Validated Stability-Indicating Method for Alendronate Sodium Employing Zwitterionic Hydrophilic Interaction Chromatography Coupled with Charged Aerosol Detection

Alendronate sodium is widely used in the treatment of osteoporosis and Paget’s disease. The HPLC method development for alendronate sodium, in particular, is challenging owing to the absence of chromophoric group and its high polarity. In the present study, a short and simple isocratic method was developed involving hydrophilic interaction liquid chromatography, coupled with a charged aerosol detector. The developed method was validated according to the ICH Q2(R1) guideline and was successfully applied for the analysis of a marketed formulation containing the drug.

     

Quantitation of memantine hydrochloride bulk drug and its tablet formulation using proton nuclear magnetic resonance spectrometry

The use of quantitative nuclear magnetic resonance spectrometry for the determination of non‐UV active memantine hydrochloride with relative simplicity and precision has been demonstrated in this study. The method was developed on a 500 MHz NMR instrument and was applied to determination of the drug in a tablet formulation. The analysis was performed by taking caffeine as an internal standard and D₂O as the NMR solvent. The signal of methyl protons of memantine hydrochloride appeared at 0.75 ppm (singlet) relative to the signal of caffeine (internal standard) at 3.13 ppm (singlet). The method was found to be linear (r² = 0.9989) in the drug concentration range of 0.025 to 0.80 mg/ml. The maximum relative standard deviation for accuracy and precision was <2. The limits of detection and quantification were 0.04 and 0.11 mg/ml, respectively. The robustness of the method was revealed by changing nine different parameters. The deviation for each parameter was also within the acceptable limits. The study highlighted possibility of direct determination of memantine hydrochloride in pure form and in its marketed tablet formulation by the use of quantitative NMR, without the need of derivatization, as is the requirement in HPLC studies. Copyright © 2016 John Wiley & Sons, Ltd.     

Study of the forced degradation behavior of prasugrel hydrochloride by liquid chromatography with mass spectrometry and liquid chromatography with NMR detection and prediction of the toxicity of the characterized degradation products

Prasugrel was subjected to forced degradation studies under conditions of hydrolysis (acid, base, and neutral), photolysis, oxidation, and thermal stress. The drug showed liability in hydrolytic as well as oxidative conditions, resulting in a total of four degradation products. In order to characterize the latter, initially mass fragmentation pathway of the drug was established with the help of mass spectrometry/time‐of‐flight, multiple stage mass spectrometry and hydrogen/deuterium exchange data. The degradation products were then separated on a C₁₈ column using a stability‐indicating volatile buffer method, which was later extended to liquid chromatography‐mass spectrometry studies. The latter highlighted that three degradation products had the same molecular mass, while one was different. To characterize all, their mass fragmentation pathways were established in the same manner as the drug. Subsequently, liquid chromatography‐nuclear magnetic resonance (NMR) spectroscopy data were collected. Proton and correlation liquid chromatography with NMR spectroscopy studies highlighted existence of diastereomeric behavior in one pair of degradation products. Lastly, toxicity prediction by computer‐assisted technology (TOPKAT) and deductive estimation of risk from existing knowledge (DEREK) software were employed to assess in silico toxicity of the characterized degradation products.     

LC and LC-MS study on establishment of degradation pathway of glipizide under forced decomposition conditions

Forced degradation studies on glipizide are conducted under the conditions of hydrolysis, oxidation, photolysis, and dry heat. The solutions are subjected to liquid chromatographic (LC) investigations to establish the number of products formed in each condition. The degradation products are characterized through isolation and subsequent NMR, IR, and MS spectral analyses, or through LC-mass spectrometry (MS) fragmentation pattern study. The drug is shown to degrade in 0.1M HCl at 85 degrees C to two products: 5-methyl-N-[2-(4-sulphamoylphenyl)ethyl]pyrazine-2-carboxamide (II) and methyl N-[4-[2-{(5-methyl-2-pyrazinoyl)amino}ethyl] phenyl]sulfonyl carbamate (III). The latter, a methyl ester, is formed only in the presence of methanol (used as a solubilizer), and does not appear on use of acetonitrile. III is shown to convert to II on continued heating in acid. The drug degrades slowly in water at the same temperature, and both II and III could be seen in the chromatograms until the end of the study. The heating of the drug in alkali (0.1M NaOH) at 85 degrees C yields 5-methyl-2-pyrazinecarboxylic acid (IV), along with a small quantity of 4-(2-aminoethyl) benzenesulfonamide (I). On extended heating in the same condition, a new product, 4-(2-aminoethyl)-N,N-bis[(cyclohexylamino)carbonyl] benzenesulfonamide (VI) is formed in small quantities. At the lower temperature of 40 degrees C, the drug converts under each hydrolytic condition and in both the absence and presence of light to products II, III, or IV, along with a new product, 1-cyclohexyl-3-[[4-(2aminoethyl)phenyl] sulfonyl]urea (V). The light catalyzes formation of V, and it is formed until one or two weeks, after which its level decreases. The drug remains stable in 30% H2O2, except that products II and III appear as small peaks due to acidic character of the peroxide solution. Also, the drug remains unaffected in solid state under thermal and photolytic stress conditions. Based on the results, a more complete picture on degradation pathway of the drug is obtained, highlighting a clear advantage of the approach suggested by International Conference on Harmonization.     

Liquid chromatography/mass spectrometric studies on atorvastatin and its stress degradation products

A comprehensive mass fragmentation pathway of atorvastatin, which has not been reported so far, was established by subjecting the drug to multi-stage mass spectrometric (MSn) studies. It was used along with liquid chromatography/mass spectrometric (LC/MS) and liquid chromatography/time-of-flight mass spectrometric (LC/TOFMS) analyses to identify the drug degradation products formed under stress conditions of hydrolysis, oxidation and photolysis. Other than lactone, which is a reported hydrolysis product, six unknown hydrolytic products could be identified, viz., dehydrated drug, dehydrated drug lactone, and diastereomers of the drug, drug lactone, dehydrated drug, and dehydrated drug lactone. Among the two products separated under oxidative conditions, one was lactone, again formed as a result of drug hydrolysis in an acidic environment of peroxide solution. The other was similar to a reported oxidative product. Under photolytic conditions in solution, one new product could be identified, while most of the others matched with those known from the literature. Hence overall a more complete degradation pathway of the drug was established than known at present, by using a stress testing approach and employing LC/MS techniques.     

Ultraviolet-photodiode array and high-performance liquid chromatographic/mass spectrometric studies on forced degradation behavior of glibenclamide and development of a validated stability-indicating method

A forced degradation study on glibenclamide was performed under conditions of hydrolysis, oxidation, dry heat, and photolysis and a high-performance column liquid chromatographic-ultraviolet (HPLC-UV) method was developed to study degradation behavior of the drug under the forced conditions. The degradation products formed under different forced conditions were characterized through isolation and subsequent infrared/nuclear magnetic resonance/mass spectral analyses, or through HPLC/mass spectrometric (HPLC/MS) studies. The drug degraded in 0.1 M HCI and water at 85 degrees C to a major degradation product, 5-chloro-2-methoxy-N-2-(4-sulfamoylphenyl)ethyl]benzamide (III), and to a minor product, 1-cyclohexyl-3-[[4-(2-aminoethyl)-phenyl]sulfonyl]urea (IV). Upon prolonged heating in the acid, the minor product IV disappeared, resulting in formation of 5-chloro-2-methoxy-benzoic acid (II) and an unidentified product (I). Heating of the drug in 0.1 M NaOH at 85 degrees C yielded II and IV as the major products and I and III as the minor products. The drug and the degradation products formed under different conditions were optimally resolved on a C18 column using ammonium acetate buffer (0.025 M, pH 3.5)-acetonitrile (45 + 55) mobile phase at a flow rate of 0.6 mL/min, with detection at 230 nm. The method was validated for linearity, precision, accuracy, and specificity. Limit of detection (LOD) and limit of quantitation (LOQ) values were also determined. The method could be successfully applied for simultaneous quantification of glibenclamide and the major product, III. The response of the method was linear in a narrow [0.4-10 micro/mL, correlation coefficient (r2) = 0.9982] and a wide (0.4-500 microg/mL, r2 = 0.9993) concentration range for glibenclamide, and in the concentration range of 0.025-50 microg/mL (r2 = 0.9998) for III. The method proved to be precise and accurate for both glibenclamide and III. It was specific for the drug and also selective for each degradation product, and LOQ values for the drug were 0.1 and 0.4 microg/mL, whereas those for III were 0.010 and 0.025 microg/mL, respectively.     

Explanation through density functional theory of the unanticipated loss of CO2 and differences in mass fragmentation profiles of ritonavir and its rCYP3A4‐mediated metabolites

In the present study, the metabolism of ritonavir was explored in the presence of rCYP3A4 using a well‐established strategy involving liquid chromatography–mass spectrometry (LC–MS) tools. A total of six metabolites were formed, of which two were new, not reported earlier as CYP3A4‐mediated metabolites. During LC–MS studies, ritonavir was found to fragment through six principal pathways, many of which involved neutral loss of CO₂, as indicated through 44‐Da difference between masses of the precursors and the product ions. This was unusual as the drug and the precursors were devoid of a terminal carboxylic acid group. Apart from the neutral loss of CO₂, marked differences were also observed among the fragmentation pathways of the drug and its metabolites having intact N‐methyl moiety as compared to those lacking N‐methyl moiety. These unusual fragmentation behaviours were successfully explained through energy distribution profiles by application of the density functional theory. Copyright © 2014 John Wiley & Sons, Ltd.