Application of LC-NMR and LC-MS to the identification of degradation products of a protease inhibitor in dosage formulations

LC-NMR and LC-MS were applied to the characterization of six degradation products of a protease inhibitor, N-hydroxy-1,3-di-[4-ethoxybenzenesulphonyl]-5,5-dimethyl-[1,3]c yclohexyldiazine-2-carboxamide, in a dosage formulation. A reversed-phase HPLC method was developed for the separation of the parent compound and its six degradation products. LC-MS was then utilized to obtain the molecular weight and fragmentation information using an electrospray ionization (ESI) interface in the positive ion mode. LC-NMR was employed to acquire detailed structural information using a selective solvent suppression pulse sequence in the stop flow mode. This work demonstrated the usefulness of this integrated approach for the rapid and unambiguous identification of drug compounds and their degradation products in dosage formulations.     

Simultaneous high-performance liquid chromatographic determination of SCH 59884 (phosphate ester prodrug of SCH 56592), SCH 207962 and SCH 56592 in dog plasma.

SCH 59884 is an IV prodrug of SCH 56592, the broad-spectrum azole antifungal agent that is active both orally and intravenously in animal models of infection. SCH 56592 is in phase III clinical trials for the treatment of serious systemic fungal infections. SCH 59884 is a carboxylate ester of SCH 56592 with gamma-butyric acid phosphate. Following IV administration of SCH 59884, the compound is rapidly dephosphorylated to SCH 207962 which is then hydrolyzed to SCH 56592. A high-performance liquid chromatographic (HPLC) method was developed for the simultaneous determination of SCH 59884, SCH 207962 and SCH 56592 in plasma of dogs, a species used for safety evaluation. The HPLC analysis involved protein precipitation with methanol followed by separation on a C-18 column and quantitation by UV absorbance at 260 nm. The lower limits of quantification were 0.1 microg/ml for SCH 59884 and 0.05 microg/ml for SCH 207962 and SCH 56592 in dog plasma. The linearity for the three compounds was satisfactory as indicated by correlation coefficients (r) of >0.98, back-calculated concentrations and visual examination of the calibration curves. The precision and accuracy were satisfactory as shown by coefficients of variation (CV) ranging from 2.4 to 10.6%, and bias values ranging from -8.4 to 13.3%. Moreover, SCH 59884 and SCH 207962 were stable in dog plasma after being subjected to three freeze-thaw cycles. SCH 56592 had been shown earlier to be stable under these conditions. The assay was shown to be specific, accurate, precise, and reliable for use in pharmacokinetic and toxicokinetic studies.     

Determination and characterization of degradation products of anastrozole by LC-MS/MS and NMR spectroscopy

Two new degradation products for Anastrozole active pharmaceutical ingredient (ANZ) have been identified and reported in this paper. The ANZ was subjected to thermal, photolytic, oxidative and base stress conditions prescribed by ICH guidelines. Separation of ANZ from its existing impurities and the two new impurities was achieved by using on Oyster ODS-3 (100 mm × 4.6 mm × 3.0 μm) column with an isocratic mixture of 10 mM ammonium formate and acetonitrile in the ratio 60:40 (v/v). The flow rate was 0.5 ml min⁻¹. The elution was monitored at 215 nm. An isocratic stability indicating reverse phase liquid chromatographic (RP-LC) and LC-MS/MS method was developed for the determination of purity and assay of ANZ through forced degradation studies. The two new impurities detected were further subjected to spectroscopic studies. Based on the results obtained from the different spectroscopic studies, these impurities have been characterized as 2,2′-(5-((1H-1,2,4-triazol-1-yl)methyl)-1,3-phenylene)bis(2-methylpropanoicacid) (Diacid) and 2-(3-((1H-1,2,4-triazol-1-yl)methyl)-5-(2-cyanopropan-2-yl)phenyl)-2-methylpropanoicacid (Monoacid). ANZ was found to degrade in base, slightly in oxidative degradation conditions. The degradation products were well resolved from main peak and its impurities thus proved the stability, indicating power of the method. The developed method was validated as per International Conference on Harmonization (ICH) guidelines with respect to specificity, limit of detection, limit of quantitation, precision, linearity, accuracy, robustness and system suitability.     

抗真菌剂两性霉素B的结构修饰

自两性霉素B作为抗真菌剂应用于临床以来,便受到了人们的广泛关注,目前仍为治疗全身或深部真菌病的首选药物.但其毒副作用,水溶件差等缺点存在,使得该抗生素的临床应用受到了一定的限制,因此开发新犁两性霉素B衍生物具有重要的研究和应用价值,近年人们对两性霉素B进行了大量的结构修饰研究,并从中获得了一系列有价值的衍生物.本文对两性霉素B结构修饰的最新研究进展作了一个简要的综述,并重点对几个毒副作用明显降低的两性霉素B衍生物作了较为详细的介绍.     

头孢硫脒降解产物的LC-MS分析

目的分析确定头孢硫脒的降解产物。方法用LC—MS分析头孢硫脒长期留存样品和冻干样品的降解情况。结果主要的两个有关物质一为头孢酯脒，二为去乙酰头孢硫脒。结论为头孢硫脒的质量控制和稳定性研究提供了重要依据。     

Photolytic and oxidative degradation of an antiemetic agent,RG 12915

RG 12915 (I) undergoes dechlorination and substitution in aqueous solutions when exposed to an artificial daylight fluorescent light. The free-radical photodecomposition exhibits apparent first-order kinetics at all concentrations and the reaction proceeds faster in dilute solutions. The drug substance is also susceptible to oxidative degradation. Oxidation occurs on the benzofuran moiety as well as on the quinuclidine moiety. Auto-oxidation on the benzofuran moiety produces a corresponding hydroperoxide. Subsequent decomposition of the hydroperoxide gives rise to the secondary oxidation product, the hydroxy compound. The oxidative free-radical reaction shows an induction period, followed by a period of accelerating degradation and eventual leveling off. The oxidation reaction proceeds faster in concentrated solutions and at lower pH, and is accelerated by cupric ion. EDTA prevents the oxidative degradation of the drug substance. Propyl gallate inhibits the oxidation of the benzofuran moiety but not N-oxide formation.     

土霉素加速破坏降解产物的LC-MS研究

目的建立土霉素及降解产物的LC—MS方法，分析土霉素在各种加速破坏条件下的降解产物，探讨其可能的降解途径。方法通过加速实验制备实验样品，利用分析柱，经柱后分流，电喷雾电离和选择性离子检测，分析降解产物和降解途径。结果在弱酸、强酸、强碱、加热和氧化5种破坏条件下．土霉素可以产生多种降解产物，其主要降解产物为4-差向土霉素(4-epi-oxytetracycline EOTC)、脱水土霉素(anhydrooxytetracycline AOTC),α-原土霉素(α—Apo—oxytetracycline α—APOTC)、β-原土霉素(β-Apo—oxytetracycline β-APOTC)、异土霉素(isooxytetracycline IOTC)。结论建立的LC—MS方法可用于该类降解产物及降解途径研究，所确定的降解产物及降解途径为该品种质量控制和稳定性研究提供了重要依据。     

The effect of antacid and cimetidine on the oral absorption of the antifungal agent SCH 39304

The single-dose pharmacokinetics of the antifungal agent SCH 39304 (Schering-Plough Corp., Kenilworth, NJ) were assessed alone and in combination with antacid and cimetidine. On three separate occasions nine healthy men received a single oral 50 mg dose of SCH 39304 either alone, with 60 mL antacid, or with oral cimetidine 300 mg four times a day for 4 days. Concomitant antacid or cimetidine administration had no significant effect on any of the SCH 39304 pharmacokinetic parameters studied. The oral absorption of SCH 39304, as assessed by the area under the plasma concentration-time curve (AUC) and the amount of drug recovered unchanged in the urine, was not affected by either antacid or cimetidine. The AUC0-1 for the drug given alone was 80.5 +/- 15.8 micrograms.hr/mL, compared to 81.4 +/- 12.7 and 79.7 +/- 9.6 micrograms.hr/mL with concomitant antacid and cimetidine, respectively. The amount of drug excreted in the urine (Ae0-1) was 22.7 +/- 5.1, 24.2 +/- 9.2, and 23.6 +/- 7.6 mg when the drug was given alone, with antacid, and with cimetidine, respectively. Antacid coadministration delayed absorption as evidenced by an increase in the tmax in 7 out of 9 subjects, although this did not reach statistical significance (P = .082, Wilcoxon test). We conclude that concomitant antacid or cimetidine does not alter the oral absorption or pharmacokinetic disposition of single-dose SCH 39304.     

LC-UV-PDA and LC-MS studies to characterize degradation products of glimepiride

Degradation products of glimepiride formed under different forced conditions have been characterized through LC-UV-PDA and LC-MS studies. Glimepiride was subjected to forced decomposition under the conditions of hydrolysis, oxidation, dry heat and photolysis, in accordance with the ICH guideline Q1A(R2). The reaction solutions were chromatographed on reversed phase C8 (150 mm x 4.6mm i.d., 5 microm) analytical column. In total, five degradation products (I-V) were formed under various conditions. The drug degraded to products II and V under acid and neutral hydrolytic conditions while products I, III and IV were formed under the alkaline conditions. The products II and V were also observed on exposure of drug to peroxide. No additional degradation product was shown up under photolytic conditions. All the products, except I, could be characterized through LC-PDA analyses and study of MS fragmentation pattern in both +ESI and -ESI modes. Product I could not be identified, as it did not ionize under MS conditions. The products II, III and V matched, respectively, to impurity B (glimepiride sulfonamide), impurity J and impurity C (glimepiride urethane) listed in European Pharmacopoeia. The product IV was a new degradation product, characterized as [[4-[2-(N-carbamoyl)aminoethyl]phenyl]sulfonyl]-3-trans-(4-methylcyclohexyl) urea. The degradation pathway of the drug to products II-V is proposed, which is yet unreported.     

Normal phase LC-MS determination of retinoic acid degradation products

The degradation products formed when 13-cis retinoic acid (13-cis RA) and all-trans RA were exposed to fluorescent light and air were investigated. These retinoids are known to undergo Z-E isomerization (due to the existence of four unsaturated double bonds) and oxidation when exposed to light and air. Analysis by LC was carried out on a 25 cm × 4.6 mm Zorbax Rx-SIL (5 μm) with a mobile phase (1.4 ml min⁻¹) of heptane-THF-acetic acid (96.5:3.5:0.015) and an in-line UV (365 nm) detector. The LC eluate was coupled through a Vestec universal interface to a Finnigan 4023 mass spectrometer. El-mass spectra were obtained at 77 eV from m/z 200 to 350 with multiplier voltage of 1200 V. Solid samples of 13-cis RA and all-trans RA exposed to light and air and also solutions of these retinoids in the mobile phase exposed to the same conditions were used for the analysis. Tentative identities of the degradation products from the mass spectra suggest the isomerization of the retinoids (Z-E isomerism) and the formation of the 5,6-epoxides of these isomers. Identities of the 5,6-epoxides were confirmed with chromatographic and mass spectral data from synthetic samples of the epoxides. Isomerization occurred more readily in solution than in the solid form and the 13-cis RA isomer oxidized more readily than the all-trans isomer.     

LC-MS/MS法检测西维来司他原料药中的降解产物

目的：探讨应用液相色谱-串联质谱法（LC-MS/MS）检测西维来司他原料药中的降解产物.方法：以乙腈-水-甲酸（40：60：0.1）为流动相,待测物经Hypersil C18柱分离,电喷雾串联质谱在线检测,获得相关的色谱和质谱信息.结果：在所应用的条件下,西维来司他与其降解产物达到了很好分离,主成分和其降解产物峰的保留时间分别为18.46和3.36min,同时通过两者的质谱特征获得了降解产物的结构信息.结论：所建立的方法能快速、准确地分离鉴定西维来司他原料药中的降解产物,从而可以对其原料药进行质量控制.     

Determination of degradation products of sumatriptan succinate using LC-MS and LC-MS-MS

Acid, base, heat, oxidation and UV irradiation stress methods were applied to study the stability of the bulk drug form of sumatriptan succinate. Liquid chromatography coupled with mass spectrometry (LC-MS and LC-MS-MS) was used to analyze the degraded samples and tentative structural identifications were assigned based upon known reactivity of the drug, molecular weight measurements and MS-MS fragmentation patterns. Sumatriptan succinate was found to be stable to exposure of acid, base, oxidation and UV irradiation at ambient conditions, but was found to degrade under acidic, basic and oxidative conditions when heated to 90 degrees C.     

LC, LC-MS/TOF and MS(n) studies for the identification and characterization of degradation products of nelfinavir mesylate

The objective of the present investigation was to separate, identify and characterize the major degradation products (DPs) of nelfinavir mesylate generated under hydrolytic, oxidative, photolytic and thermal stress conditions as advised in International Conference on Harmonization (ICH) guideline Q1A(R2). The drug was found to degrade under acidic, basic, oxidative and photolytic stress, while it was stable in neutral and thermal stress conditions. A total of three degradation products were formed, which were separated on a C-18 column employing a gradient HPLC method. A complete mass fragmentation pathway of the drug was first established with the help of multi-stage (MS(n)) and MS/TOF accurate mass studies. Then stressed samples were subjected to LC-MS/TOF studies, which provided their fragmentation pattern and accurate masses. The mass spectral data were employed to characterize the DPs and assign structures to them. The total information was also used to establish the degradation pathway of the drug. The degradation products were identified as 3-hydroxy-N-((2R,3R)-3-hydroxy-1-(phenylthio)butan-2-yl)-2-methylbenzamide and (3S,4aS,8aS)-N-tert-butyl-2-((2R,3R)-2-hydroxy-3-(3-hydroxy-2-methylbenzamido)-4-(phenylsulfinyl)butyl)decahydroisoquinoline-3-carboxamide.     

Isolation and structure determination of oxidative degradation products of atorvastatin

Methods were developed for the preparation and isolation of four oxidative degradation products of atorvastatin. ATV-FX1 was prepared in the alkaline acetonitrile solution of atorvastatin with the addition of hydrogen peroxide. The exposition of aqueous acetonitrile solution of atorvastatin to sunlight for several hours followed by the alkalization of the solution with potassium hydroxide to pH 8–9 gave ATV-FXA. By the acidification of the solution with phosphoric acid to pH 3 ATV-FXA1 and FXA2 were prepared. The isolation of oxidative degradation products was carried out on a reversed-phase chromatographic column Luna prep C18(2) 10 μm applying several separation steps. The liquid chromatography coupled with a mass spectrometer (LC-MS), high resolution MS (HR-MS), 1D and 2D NMR spectroscopy methods were applied for the structure elucidation. All degradants are due to the oxidation of the pyrrole ring. The most probable reaction mechanism is intermediate endoperoxide formation with subsequent rearrangement and nucleophilic attack by the 5-hydroxy group of the heptanoic fragment. ATV-FX1 is 4-[1b-(4-Fluoro-phenyl)-6-hydroxy-6-isopropyl-1a-phenyl-6a-phenylcarbamoyl-hexahydro-1,2-dioxa-5a-aza-cyclopropa[a]inden-3-yl]-3-(R)-hydroxy-butyric acid and has a molecular mass increased by two oxygen atoms with regard to atorvastatin. ATV-FXA is the regioisomeric compound, 4-[6-(4-Fluoro-phenyl)-6-hydroxy-1b-isopropyl-6a-phenyl-1a-phenylcarbamoyl-hexahydro-1,2-dioxa-5a-aza-cyclopropa[a]inden-3-yl]-3-(R)-hydroxy-butyric acid. Its descendants ATV-FXA1 and FXA2 appeared without the atorvastatin heptanoic fragment and are 3-(4-Fluoro-benzoyl)-2-isobutyryl-3-phenyl-oxirane-2-carboxylic acid phenylamide and 4-(4-Fluoro-phenyl)-2,4-dihydroxy-2-isopropyl-5-phenyl-3,6-dioxa-bicyclo[3.1.0]hexane-1-carboxylic acid phenylamide, respectively. Quantitative NMR spectroscopy was employed for the assay determination of isolated oxidative degradation products. The results obtained were used for the determination of the UV response factors relative to atorvastatin.     

Structure elucidation and characterization of daunorubicin degradation products

The thermal degradation of the cytostatic drug, daunorubicin, in aqueous solution was studied. Degradation products were extracted with chloroform from the degradation mixtures and separated by thin-layer chromatography. The predominant degradation products were identified and characterized with several spectroscopic techniques. All identified compounds were aglycones.     

The degradation of the antitumor agent gemcitabine hydrochloride in an acidic aqueous solution at pH 3.2 and identification of degradation products

A study of the degradation kinetics of gemcitabine hydrochloride (2'-deoxy-2',2'-difluorocytidine) in aqueous solution at pH 3.2 was conducted. The degradation of gemcitabine followed pseudo first-order kinetics, and rate constants were determined at four different temperatures. These rates were used to construct an Arrhenius plot from which degradation rates at lower temperatures were extrapolated and activation energy calculated. Four major degradation products were identified. Only one of these degradation products, the uridine analogue of gemcitabine, was a known degradation product of gemcitabine and was identified by comparison with synthesized material. The other three degradation products were isolated and characterized by spectroscopic techniques. Two of these products were determined to be the diastereomeric 6-hydroxy-5, 6-dihydro-2'-deoxy-2',2'-difluorouridines, and the other product was determined to be O(6),5'-cyclo-5,6-dihydro-2'-deoxy-2', 2'-difluorouridine. The mechanisms of formation of these degradation products are discussed.     

The application of LC-NMR and LC-MS for the separation and rapid structure elucidation of an unknown impurity in 5-aminosalicylic acid

This work has demonstrated the usefulness of combining liquid chromatography–nuclear magnetic resonance spectroscopy (LC–NMR) and liquid chromatography–mass spectrometry (LC–MS) methodologies for a rapid identification of an unknown impurity (N1) in the drug 5-aminosalycilic acid. Complementary information obtained from the two methods has revealed plenty of structural information and led to the fast on-line structure determination of N1 prior to its isolation and purification. The analysis of LC–NMR and LC–MS spectra revealed that N1 and 5-aminosalycilic acid are structurally closely related compounds. The structure of N1 was later confirmed by high-resolution NMR spectroscopy of the isolated compound and the atom assignment was made. The approach described here has potential for 5-aminosalycilic acid impurity profiling and monitoring the production process.     

HPLC determination of cisatracurium besylate and propofol mixtures with LC-MS identification of degradation products

A stability-indicating HPLC method was developed to simultaneously determine cisatracurium besylate and propofol in mixtures. The effects of organic modifier, ionic strength and the pH of the mobile phase on resolution and retention were investigated. A baseline separation was achieved on an octadecylsilane column with an isocratic mobile phase of acetonitrile–ammonium formate (pH 5.2; 0.3 M) (50:50, v/v). Cisatracurium and propofol were confirmed by both retention time and mass-to-charge ratio using LC-MS. The degradation products of cisatracurium were identified by ESI positive-ion detection as Hofmann elimination and ester hydrolysis products of cisatracurium. There were no propofol degradation products observed. The quantitation of the two drugs was accomplished using UV detection at 280 nm. This method showed linearity for cisatracurium besylate and propofol in the 8–128 and 37–592 μg ml⁻¹ ranges, respectively. Accuracy and precision were in the 0.4–1.4 and 0.4–2.9% ranges respectively, for both analytes.