Identification, Characterization and HPLC Quantification of Process-Related Impurities in Bepotastine Besilate Bulk Drug.

Bepotastine besilate (here after referred to as BTST), chemically known as ({d(S)4[4[(4chlorophenyl) (2pyridyl) methoxy] piperidino} butyric acid monobenzene sulphonate), is a second-generation antihistamine drug. To the best of our knowledge, no studies concerning the isolation or identification of process-related impurities have been reported so far. The current study reports the development and validation of a stability-indicating RP-HPLC method for the separation and identification of 5 potential impurities in bepotastine besilate. In this experiment, the structures of 3 process-related impurities were found to be new compounds. They were characterized and confirmed by NMR and MS spectroscopy analyses. These 3 new compounds were proposed to be (S)-4-[(phenyl)-2-pyridinylmethoxy]-1-piperidinebutanoic acid,(Imp-A); 4-[(S)-(4-chlorophenyl)-2-pyridinylmethoxy]-1- piperidinebutyric acid, N-oxide (Imp-B) and (S)-4-[(4- chlorophenyl)-2-pyridinylmethoxy]-1-piperidylethane (Imp-C). In addition, an efficient optimized chromatographic method was performed on a Shimadzu Inertsil C8-3 column (150 mm×4.6 mm, 3 µm) to separate and quantify these 5 impurities. It was using 15 mmol ammonium formate buffer in water (pH adjusted to 3.8 with formic acid) and acetonitrile as the mobile phase in gradient mode. The method was developed to separate and quantify these 5 impurities obtained in the range of 0.05-0.75 µg/mL. It was validated and proven to be selective, accurate and precise and suitable. It is the first publication of identification and characterization data of the 3 new compounds. It is also the first effective HPLC method for separation and quantification of all of process-related impurities in bepotastine besilate.

The Chiral Bioconversion and Pharmacokinetic Analysis of Trelagliptin in Beagle Dog Plasma by LC-MS/MS.

A simple and enantioselective method was developed and validated for the simultaneous determination of (R)- and (S)-trelagliptin in beagle dog plasma by chiral liquid chromatography tandem mass spectrometry. Trelagliptin enantiomers and (R)-rabeprazole (as internal standard, IS) were extracted from plasma samples by liquid-liquid extraction and separated on a CHIRALCEL OX-3R column using acetonitrile-5 ammonium bicarbonate as the mobile phase in gradient elution mode. The multiple reactions monitoring transitions of m/z 358.1→341.2 and 359.9→150.1 were used to quantify trelagliptin enantiomers and IS, respectively. This method was validated for sensitivity, specificity, linearity, precision, accuracy and stability of specific analytes under various conditions. And it was successfully applied to evaluating the pharmacokinetic profile of trelagliptin enantiomers in beagle dogs after single intravenous administration of (R)-trelagliptin injection (at 1 mg/kg) and oral administration (at 6.7 mg/kg). In this study, no chiral bioconversion of (R)-trelagliptin to (S)-trelagliptin in beagle dog plasma was observed. The absolute bioavailability of (R)-trelagliptin was identified to be 128.2%.

[Simultaneous determination of leucovorin and 5-methyl-tetrahydrofolate diastereoisomers in human plasma by high-performance liquid chromatography-tandem mass spectrometry coupled with ultrafiltration centrifugation-based pretreatment and its application to a pharmacokinetic study].

A simple, sensitive, and stable high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and validated for the simultaneous determination of leucovorin and 5-methyltetrahydrofolate diastereomers in human plasma using methotrexate as the internal standard. The analytes and the internal standard were extracted from plasma samples by simple ultrafiltration centrifugation-based extraction. The separation was achieved on a chiral HSA column (150 mm×4 mm, 5 µm) using mobile phases containing 10 mmol pH 8.0 ammonium acetate and acetonitrile in gradient mode. The method showed good linearities in the ranges of 25-5000 µg/L and 12.5-3000 µg/L for leucovorin and 5-methyltetrahydrofolate diastereoisomers, respectively. The method was fully validated with respect to sensitivity, precision, accuracy, matrix effect, extraction recovery, and stability of analytes under various conditions. The method was successfully applied to a pharmacokinetic study of 125 mg/m2 6R,S-leucovorin and 62.5 mg/m2 6S-leucovorin. The results showed that the maximum observed concentrations (Cmax) of 6S-leucovorin and L-5-methyltetrahydrofolate were (3137.917±408.837) and (1679.633±244.132) µg/L, respectively, and the areas under the curve from the time of dosing to the last measurable concentration (AUC0-t) were (7504.883±1185.101) and (14001.214±2868.949) µg/L in the 125 mg/m2 6R,S-leucovorin dose group. The Cmax values of 6S-leucovorin and L-5-methyltetrahydrofolate were (3187.917±387.298) and (1739.204±224.755) µg/L, respectively, and AUC0-t values were (7426.664±854.825) and (14884.331±1843.353) µg/L in the 62.5 mg/m2 6S-leucovorin dose group. There were no significant diffe-rences in the main pharmacokinetic parameters between the two dose groups, and the pharmacokinetic characteristics as well as the rate and extent of absorption were consistent. This method can provide technical support for future bioequivalence studies of sodium leucovorin.

Pharmacokinetics of cligosiban in dog plasma after oral administration by liquid chromatography electrospray ionization tandem mass spectrometry.

In this study, a simple and reliable liquid chromatography coupled with Q-Exactive-Orbitrap-MS was developed and validated for detecting and quantifying cligosiban and its metabolites in dog plasma after oral administration. The plasma samples were pretreated with acetonitrile and separated on a Diamonsil C18 column (4.6 × 100 mm, i.d. 3 µm) with 0.1% formic acid in water and acetonitrile as mobile phase. The method was validated according to the guidance of the US Food and Drug Administration. The assay was linear over the tested concentration ranges with coefficients of correlation >0.995. The extraction recovery was >83.23% with RSD <15%. Precision was <9.31% and accuracy ranged from -4.40 to 10.20%. The method was free of matrix effects. Under the conditions used, four metabolites were detected and their identities were identified by accurate masses and fragment ions. M1 and M3 were further confirmed by reference standards. The biotransformation pathways included demethylation and glucuronidation. The validated method was further applied to quantify cligosiban, M1 and M3 in dog plasma. After oral administration, cligosiban was detectable in dog plasma and reached the maximum concentration at ~1.67 ± 0.58 h post-dose. It was rapidly eliminated with a half-life of 3.48 ± 0.80 h. M1 showed high plasma exposure with its area under the curve being 23.31% of that of cligosiban.

[Determination of trihexyphenidyl in human plasma by ultra high performance liquid chromatography-tandem mass spectrometry].

A sensitive and high throughput method by ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was established for the determination of trihexyphenidyl in human plasma. The method was used to evaluate the bioequivalence of the test preparation and reference preparation, and to investigate the effect of food on the pharmacokinetic behavior of trihexyphenidyl. The trihexyphenidyl and internal standard trihexyphenidyl-d11 were extracted from human plasma by protein precipitation using methanol as the precipitant. Chromatographic separation was achieved on a Waters UPLC BEH C8 column (50 mm×2.1 mm, 1.7 µm) with 0.1% (v/v) formic acid aqueous solution containing 5 mmol/L ammonium acetate and acetonitrile-water (95:5, v/v) as the mobile phases. The analytes were detected by an electrospray ionization source in positive ion and multiple reaction monitoring modes. The linear range of trihexyphenidyl was 0.1-40 ng/mL. This test involved 30 healthy male and female subjects with a single oral administration of a 2-mg trihexyphenidyl hydrochloride tablet each. The 90% confidence intervals under fasting conditions of peak plasma concentration (Cmax), area under the plasma concentration-time curve (AUC0-t) and area under the plasma concentration-time curve from zero to infinity (AUC0-∞) were 82.2%-99.4%, 82.3%-97.3% and 83.4%-97.9%, and these pharmacokinetics parameters under postprandial conditions were 100.8%-122.8%, 96.8%-112.4% and 96.6%-112.1%, which were all in the range of 80.0%-125.0%, indicating that the test tablets and reference tablets were bioequivalent.

A Validated Quantification Method for Brexpiprazole in Dog Plasma.

A sensitive, efficient and stable bioanalytical method has been developed and validated for determination of brexpiprazole in dog plasma with UPLC-MS-MS for the first time. Brexpiprazole and internal standard were extracted from plasma samples by liquid-liquid extraction and separated on an Acquity UPLC BEH C18 column. A gradient elution program was developed employing methanol and 10 mM ammonium acetate aqueous solution as mobile phases. The method was validated for parameters of selectivity, LLOQ, linearity, accuracy, precision, matrix effects and stability in accordance with the regulatory guidance on bioanalytical method validation. The validated method was applied in evaluating the pharmacokinetic profiles of brexpiprazole in beagle dogs after a single-dose oral administration of a 4 mg tablet.

Identification, Characterization, and Quantification of Impurities of Safinamide Mesilate: Process-Related Impurities and Degradation Products.

The characterization of process-related impurities and degradation products of safinamide mesilate (SAFM) in bulk drug and a stability-indicating HPLC method for the separation and quantification of all the impurities were investigated. Four process-related impurities (Imp-B, Imp-C, Imp-D, and Imp-E) were found in the SAFM bulk drug. Five degradation products (Imp-A, Imp-C, Imp-D, Imp-E, and Imp-F) were observed in SAFM under oxidative conditions. Imp-C, Imp-D, and Imp-E were also degradation products and process-related impurities. Remarkably, one new compound, identified as (S)-2-[4-(3-fluoro-benzyloxy) benzamido] propanamide (i.e., Imp-D), is being reported here as an impurity for the first time. Furthermore, the structures of the aforementioned impurities were characterized and confirmed via IR, NMR, and MS techniques, and the most probable formation mechanisms of all impurities proposed according to the synthesis route. Optimum separation was achieved on an Inertsil ODS-3 column (250 × 4.6 mm, 5 µm), using 0.1% formic acid in water (pH adjusted to 5.0) and acetonitrile as the mobile phase in gradient mode. The proposed method was found to be stability-indicating, precise, linear, accurate, sensitive, and robust for the quantitation of SAFM and its process-related substances, including its degradation products.

Structural elucidation of the impurities in Enzalutamide bulk drug and the development, validation of corresponding HPLC method.

As the first approved androgen receptor(AR) signalling inhibitor, Enzalutamide was approved by the US Food and Drug Administration as an anticancer drug used to treat castration-resistant prostate cancer in 2012. In this manuscript, six potential impurities of Enzalutamide including process impurities and degradation products were studied. The structures of six impurities obtained by synthesis were characterized and confirmed by IR, NMR and MS techniques. In addition, an efficient chromatographic method to separate and quantify these impurities was developed, which achieved on Inertsil ODS-3 column (250mm×4.6mm,5µm) in gradient mode with a mixture of acetonitrile and the ammonium acetate buffer (10mM, pH adjusted to 4.0 with glacial acetic acid). The method was validated with respect to specificity, precision, accuracy, and sensitivity and satisfactory result was achieved. The method was demonstrated to be applicable in routine quality control and stability evaluation of Enzalutamide.

Quantitative analysis of ripasudil hydrochloride hydrate and its impurities by reversed-phase high-performance liquid chromatography after precolumn derivatization: Identification of four impurities.

We report the development and validation of a stability-indicating reversed-phase high-performance liquid chromatography method with precolumn derivatization for the separation and identification of the impurities of ripasudil hydrochloride hydrate, a novel protein kinase inhibitor. 2,3,4,6-Tetra-O-acetyl-ß-d-glucopyranosyl isothiocyanate was chosen as the derivatizing reagent and triethylamine was added as catalyst. 200 µL sample solution (1 mg/mL), 600 µL derivatizing reagent (1 mg/mL), and 200 µL triethylamine solution (1%, v/v) were mixed and reacted at 40°C for 30 min. The separation was achieved on an Inertsil C18 ODS-3 (250 mm × 4.6 mm, 5 µm) column using mobile phases including 10 mmol monopotassium phosphate buffer (pH 3.0) and methanol in gradient mode. The column temperature was adjusted at 25°C and the flow rate at 1 mL/min. The detection was carried out at 220 nm. Different precolumn derivatization conditions as well as the high-performance liquid chromatography conditions were optimized. Ripasudil hydrochloride hydrate and its four impurities were detected and quantitated, among which two new compounds were characterized. The proposed method was validated and proven to be selective, accurate, and precise and suitable for the quantitative analysis of ripasudil hydrochloride hydrate.

Identification, characterization and HPLC quantification of process-related impurities in Trelagliptin succinate bulk drug: Six identified as new compounds.

A sensitive, selective and stability indicating reversed-phase LC method was developed for the determination of process related impurities of Trelagliptin succinate in bulk drug. Six impurities were identified by LC-MS. Further, their structures were characterized and confirmed utilizing LC-MS/MS, IR and NMR spectral data. The most probable mechanisms for the formation of these impurities were also discussed. To the best of our knowledge, six structures among these impurities are new compounds and have not been reported previously. The superior separation was achieved on an InertSustain C18 (250mm×4.6mm, 5µm) column in a gradient mixture of acetonitrile and 20mmol potassium dihydrogen phosphate with 0.25% triethylamine (pH adjusted to 3.5 with phosphate acid). The method was validated as per regulatory guidelines to demonstrate system suitability, specificity, sensitivity, linearity, robustness, and stability.

Chiral Separation and Thermodynamic Investigation of Ezetimibe Optical Isomers on a Chiralpak IC Column.

A liquid chromatographic separation method of ezetimibe optical isomers on the immobilised-type Chiralpak IC chiral stationary phase, onto which cellulose tris-(3,5-dichlorophenylcarbamate) was covalently grafted as a chiral selector, was developed under normal-phase conditions. The optimized chromatographic conditions were as follows: n-hexane-isopropanol-diethylamine (90:10:0.1, v/v) as mobile phase, 1.0 mL min-1 as flow rate, 256 nm as UV detection wavelength and 25°C as column temperature. Several factors concerning the enantioseparation were studied, including quality and quantity of different polar organic modifiers, additives, and flow rate and column temperature. Separation and resolution in the temperature range of 15-35°C was investigated. Apparent thermodynamic parameters were calculated from the Van't Hoff plots and used to explain the strength of interactions between the optical isomers and chiral selector. The validated method is simple, rapid and robust. Four ezetimibe optical isomers can be effectively separated from each other and the resolutions are 3.5, 2.7 and 2.5 successively. The method has been demonstrated to be applicable in routine quality control of Ezetimibe.

Identification, characterization, and high-performance liquid chromatography quantification of process-related impurities in vonoprazan fumarate.

High-performance liquid chromatography analysis of vonoprazan fumarate, a novel proton pump inhibitor drug revealed six impurities. These were identified by liquid chromatography with mass spectrometry. Further, the structures of the impurities were confirmed by synthesis followed by characterization by mass spectrometry, NMR spectroscopy, and infrared spectroscopy. On the basis of these data and knowledge of the synthetic scheme of vonoprazan fumarate, the previously unknown impurity was identified as 1-[5-(2-fluorophenyl)-1-(pyridin-3-ylsulfonyl)-1H-pyrrol-3-yl]-N-methyldimethylamine, which is a new compound. The possible mechanisms by which these impurities were formed were also discussed. A high-performance liquid chromatography method was optimized in order to separate, selectively detect, and quantify all process-related impurities of vonoprazan fumarate. The presented method has been validated in terms of linearity, limits of detection, and quantification, and response factors and, therefore, is highly suitable for routine analysis of vonoprazan fumarate related substances as well as stability studies.

Stability-indicating reversed-phase HPLC method development and characterization of impurities in vortioxetine utilizing LC-MS, IR and NMR.

The current study reports the development and validation of a stability-indicating reversed phase HPLC method for the separation and identification of potential impurities in vortioxetine, a recently developed antidepressant. The structures of a new compound and four process-related impurities formed during the synthesis were characterized and confirmed by NMR, MS, and IR spectroscopy analyses. The most probable formation mechanisms of the impurities identified were proposed. Based on the characterization data, the new compound was proposed to be 1-[4-[(2,4-dimethylphenyl)thio]phenyl]-piperazine. In addition, an efficient chromatographic method was optimized to separate and quantify the impurities, which were obtained in the 0.05-0.75 µg/mL range. The developed HPLC method was validated with respect to accuracy, precision, linearity, robustness, and limits of detection and quantitation.

Structural Analysis and Quantitative Determination of Clevidipine Butyrate Impurities Using an Advanced RP-HPLC Method.

Eleven potential impurities, including process-related compounds and degradation products, have been analyzed by comprehensive studies on the manufacturing process of clevidipine butyrate. Possible formation mechanisms could also be devised. MS and NMR techniques have been used for the structural characterization of three previously unreported impurities (Imp-3, Imp-5 and Imp-11). To separate and quantify the potential impurities in a simultaneous fashion, an efficient and advanced RP-HPLC method has been developed. In doing so, four major degradation products (Imp-2, Imp-4, Imp-8 and Imp-10) can be observed under varying stress conditions. This analytical method has been validated according to ICH guidelines with respect to specificity, accuracy, linearity, robustness and stability. The method described has been demonstrated to be applicable in routine quality control processes and stability evaluation studies of clevidipine butyrate.

Liquid Chromatographic Separation and Thermodynamic Investigation of Mirabegron Enantiomers on a Chiralpak AY-H Column.

Liquid chromatographic separation of mirabegron enantiomers on Chiralpak AY-H, a column coated with amylose tris-(5-chloro-2-methylphenylcarbamate) as a chiral stationary phase, was studied under normal phase conditions. The influence of ethanol content (30-45%) and column temperature (20-40°C) on retention, resolution and separation were evaluated. Apparent thermodynamic parameters deduced from Van't Hoff plots were used to understand chiral separation mechanisms, and the chiral separation was enthalpy driven. The optimized chromatographic conditions were using a mixture solution of n-hexane, ethanol and diethyl amine (55 : 45 : 0.1, v/v/v) as a mobile phase at a flow rate of 1.0 mL/min. The column temperature and UV detector were set at 35°C and 254 nm, respectively. The method was validated to be simple, accuracy, sensitive and robust according to the ICH guidelines, and it was suitable for the routine quality control of mirabegron enantiomers for pharmaceutical industries.

Determination of clevidipine and its primary metabolite in rat plasma by a dispersive liquid-liquid microextraction method.

Dispersive liquid-liquid microextraction based on the solidification of a floating organic droplet combined with high-performance liquid chromatography with ultraviolet detection (HPLC-UV) was developed for the determination of clevidipine and its primary metabolite in Sprague-Dawley rat plasma samples. During the extraction procedure, to facilitate an efficient procedure, the plasma protein was precipitated first by using a mixture of a zinc sulfate solution and acetonitrile. Several extraction parameters were carefully evaluated and optimized, including the type and volume of the extraction solvent, salt effect, pH and extraction time. Under the optimum conditions, the limits for quantification were 2.5 and 5.0 ng mL(-1) for clevidipine and its primary metabolite, respectively. Sufficient linearity of clevidipine or its primary metabolite was observed with the coefficient of correlation (r) above 0.9979. The method showed good precision and accuracy, with intra- and inter-assay coefficients of variation <6.1% at all concentrations. The proposed pretreatment technique with HPLC was successfully applied to the determinate of clevidipine and its primary metabolite in rat plasma samples. Furthermore, the method, which requires less time than conventional techniques, is environmentally friendly and requires only a small amount of low-toxicity extraction solvent.

Bioavailability comparison of a new form of vilazodone XVII to IV in beagles using liquid chromatography/mass spectrometry.

Vilazodone hydrochloride (CAS 163521-12-8) is polymorphic and has 15 crystal forms, referred to as I-XI and XIII-XVI. In the study, we prepared and performed structural identification of a new crystal form named XVII. To investigate this in vivo, a rapid and sensitive method based on liquid-liquid extraction, followed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed and validated for the determination of vilazodone hydrochloride in dog plasma. This HPLC-MS/MS method was successfully applied to a bioavailability comparison of two crystal forms of vilazodone hydrochloride (IV and XVII) in six healthy beagles using a single-dose, two-way crossover design. The maximum plasma concentration (C(max)), the time taken to reach C(max), and the area under the concentration-time curve were determined following oral administration of 10 mg vilazodone hydrochloride (IV or XVII) to beagles. These analyses revealed no significant bioavailability differences between vilazodone hydrochloride forms IV and XVII in dogs.

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.

Structural elucidation of potential impurities in Azilsartan bulk drug by HPLC.

During the synthesis of Azilsartan (AZS), it was speculated that 15 potential impurities would arise. This study investigated the possible mechanism for the formation of 14 of them, and their structures were characterized and confirmed by IR, NMR, and MS techniques. In addition, an efficient chromatographic method was developed to separate and quantify these impurities, using an Inertsil ODS-3 column (250 x 4.6 mm, 5 pm) in gradient mode with a mixture of acetonitrile and the potassium dihydrogen orthophosphate buffer (10 mM, pH adjusted to 3.0 with phosphoric acid). The HPLC method was validated for specificity, precision, accuracy, and sensitivity. LOQ of impurities were in the range of 1.04-2.20 ng. Correlation coefficient values of linearity were >0.9996 for AZS and its impurities. The mean recoveries of all impurities in AZS were between 93.0 and 109.7%. Thus, the validated HPLC method is suitable for the separation and quantification of all potential impurities in AZS.

Determination of fenticonazole enantiomers by LC-ESI-MS/MS and its application to pharmacokinetic studies in female rats.

A simple, rapid, and specific high-performance liquid chromatograph coupled with a tandem mass spectrometry method has been developed and validated for the determination of fenticonazole (CAS 72479-26-6) enantiomers in rat plasma. Simple protein precipitation by acetonitrile was utilized for extracting analytes from the plasma samples. Chromatography separation was performed on a C18 analytical column (150 mm x 2.0 mm, 5 microm) with a mobile phase consisting of methanol-10 mM aqueous ammonium acetate (adjusted to pH 3.5 with acetic acid) (90:10, v/v) at a flow rate of 0.2 ml/min. Detection was carried out on a triple quadrupole mass spectrometer equipped with electrospray ionization (ESI) source, and operated in multiple-reaction monitoring (MRM) mode. The calibration curves were linear over the range 0.5 -200 ng/ml (r > 0.99). The relative recoveries of R-(-)-fenticonazole and its enantiomer were better than 85%. The intra- and inter-day precisions (R.S.D.%) and deviations of the assay accuracies were less than 10%. This newly developed and validated method was successfully applied to pharmacokinetic studies after administration at a single dose of 20 mg/ kg R-(-)-fenticonazole nitrate and its enantiomer to female rats per vagina. The Cmax value of S-(+)-fenticonazole was greater than that of R-(-)-fenticonazole by 1.36-fold, whereas, the t(1/2) beta and MRT values of R-(-)-fenticonazole were longer than those of its enantiomer by 1.95- and 1.24-fold. The results indicated that S-(+)-fenticonazole was faster in absorption and elimination in female rat. But, the Tmax and AUC(0-12) values for each of fenticonazole enantiomers were not significantly different.