most significantly, this sensitive method has been successfully transferred to six other near-IR instruments without resorting to sophisticated multivariate calibration transfer strategies.

Determination of streptomycin sulfate and dihydrostreptomycin sulfate by high-performance liquid chromatography

An isocratic, paired-ion reversed-phase high-performance liquid chromatographic method for the determination of streptomycin and dihydrostreptomycin has been developed. The method employs a microparticulate reversed-phase column (μBondapak C18, LiChrosorb RP-18 or Ultrasphere Ion Pair), and a mobile phase composed of 0.02 M sodium hexanesulfonate and 0.025 M tribasic sodium phosphate in acetonitrile-water (8:92, v/v) at pH 6.0 with detection by ultraviolet absorbance at 195 nm. Resolution and simultaneous quantitation of streptomycin A, dihydrostreptomycin A, streptomycin B, streptidine and process-related substances can be achieved in less than 25 min, with a relative standard deviation of ca. 1.1% for the assay of either streptomycin sulfate or dihydrostreptomycin sulfate. The chromatographic bioequivalency data for streptomycin sulfate and dihydrostreptomycin sulfate were statistically identical with results obtained by the officially recognized microbiological assay. The method is designed for applicability to the analysis of other aminoglycoside antibiotics and antituberculous agents.

High-performance Liquid Chromatographic Determination of (Z)- and (E)-doxepin Hydrochloride Isomers

A high-performance liquid chromatographic method for the determination of (Z)- and (E)-doxepin hydrochloride isomers was developed. The analysis employs a column packed with spherical silica microparticles (5-6 μm), and a mobile phase of acetonitrile-chloro-form-diethylamine (750:250:0.2) permits baseline resolution and simultaneous determination of the (Z)- and (E)-doxepin isomers. Process-related substances do not interfere. The method is accurate and precise (the relative standard deviation was 0.3% for both isomers). The simple procedure is highly suitable for routine doxepin hydrochloride analysis.