Novel, Precise, Accurate Ion-Pairing Method to Determine the Related Substances of the Fondaparinux Sodium Drug Substance: Low-Molecular-Weight Heparin.

Fondaparinux sodium is a synthetic low-molecular-weight heparin (LMWH). This medication is an anticoagulant or a blood thinner, prescribed for the treatment of pulmonary embolism and prevention and treatment of deep vein thrombosis. Its determination in the presence of related impurities was studied and validated by a novel ion-pair HPLC method. The separation of the drug and its degradation products was achieved with the polymer-based PLRPs column (250 mm × 4.6 mm; 5 µm) in gradient elution mode. The mixture of 100 mM n-hexylamine and 100 mM acetic acid in water was used as buffer solution. Mobile phase A and mobile phase B were prepared by mixing the buffer and acetonitrile in the ratio of 90:10 (v/v) and 20:80 (v/v), respectively. Mobile phases were delivered in isocratic mode (2% B for 0-5 min) followed by gradient mode (2-85% B in 5-60 min). An Evaporative Light Scattering Detector (ELSD) was connected to the LC system to detect the responses of chromatographic separation. Further, the drug was subjected to stress studies for acidic, basic, oxidative, photolytic, and thermal degradations as per ICH guidelines and the drug was found to be labile in acid, base hydrolysis, and oxidation, while stable in neutral, thermal, and photolytic degradation conditions. The method provided linear responses over the concentration range of the LOQ to 0.30% for each impurity with respect to the analyte concentration of 12.5 mg/mL, and regression analysis showed a correlation coefficient value (r(2)) of more than 0.99 for all the impurities. The LOD and LOQ were found to be 1.4 µg/mL and 4.1 µg/mL, respectively, for fondaparinux. The developed ion-pair method was validated as per ICH guidelines with respect to accuracy, selectivity, precision, linearity, and robustness.

Development of an Ion Chromatography Method for Analysis of Organic Anions (Fumarate, Oxalate, Succinate, and Tartrate) in Single Chromatographic Conditions.

A single organic counterion analysis method was developed by using an ion chromatography separation technique and conductivity detector. This allows the rapid characterization of an API to support clinical studies and to fulfil the regulatory requirements for the quantitation of fumarate, oxalate, succinate, and tartrate counterions in active pharmaceutical ingredients (quetiapine fumarate, escitalopram oxalate, sumatriptan succinate, and tolterodine tartrate). The method was developed by using the Metrohm Metrosep A Supp 1 (250 × 4.0 mm, 5.0 µm particle size) column with a mobile phase containing an isocratic mixture of solution A: 7.5 mM sodium carbonate and 2.0 mM sodium bicarbonate in Milli-Q water and solution B: acetonitrile. The flow rate was set at 1.0 mL/min and the run time was 25 minutes. The developed method was validated as per ICH guidelines, and the method parameters were chosen to ensure the spontaneous quantitation of all four anions. The method was validated for all four anions to demonstrate the applicability of this method to common anions present in various APIs.

Stress Degradation Behavior of Atorvastatin Calcium and Development of a Suitable Stability-Indicating LC Method for the Determination of Atorvastatin, its Related Impurities, and its Degradation Products.

A rapid, reversed-phase liquid chromatographic method was developed for the quantitative determination of Atorvastatin calcium, its related substances (12 impurities), and degradation impurities in bulk drugs. The chromatographic separation was achieved on a Zorbax Bonus-RP column by employing a gradient elution with water-acetonitrile-trifluoroacetic acid as the mobile phase in a shorter run time of 25 min. The flow rate was 1.0 mL/min and the detection wavelength was 245 nm. The drug substance was subjected to stress studies such as hydrolysis, oxidation, photolysis, and thermal degradation, and considerable degradation was observed in acidic hydrolysis, oxidative, thermal, and photolytic stress conditions. The formed degradation products were reported and were well-resolved from the Atorvastatin and its related substances. The stressed samples were quantified against a qualified reference standard and the mass balance was found to be close to 99.5% (w/w) when the response of the degradant was considered to be equal to the analyte (i.e. Atorvastatin), which demonstrates the stability-indicating capability of the method. The method was validated in agreement with ICH requirements. The method developed here was single and shorter (25 min method for the determination of all 12 related impurities of Atorvastatin and its degradation products), with clearly better resolution and higher sensitivity than the European (85 min method for the determination of six impurities) and United States pharmacopeia (115 min and 55 min, two different methods for the determination of six related substances).

Identification of degradation products in stressed tablets of Rabeprazole sodium by HPLC-hyphenated techniques.

Three unknown impurities of Rabeprazole, a proton pump inhibitor, were formed in the formulated drug under the stress conditions, [40 degrees C/75% relative humidity (RH) for 6 months] with relative retention times (RRTs) 0.17, 0.22 and 0.28. The Impurity-I (0.17 RRT) was isolated using preparative HPLC and characterized by NMR and MS. The other two impurities, Impurity-II (RRT 0.22) and Impurity-III (RRT 0.28) could not be isolated, hence they are characterized by HPLC-hyphenated techniques, LC-NMR and high-resolution LC-MS. On the basis of the spectral data, the Impurity-I, Impurity-II and Impurity-III were characterized as 1-(1H-benzo[d]imidazol-2-yl)-3-methyl-4-oxo-1,4-dihydropyridine-2-carboxylic acid, 1H-benzo [d] imidazole-2-sulfonic acid and 4-(3-methoxy propoxy)-3-methyl-2-pyridine carboxylic acid, respectively.

Identification, synthesis, isolation and spectral characterization of potential impurities of montelukast sodium.

During the process development of montelukast sodium, three polar impurities and one non-polar impurity with respect to montelukast sodium were detected by simple reverse phase high-performance liquid chromatography (HPLC). Initially, all the four impurities were identified by the liquid chromatography-mass spectrometry (LC-MS) data and out of four impurities, three have been prepared by the synthetic method and remaining one is isolated by preparative HPLC. Based on the spectral data (IR, (1)H NMR, (13)C NMR and MS), the structure of these impurities 1-4 were characterised as 1-[[[(1R)-1-[3-[(1E)-2-(7-chloro-2-quinolinyl)ethenyl]phenyl-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropane acetamide (impurity-1), {1-[1-{3-[2-(7-chloro-quinolin-2-yl)-vinyl]-phenyl}-3-(2-isopropenyl-phenyl)-propylsulfanylmethyl]-cyclopropyl}-acetic acid (impurity-2), 1-[[[(1R)-1-[3-[(1E)-2-(7-chloro-2-quinolinyl)ethyl]phenyl-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetic acid (impurity-3) and 1-[[[(1R)-1-[3-[(1E)-2-(2-quinolinyl)ethenyl]phenyl-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetic acid (impurity-4).

(3S,4R)-4-(4-Fluoro-phen-yl)-3-(hydroxy-meth-yl)piperidinium chloride.

The title compound, C(12)H(17)FNO(+)·Cl(-), is a degradation impurity of paroxetine hydro-chloride hemihydrate (PAXIL), an anti-depressant belonging to the group of drugs called selective serotonin reuptake inhibitors (SSRIs). Similar to the paroxetine hydro-chloride salt with protonation having taken place on the basic piperidine ring, the degradation impurity also exists as the hydro-chloride salt. The cyclic six-membered piperidinium ring adopts a chair conformation with the hydroxy-methyl and 4-fluoro-phenyl groups in the equatorial positions. The ions form a tape along the b axis through charge-assisted N(+)-H⋯Cl(-) hydrogen bonds; these tapes are connected by O-H⋯Cl(-) hydrogen bonds along the a axis.

Identification and characterization of potential impurities of amlodipine maleate.

Six impurities ranging from 0.43 to 1.42% in amlodipine maleate were detected by a simple isocratic reverse-phase high performance liquid chromatography (HPLC). LC-MS was performed to identify the mass of the impurities. Based on the spectral data (IR, NMR and MS), the structures of these impurities were characterized as 3-ethyl 5-methyl 4-(2-chlorophenyl)-2-[2-(1,3-dioxo-2,3-dihydro-1H-2-isoindolyl)ethoxymethyl]-6-methyl-1,4-dihydro-3,5-pyridinedicarboxylate (impurity I); 5-ethyl 3-methyl 4-(2-chlorophenyl)-2-methyl-6-[2-(2-methylcarbamoylphenyl-carboxamido)ethoxymethyl]-1,4-dihydro-3,5-pyridinedicarboxylate (impurity II); besylate salt of 3-ethyl 5-methyl 2-(2-aminoethoxymethyl)-4-(2-chlorophenyl)-6-methyl-3,5-pyridinedicarboxylate (impurity III); dimethyl 2-(2-aminoethoxymethyl)-4-(2-chlorophenyl)-6-methyl-1,4-dihydro-3,5-pyridinecarboxylate (impurity IV); 3-ethyl 5-methyl 2-(2-aminoethoxymethyl)-4-(4-chlorophenyl)-6-methyl-1,4-dihydro-3,5-pyridinedicarboxylate (impurity V); diethyl 2-(2-aminoethoxymethyl)-4-(2-chlorophenyl)-6-methyl-1,4-dihydro-3,5-pyridinedicarboxylate (impurity VI).

Isolation and characterization of impurities in docetaxel.

During the process development of docetaxel, two polar impurities (Impurities I and II) and two non-polar impurities (Impurities III and IV) were detected by high performance liquid chromatography (HPLC). All the impurities were isolated by Medium Pressure Liquid Chromatography (MPLC). The Impurities I, II, III and IV were identified as 13-[(4S,5R)-2-oxo-4-phenyl-oxazolidine-5-carboxy]-10-deacetyl baccatin III ester, 2'-epi docetaxel, 7-epi docetaxel and 13-[(4S,5R)-2-oxo-4-phenyl-oxazolidine-3,5-dicarboxyl-3-tert-butyl)]-10-deacetyl baccatin III ester, respectively, based on one- (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy data. The Impurity IV was crystallized and the structure was solved by single crystal X-ray diffraction (XRD). Two impurities (Impurities II and III) were found to be process related, while the remaining two impurities (Impurities I and IV) turned out to be isomers. The formation of these impurities was discussed.

Identification and characterization of potential impurities of donepezil.

Five unknown impurities ranging from 0.05 to 0.2% in donepezil were detected by a simple isocratic reversed-phase high performance liquid chromatography (HPLC). These impurities were isolated from crude sample of donepezil using isocratic reversed-phase preparative high performance liquid chromatography. Based on the spectral data (IR, NMR and MS), the structures of these impurities were characterised as 5,6-dimethoxy-2-(4-pyridylmethyl)-1-indanone (impurity I), 4-(5,6-dimethoxy-2,3-dihydro-1H-2-indenylmethyl) piperidine (impurity II), 2-(1-benzyl-4-piperdylmethyl)-5,6-dimethoxy-1-indanol (impurity III) 1-benzyl-4(5,6-dimethoxy-2,3-dihydro-1H-2-indenylmethyl) piperidine (impurity IV) and 1,1-dibenzyl-4(5,6-dimethoxy-1-oxo-2,3-dihydro-2H-2-indenylmethyl)hexahydropyridinium bromide (impurity V). The synthesis of these impurities and their formation was discussed.

Application of LC-MS/MS for the identification of a polar impurity in mosapride, a gastroprokinetic drug.

In the impurity profile of mosapride a polar impurity (0.1%) was detected in HPLC with respect to mosapride. Based on the mass spectral data obtained by LC-MS/MS analysis this impurity structure was characterised as 4-amino-5-chloro-2-ethoxy-N-[[(4-benzyl)-2-morphinyl] methyl] benzamide. It is interesting to note that this impurity is potent analogue of mosapride, which will have much higher gastroprokinetic activity than metoclopramide. This impurity was synthesised from an unambiguous route and confirmed the structure by collecting various spectral data and the formation is discussed. To our knowledge this compound was not reported as process impurity elsewhere.

Structural studies on the impurities of troglitazone.

The impurity profile study of troglitazone has been carried out primarily by (liquid chromatography-mass spectrometry) LC-MS. Four process-related impurities have been detected by LC-MS and were confirmed by co-injection with authentic samples. Apart from the process-related impurities, two polar by-products were characterized by mass spectral data and comparison with reference samples, while one non-polar by-product and one degradation product have been isolated by means of preparative HPLC and characterized by 2D NMR and mass spectral study. Single-crystal X-ray diffraction studies have been carried out on the degradation product. The formation and characterization of these by-products and degradation product are discussed.

Isolation and characterization of process-related impurities in linezolid.

Two unknown impurities in linezolid bulk drug at levels below 0.1% (ranging from 0.05 to 0.1%) were detected by a simple isocratic reverse phase high performance liquid chromatography (HPLC). These impurities were isolated from crude sample of linezolid using reverse phase preparative HPLC. Based on the spectroscopic data (IR, NMR and MS) the structures of the impurities were characterized as (S)-N-[[-(3-(3-fluoro-4-(4-morpholinyl)phenyl]-2-oxo-5-oxazolidinyl]methyl] acetate(I) and (S)-N-[[-(3-(3-fluoro-4-(4-morpholinyl)phenyl]-2-oxo-5-oxazolidinyl]methyl] chloride(II). The synthesis from an unambiguous route and the formation of impurities was discussed.

Isolation and characterisation of process-related impurities in rofecoxib.

Two unknown impurities in rofecoxib bulk drug at levels below 0.1% were detected by a simple isocratic reverse phase high performance liquid chromatography (HPLC). These impurities were isolated from crude sample of rofecoxib using reverse phase preparative HPLC. (1)H, (13)C and Mass spectroscopic investigations revealed the structures of the impurities as 4-[4-(methylsulphonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one (I) and 4-[4-(methylsulphonyl)phenyl]-3-phenyl-2,5-furandione (II), respectively. These structures were further confirmed by prepared synthetic standards of the impurities. The tentative mechanism for the formation of these impurities was discussed.