The use of HPLC/MS, GC/MS, NMR, UV and IR to identify a degradation product of eperisone hydrochloride in the tablets

Understanding drug degradation in the pharmaceutical dosage forms is critical as it has significant impacts on drug efficacy, safety profile and storage conditions. In this study, analytical techniques, such as high-performance liquid chromatograph mass spectrometry (HPLC/MS), gas chromatograph mass spectrometry (GC/MS) and nuclear magnetic resonance (NMR) had been applied to the identification of a drug degradation product which grew over time in the stability study of eperisone hydrochloride tablets. The target unknown degradation product is ionizable by atmospheric pressure chemical ionization (APCI) in a positive mode to determine its relative molecular weight. GC/MS with electron impact ionization (EI) was employed to determine the fragmentation pattern of this unknown compound. Structure elucidation of eperisone and its unknown degradation product by spectral data had been discussed in detail. The isolated unknown was analyzed by NMR, UV and IR, from which the structure of the degradation product was further confirmed as 1-(4-ethylphenyl)-2-methyl-2-propen-1-one.     

Application of LC/MS and related techniques to high-throughput drug discovery

The broad coordinated implementation of common platform technologies, such as LC/MS, can be a key factor in attaining increased throughput, sensitivity and data quality for pharmaceutical discovery. These platform technologies are the key tools that Medicinal Analytical Chemists rely upon to address the ever-changing needs of a drug discovery team. Despite the recent advances in key areas of sensitivity and speed for LC/MS, additional progress to address these never-ending analytical problems can be anticipated. This review will highlight current status and future advances for the applications of LC/MS and related techniques to high-throughput drug discovery.     

A multidisciplinary approach to identify a degradation product in a pharmaceutical dosage form

An unknown degradation product found in non-MS compatible HPLC analysis was studied using a multidisciplinary approach. The unknown was separated and isolated from other components in the drug product by HPLC followed by ion trap MS to obtain MS(n) fragmentation patterns. Its chemical formula was determined using a high resolution time-of-flight mass spectrometer (TOF MS). Nuclear Magnetic Resonance (NMR) was used to elucidate the molecular structure. The impurity was identified as 5-hydroxymethyl furfural, which was a degradation product of lactose, one of the excipients used in the formulation of this dosage form.     

Identification of a degradation product in stressed tablets of olmesartan medoxomil by the complementary use of HPLC hyphenated techniques

An unknown degradation product (DP-1) increased in olmesartan medoxomil (OLM) tablets stored at 40 degrees C/75% r.h., reaching 0.72% after 6 months. The molecular weight and fragment information obtained by LC-MS suggested that DP-1 was a dehydrated dimer of olmesartan (OL) and the presence of ester carbonyl group was indicated by solvent-elimination LC-IR analysis. LC-(1)H NMR confirmed the structure of DP-1 as an esterified dimer of OL. Rapid and accurate identification of the degradation product was achieved by the complementary use of HPLC hyphenated techniques without complicated isolation or purification processes.     

LC/MS/MS与GC/MS法分析鉴定小鼠尿中沙美特罗的代谢产物

目的:鉴定沙美特罗在小鼠尿中的主要代谢产物.方法:ig给药后,收集小鼠尿液,经固相提取,葡萄糖醛酸酶水解,进行LC/MS/MS分析和硅烷化后进行GC/MS分析同时分离鉴定沙美特罗代谢产物.结果和结论:在给药后尿样中发现沙美特罗原型和4种代谢产物M1～M4,其结构推测为19-羟基沙美特罗(M1)、2-羰基沙美特罗(M2)、19-羰基沙美特罗(M3)和19-羟基-8-甲氧基沙美特罗(M4).     

Structural elucidation of a process-related impurity in ezetimibe by LC/MS/MS and NMR

A major process-related impurity associated with the synthesis of ezetimibe was detected by LC–MS. The isolated impurity was found not to have been previously reported. Based on LC/MS/MS studies and accurate mass data, the structure of that impurity was proposed to be 2-(4-hydroxybenzyl)-N,5-bis-(4-fluorophenyl)-5-hydroxypentanamide. The postulated structure was unambiguously confirmed with the help of the NMR and IR analyses of a synthetically obtained sample. The chemical shift of the labile proton of that new entity was assigned by a 2D-NOESY NMR experiment. A rationalization for the formation of this impurity is provided.     

Recent advances in use of LC/MS/MS for quantitative high-throughput bioanalytical support of drug discovery

LC/MS/MS based bioanalysis using atmospheric pressure ionization (API)-style interfaces has now been applied for over a decade. This technology, which initially found application for clinical bioanalysis, is now firmly established as the primary bioanalytical tool for ADME studies related to drug discovery and lead optimization (LO). This review focuses on recent advances in LC/MS/MS based bioanalysis in support of drug discovery and LO. The initial part of the article reviews the principal components of LC/MS/MS bioanalysis: sample preparation, chromatography, ionization and mass analysis. In each section, factors affecting high throughput bioanalysis are addressed. Because of the importance of on-line column switching methods to discovery bioanalysis, the section on sample preparation is divided into off-line and on-line approaches. In addition, the discussion of chromatography is limited to reversed phase liquid chromatography with emphasis given to the trend towards high-flow gradient elution techniques. The latter part of the review focuses on considerations for experimental design. In this section, pooling methods such as cassette dosing are discussed along with more highly integrated strategies linking bioanalysis with protocol generation and sample collection. The article concludes by briefly reviewing factors, which affect bioanalytical precision and accuracy, such as ion suppression, analyte stability and metabolite interference.     

Identification and characterization of process related impurities in chloroquine and hydroxychloroquine by LC/IT/MS,LC/TOF/MS and NMR

The focus of this study is identification and characterization of major unknown impurities in chloroquine (CQ) and hydroxychloroquine (HCQ) bulk drug samples using liquid chromatography/ion trap mass spectrometry (LC/IT/MS) and liquid chromatography/time of flight mass spectrometry (LC/TOF/MS). The newly developed LC/MS method was employed for the analysis of both the drugs. The analysis revealed the presence of two impurities in each of the drugs. The impurities are designated as CQ-I, CQ-II (for chloroquine); HCQ-I and HCQ-II (for hydroxychloroquine). Three of the impurities, CQ-II, HCQ-I and HCQ-II were unknown have not been reported previously. Accurate masses of the impurities were determined by using Q-TOF mass spectrometer and fragmentation behavior was studied by an ion trap mass spectrometer. Based on the spectrometric data and synthetic specifics the structures of CQ-II, HCQ-I and HCQ-II were proposed as 1,4 pentanediamine, N⁴(7-chloro-4-quinolinyl), N⁴-chloromethyl, N⁴-ethylamine; 2-(4-(7-chloroquinolin-4-ylamino) pentylamino) ethanol and [[4-[(7-chloro-4-quinolyl) amino] N-pentyl] N-chloromethyl-N-ethylamino] ethanol respectively. The impurities were isolated by semi-preparative HPLC and structures were confirmed by NMR spectroscopy. The formation and through characterization of known CQ-I impurity is also discussed.     

The use of mixed-mode ion-exchange solid phase extraction to characterize pharmaceutical drug degradation

Solid phase extraction (SPE) has been utilized extensively in the pharmaceutical industry for the isolation of pharmaceuticals from interfering biological matrices and the purification and concentration of impurities and degradation products present in analytical samples. The work described herein involves the novel use of mixed-mode ion-exchange solid phase extraction to characterize degradation products of several pharmaceutical drugs, thereby giving important clues to their structure and sites of reactivity. Several examples of the use of mixed-mode ion-exchange solid phase extraction to illustrate the utility of this technique are presented.     

Use of hyphenated LC-MS/MS technique for characterization of impurity profile of quetiapine during drug development

As a part of an integrated quality concept in drug development, the multidimensional evaluation of impurity profiles by LC-MS/MS is presented for quetiapine--an active pharmaceutical ingredient (API). LC-UV is commonly employed for the determination of impurities and degradation products. In this work LCMS/MS technique is proposed as a modern alternative for the characterization of these compounds. The use of this technique allowed to develop methods for the separation and identification of the impurities resulting from both, synthesis and degradation processes.     

Micro LC/MS in drug analysis and metabolism studies

LC/MS has become a routine research tool in some laboratories. Although no single approach to LC/MS presently is without limitations, each approach offers encouraging results and prompts continued improvements. The remaining hurdles appear to be the introduction of total LC effluent into the MS, increased sensitivity, and the ability to analyze higher molecular weight, polar molecules. The micro LC/MS results discussed in this paper offer an opportunity for significantly increased sensitivity by allowing total micro LC effluent introduction into the MS. It remains to be seen whether the development of micro LC column and equipment technology will allow practical micro LC/MS. It may someday provide improved separation of drugs, metabolites, and their conjugates from high levels of endogenous materials in reasonable time periods. Currently, the impressive efficiencies demonstrated for micro LC columns involve mixtures containing low molecular weight solutes that are perhaps more amenable to GC analysis. The analyst routinely involved with problem solving must deal with complex mixtures composed of components with large concentration differences. The future of micro LC, and hence micro LC/MS, will depend upon how well the technique helps solve problems. We believe the future is bright for micro LC/MS. The techniques may require some fine tuning of operating procedures, just as with capillary GC/MS, but certainly the potential for increased efficiency and sensitivity is worth the effort. Currently we are testing a new micro LC/MS DLI probe wherein the exit of the micro column is within 1 cm of the MS ion source [82]. The micro LC column is contained within the DLI probe and should offer the lowest dead volume and the least extracolumn effects yet achieved by LC/MS. Initial testing of this new probe is under way, and experimental results will be reported subsequently. The analytical potential of micro LC/MS is receiving considerable interest. Practical micro LC performance can provide increased capabilities to all types of LC/MS reported to date, and perhaps offer new insight into alternative methods of LC/MS not yet reported. We look forward to learning of these breakthroughs as they become available.     

Characterization of metabolites of xylazine produced in vivo and in vitro by LC/MS/MS and by GC/MS.

The metabolic fate of xylazine, 2-(2,6-dimethylphenylamino)-5,6-dihydro-4H-1,3-thiazine, in horses is described. The major metabolites identified in the hydrolyzed horse urine were 2-(4'-hydroxy-2',6'-dimethylphenylamino)-5,6-dihydro-4H-1,3-thiazi ne, 2-(3'-hydroxy-2',6'-dimethylphenylamino)-5,6-dihydro-4H-1,3-thiazi ne, N-(2,6-dimethylphenyl)thiourea, and 2-(2',6'-dimethylphenylamino)-4-oxo-5,6-dihydro-1,3-thiazine. These metabolites were also produced by incubating xylazine with rat liver microsomes. The major metabolite produced in vitro by rat liver preparations was found to be the ring opened N-(2,6-dimethylphenyl)thiourea. The identities of these metabolites were confirmed by spectroscopic comparisons with synthetic standards. Phenolic metabolic standards were synthesized efficiently by the use of Fenton's reagent. This reagent was used to monohydroxylate multiply substituted aromatic ring systems. LC/MS/MS, with an atmospheric pressure chemical ionization source, was found to be particularly useful in confirming the presence of phenolic metabolites in hydrolyzed equine urine and microsomal extracts. These phenolic metabolites could not be analyzed by GC/MS even after derivatization with silylating agents. The advantage of LC/MS/MS was that no or little sample preparation of urine or microsomal extract was necessary prior to the analysis. A mechanism is also proposed for the formation of the major metabolite, N-(2,6-dimethylphenyl)thiourea, from xylazine.     

The use of mass spectrometry in the study of chemically-reactive drug metabolites. Application of MS/MS and LC/MS to the analysis of glutathione- and related S-linked conjugates of N-methylformamide

The S-(N-methylcarbamoyl) derivatives of glutathione, cysteine and N-acetylcysteine, the S-linked conjugates derived from a reactive metabolite of N-methylformamide (NMF), were studied in mice dosed with an equimolar mixture of NMF and deuterium-labelled NMF. Following preparation of N-benzyloxycarbonyl derivatives in aqueous media, the title conjugates were isolated, purified as their methyl esters and subjected to analysis by fast atom bombardment mass spectrometry (FAB/MS), fast atom bombardment tandem mass spectrometry (FAB/MS/MS) or thermospray liquid chromatography/mass spectrometry (TSP LC/MS). Characteristic isotope clusters in the FAB or TSP mass spectra facilitated recognition of drug metabolites, while constant neutral loss (89 u) and daughter ion scanning tandem mass spectrometry (MS/MS) experiments provided unique structural information on the conjugates of interest. It is concluded that the combined use of stable isotopes, aqueousphase derivatization and contemporary mass spectrometric techniques represents a powerful approach for the analysis of glutathione adducts and related S-linked conjugates of chemically-reactive drug metabolites.     

LC determination of impurities in methoxsalen drug substance: isolation and identification of isopimpinellin as a major impurity by atmospheric pressure chemical ionization LC/MS and NMR

A gradient elution LC method was developed to separate methoxsalen from three of its known impurities: isopimpinellin, bergapten, and ammidin. The method employs a methanol-6%THF (aq) mobile phase, phenyl column, and detection at 254 nm. The gradient LC procedure was applied to seven lots of methoxsalen from five different manufacturers. Six of the seven lots tested contained isopimpinellin as the major impurity at a concentration range of 0.2-2.5%. Identification of the impurity as isopimpinellin was accomplished by a combination of analytical and preparative LC, atmospheric pressure chemical ionization liquid chromatography/mass spectrometry, and NMR.     

The use of mass spectrometry in the study of chemically-reactive drug metabolises. Application of MS/MS and LC/MS to the analysis of glutathione- and related S-linked conjugates of N-methylformamide

The S-(N-methylcarbamoyl) derivatives of glutathione, cysteine and N-acetylcysteine, the S-linked conjugates derived from a reactive metabolite of N-methylformamide (NMF), were studied in mice dosed with an equimolar mixture of NMF and deuterium-labelled NMF. Following preparation of N-benzyloxycarbonyl derivatives in aqueous media, the title conjugates were isolated, purified as their methyl esters and subjected to analysis by fast atom bombardment mass spectrometry (FAB/MS), fast atom bombardment tandem mass spectrometry (FAB/MS/MS) or thermospray liquid chromatography/mass spectrometry (TSP LC/MS). Characteristic isotope clusters in the FAB or TSP mass spectra facilitated recognition of drug metabolites, while constant neutral loss (89 u) and daughter ion scanning tandem mass spectrometry (MS/MS) experiments provided unique structural information on the conjugates of interest. It is concluded that the combined use of stable isotopes, aqueousphase derivatization and contemporary mass spectrometric techniques represents a powerful approach for the analysis of glutathione adducts and related S-linked conjugates of chemically-reactive drug metabolites.     

LC/MS characterization of impurities and degradation products of a potent antitumor peptidic dimer,CU201

Compound CU201 [SUIM-(d-Arg-Arg-Pro-Hyp-Gly-Igl-Ser-d-Igl-Oic-Arg)₂, where SUIM = suberimidyl; Hyp = trans-4-hydroxyproline; Igl = α-(2-indanyl)-glycine; Oic = octahydroindole-2-carboxylic acid], is a dimeric analog of the potent bradykinin antagonist peptide B9430. It blocks the G_αq,11 signal of the heterotrimeric G proteins, stimulates c-Jun kinases, and induces apoptosis in lung cancer cells with neuroendocrine features. CU201 shows potent inhibition for small-cell lung cancer cells in vitro (ED₅₀ = 0.15 μM), as well as for small-cell lung cancer SHP-77 tumor growth in vivo. An HPLC method was developed, as part of a study supported by the National Cancer Institute's (NCI's) Rapid Access to Interventional Development (RAID) program, to assess the purity and stability of CU201. Impurities and degradation products were characterized by LC/MS. The identity of a major impurity, with 1 mass unit different from CU201, was confirmed by high resolution LC/MS and the investigation of model compounds. Susceptible linkages in the peptide chains were revealed by the degradation study.     

Characterization of a new rat urinary metabolite of piperine by LC/NMR/MS studies

Potential of piperine, an active alkaloid of black and long peppers, to increase the bioavailability of drugs in humans is of great clinical significance owing to its omnipresence in food. In an attempt to further study the reported differences in its metabolism in rats and humans, a new major urinary metabolite was detected in rat urine and plasma using HPLC. The metabolite was partially purified using reverse phase column chromatography on Sephadex^®-LH 20 and characterized as 5-(3, 4-methylenedioxy phenyl)-2E,4E-pentadienoic acid-N-(3-yl propionic acid)-amide with the help of LC/NMR/positive ESI–MS studies. Complete mass fragmentation pattern could be assigned with MS/MS studies. The metabolite has a unique structure compared to the previously reported metabolites in that it retains methylenedioxy ring and conjugated double bonds while the piperidine ring is modified to form propionic acid group. Mechanism of formation of the metabolite by oxidation and cleavage of piperidine ring is proposed. Kidney appears to be the major excretion route for piperine metabolites in rats as no metabolite could be detected in feces.     

Identification of oxidative degradates of the TRIS salt of a 5,6,7,8-tetrahydro-1,8-naphthyridine derivative by LC/MS/MS and NMR spectroscopy--interactions between the active pharmaceutical ingredient and its counterion

The Tris(hydroxymethyl)aminomethane (TRIS) salt of a substituted 5,6,7,8-tetrahydro-1,8-naphthyridine compound (I) in a mannitol-based formulation was stressed at various conditions. Liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) analyses of the stressed samples revealed that oxidation and dimerization were the primary degradation pathways for this compound. ¹H- and ¹³C-nuclear magnetic resonance (NMR) spectroscopy were used to characterize the isolated dimers. The aromatized degradate, N-oxide, amide, and three dimeric products were all confirmed by either LC/MS using authentic standards or NMR spectroscopy. In general, the aromatized product was always the primary degradate produced under all stress conditions. When stressed at 80 °C, the TRIS counterion also underwent thermal degradation to yield formaldehyde in situ which reacted with the parent compound to form a unique methylene-bridged dimeric product and an N-formyl degradate. A minor condensation product between the compound I and the TRIS counterion was also detected in the 80 °C stressed samples. Under 40 °C/75% RH stress conditions, TRIS derived degradates were insignificant, while dimers formed by compound I became predominant. In addition, two hydroxylated products (7-OH and 5-OH) were also detected. Mechanisms for the formation of the oxidative and dimeric degradates were proposed.     

LC/MS/MS quantitation of an anti-cancer drug in human plasma using a solid-phase extraction workstation: application to population pharmacokinetics

A liquid chromatographic/mass spectrometric (LC/MS/MS) method to quantitate an anti-cancer drug in human plasma was validated. The method has proven suitable for routine quantitation of the experimental anti-cancer compound at concentrations from 1 to 400 ng/ml. Retention times of the compound and internal standard (compounds I and II, respectively) were 1.8 and 2.1 min, respectively. No interfering endogenous peaks were observed throughout the validation process. Precision estimates for this approach were typically less than 5% relative standard deviation (RSD) across the calibration range. Other validation parameters studied included specificity, system reproducibility, limit of quantitation, accuracy, linear range, and stability of the compound and internal standard in plasma and injection solvent. This method was used to quantify drug for population pharmacokinetic studies.     

Simultaneous determination of formic acid and formaldehyde in pharmaceutical excipients using headspace GC/MS

Formic acid and its esters, as well as formaldehyde, are trace impurities that are often present in pharmaceutical excipients. These trace impurities can potentially react with amino and/or hydroxyl groups in drugs to form significant levels of degradants. To select the appropriate excipients for a stable formulation, a gas chromatography/mass spectrometry (GC/MS) method was developed and validated for the rapid screening of trace amounts of residual formic acid, its esters and formaldehyde in pharmaceutical excipients. Samples were dissolved or dispersed in acidified ethanol to convert formic acid and formaldehyde to ethyl formate and diethoxymethane, respectively. Identification was conducted using a GC/MS system under scan mode and quantified using a selected ion monitoring (SIM) mode. Evaluation of the mass spectra of ethyl formate and diethoxymethane in the samples indicated that the method is specific. The limits of quantitation of the method were 0.5 ppm for formic acid and 0.2 ppm for formaldehyde. The precision of the method was demonstrated by the acceptable R.S.D. (相似文献