Investigation of amodiaquine bulk drug impurities by liquid chromatography/ion trap mass spectrometry

Three unknown impurities in an amodiaquine bulk drug sample were detected by reversed-phase high-performance liquid chromatography with ultraviolet detection (HPLC/UV). A liquid chromatography/tandem mass spectrometry (LC/MS(n)) method is described for the investigation of these impurities. Mass spectral data were acquired on an LCQ ion trap mass analyzer equipped with an electrospray ionization (ESI) source operated in positive ion mode. The fragmentation behavior of amodiaquine and its impurities has been studied. Based on the mass spectral data and the specifics of the synthetic route, the possible structures of these impurities were elucidated as 4-[(5-chloroquinolin-4-yl)amino]-2-(diethylaminomethyl)phenol (impurity I), 4-[(7-chloroquinolin-4-yl)-amino]phenol (impurity II) and 4-[(7-chloroquinolin-4-yl)amino]-2-(diethylaminomethyl)-N(1)-oxy]phenol (impurity III). The structures were confirmed by their independent synthesis and NMR spectral assignment.     

Structural analysis of an impurity of the drug landiolol

In a course of development and preparation of landiolol (1a), a known ultra‐short‐acting β‐blocker, process quality control by HPLC and LC‐MS analysis consistently showed an impurity peak ranging from 0.05% to 0.15 % and exhibiting a molecular mass m/z 887. To identify the hitherto unknown impurity, we prepared one of the possible landiolol derivatives with the same molecular mass for proper spectral characterization (NMR and MS). Its equivalence with the unknown impurity was then confirmed by LC‐MS analysis. Ultimately, using fragmentation patterns in LC‐MS and selective two‐dimensional NMR experiments, the structure of the impurity was assigned as [(4S)‐2,2‐dimethyl‐1,3‐dioxolan‐4‐yl]methyl 3‐{4‐[(2S)‐2‐hydroxy‐3‐(3‐{4‐[(2S)‐2‐hydroxy‐3‐[(2‐{[(morpholin‐4‐yl)carbonyl]amino}ethyl)amino]propoxy]phenyl}‐N‐(2‐{[(morpholin‐4‐yl)carbonyl]amino}ethyl)propanamido)propoxy]phenyl}propanoate (2). It was found that the impurity was present in two rotameric forms at room temperature. The synthesis and NMR characterization of (2) are discussed. Copyright © 2014 John Wiley & Sons, Ltd.     

Drug impurity profiling strategies

A general scheme is set up for the estimation of the impurity profile of bulk drug substances by the complex use of chromatographic, spectroscopic and hyphenated techniques. Several examples are presented as illustrations to the scheme from the authors' laboratory involving the use of chromatographic methods such as thin-layer-(TLC), gas-(GC), analytical and preparative high-performance liquid chromatography (HPLC), spectroscopic methods such as mass spectrometry (MS) and NMR spectroscopy as well as hyphenated techniques (HPLC/diode-array UV, GC/MS and HPLC/MS). In addition to summarizing earlier work, new examples are also presented: identification of an impurity (propyl 4-[diethylcarbamoyl(methoxy)]-3-methoxy phenylglyoxylate, II) in propanidid (I) and two unsaturated impurities in allylstrenol (VII) by GC/MS and HPLC/diode-array UV as well as estimation of the impurity profile of mazipredone (III) by HPLC/MS and HPLC/diode-array UV.     

The application of sub-2-microm particle liquid chromatography-operated high mobile linear velocities coupled to orthogonal accelerated time-of-flight mass spectrometry for the analysis of ranitidine and its impurities

Impurity profiling of pharmaceutical drug substances or dosage formulations require methods involving high sensitivity and resolution from LC and MS alike as well as an acceptable analysis time. While throughput can be increased, it is usually at the expense of chromatographic resolution. The application of sub-2-microm stationary phases and high mobile linear velocities has been combined with orthogonal acceleration (oa)-TOF MS for the impurity structural characterization analysis of small-molecule pharmaceutics. A pharmaceutical drug substance was forcefully degraded and used to test the proof of concept of developing an impurity profile method by ultra performance liquid chromatography (UPLC). Optimum conditions were identified by use of method development simulation software as well as traditional approaches of method scouting with columns and a varied range of pH. Further analysis illustrated the effectiveness of applying oa-TOF MS techniques to assist in achieving exact mass coupled with MS/MS to define the structural characterization of the related substances relative to the pharmaceutical active ingredient and identification of any unknown impurity substances. The barriers with trade-offs between resolution and speed are overcome by the application of UPLC, whereas the increased sensitivity provides for superior exact mass oa-TOF MS.     

Validation of higher-throughput high-performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry assays to conduct cytochrome P450s CYP2D6 and CYP3A4 enzyme inhibition studies in human liver microsomes

In the early stage of drug discovery, thousands of new chemical entities (NCEs) may be screened before a single drug candidate can be identified for development. In order to accelerate the drug discovery process, we have developed higher-throughput enzyme assays to evaluate the inhibition of cytochrome P450 isoforms 2D6 (CYP2D6) and 3A4 (CYP3A4) in human liver microsomes. The assays are based on high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) techniques. The analysis time for each sample was reduced from approximately 20 minutes for the conventional HPLC assay to 30 seconds for the LC/MS/MS assay. For both LC/MS/MS assays, the linearity (r(2) > 0.99), precision (%CV < 15%) and accuracy (% bias <15%) for both inter- and intraday validations were satisfactory. Since the implementation of the LC/MS/MS assays, our sample throughput has increased by over 40-fold.     

Isolation and identification of a major impurity in a new bulk drug candidate by preparative LC, ESI-MS(n), LC-MS-MS, and NMR

A simple preparative liquid chromatography (LC) method is developed to isolate a major impurity in a new bulk drug candidate, 6-bromo-4-(carbamidinemethyl)-5-hydroxy-1-methyl-2-(phenylthiomethyl)-1H-indole-3-carboxylic acid ethyl ester hydrochloride monohydrate (carmidole). The carmidole solution for preparation is exposed to daylight before isolation. Based on the electrospray ionization (ESI)-mass spectroscopy (MS(n)) spectral data of the impurity fraction and carmidole, the impurity is preliminarily characterized as 6-bromo-4-(carbamidinemethyl)-5-hydroxy-1-methyl-2-methyl-1H-indole-3-carboxylic acid ethyl ester. LC-MS-MS is used to analyze a carmidole sample. The impurity, lyophilate, is obtained from the fraction of preparative LC, and the impurity standard is synthesized. By comparison of the retention times of high-performance liquid chromatography, ESI-MS(n), and (1)H-nuclear magnetic resonance of the impurity lyophilate with impurity standard and carmidole itself, the structure of the impurity is confirmed and its formation is discussed.     

Determination of the metabolites of gestrinone in human urine by high performance liquid chromatography, liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry

Gestrinone was studied by high performance liquid chromatography (HPLC) for screening and by gas chromatography/mass spectrometry (GC/MS) for confirmation. When the chromatograms of blank, spiked urine and dosed urine were compared by HPLC, two unknown metabolites were found and these were excreted as the conjugated forms. Metabolites 1 and 2 were tested by LC/MS and LC/MS/MS and both had parent ions at m/z 325. The fragment ion of metabolite 1 was at m/z 263 and ions for metabolite 2 were m/z 307 [MH - H(2)O](+), 289, 279 and 241. LC/MS/MS of m/z 263 as the parent ion of metabolite 1 gave fragment ions at m/z 245 and 217, which were assumed to be [263 - H(2)O](+) and [235 - H(2)O](+), respectively. The trimethylsilyl (TMS)-enol-TMS ether derivative of gestrinone displayed three peaks in its GC/MS chromatogram, formed by tautomerism.     

Evaluation and validation of an HPLC procedure for the determination of the positional isomeric impurity in 2-[4-(1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-butyl)-phenyl]-2-methylpropionic acid

A high-performance liquid chromatographic (HPLC) procedure was evaluated for the determination of a positional isomeric impurity in bulk 2-[4-(1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-butyl)-phenyl]-2-methylpropionic acid HCl drug substance. The use of a β-cyclodextrin bonded-phase column with a mobile phase of 20/80 (v/v) acetonitrile/water containing an ammonium acetate buffer at apparent pH 4.0 and a flow rate of 0.45 mL/min resulted in an excellent separation of the isomers. Ultraviolet detection was used at 220 nm. A recovery study of known spike levels (0.1 to 1.5% w/w) showed that the procedure was accurate. A two-day, two-column repeatability study showed consistent results with the test batch of the bulk compound. The level of impurity in the tested lot of the compound had a mean level of 0.32% (w/w) and a standard deviation of 0.038% (w/w, n = 5). The text was submitted by the author in English.     

Characterization of the trace-level impurities in the bulk drug citalopram by high-performance liquid chromatography/tandem multistage mass spectrometry

Five trace impurities were detected in the bulk drug citalopram using high-performance liquid chromatography with UV detection. A simple and sensitive method suitable for liquid chromatography tandem multistage mass spectrometry (HPLC/MS(n)) analysis was developed. Using this method, the fragmentation behavior of citalopram and the impurities was investigated. Four impurities were rapidly characterized, and an unknown impurity was elucidated as 3-(3-dimethylaminopropyl)-N-(1-(3-dimethylaminopropyl)-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-yl)-3-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-carboxamide on the basis of the MS(n) and exact mass evidence, and the proposed structure was further confirmed by nuclear magnetic resonance (NMR) experiments after preparative isolation.     

新型除草剂及其杂质的LC-MS和GC-MS分析

High performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS) have been utilized to analyze the synthesized 2-(2-arylaminomethylphenoxy)pyrimidine derivatives, which are a new kind of environmentally benign herbicides and have passed the temporary pesticide registration. The identification of main product and impurities has been achieved according to the UV and mass spectra. Moreover, one impurity, introduced by the raw material in the last step of the synthetic route, was identified by GC-MS analysis. It can be concluded that the combination of chromatography and mass spectrometry, including LC-MS and GC-MS, provided a vital tool of the pesticide science.     

高效液相色谱-四极杆飞行时间质谱法快速鉴定硝苯地平原料药中杂质

利用高效液相色谱-四极杆飞行时间质谱法(HPLC-QTOF MS)对硝苯地平原料药的4种杂质进行了在线的质谱分析。色谱柱为Kromasil C18(250 mm×4.6 mm, 5 μm),流动相为甲醇-水(60:40, v/v),检测波长为235 nm。通过紫外检测器和四极杆飞行时间质谱仪在线检测,分离并检测了硝苯地平及其杂质,获得了它们的紫外光谱和质谱数据;通过比较加合质子的硝苯地平和杂质的准分子离子的碎裂特征,直接推断出了3个杂质可能的结构,其中1个为未知杂质;采用与对照品保留时间和质谱数据的比对,确定了另外1个杂质的结构。实验表明,HPLC-QTOF MS可以快速鉴定硝苯地平中杂质的化学成分。     

Analysis of ceramides in cosmetics by reversed-phase liquid chromatography/electrospray ionization mass spectrometry with collision-induced dissociation

A rapid, sensitive and selective method involving reversed-phase liquid chromatography (LC) with electrospray ionization (ESI) mass spectrometry (MS) was employed for determination of commercial ceramides in cosmetics for quality control of the product formulation. Using this LC/ESI-MS technique, simultaneous separation and characterization of ceramides and an impurity substance were possible. Informative fragmentation patterns were obtained by employing LC/ESI-MS in both positive and negative ionization modes to identify the structures of both sphingoid base and N-acyl chains of ceramides, and also of an impurity. The combination of positive and negative mass spectra can be used for unambiguous confirmation of ceramides and for characterization of unknown species. In-source collision-induced fragmentation resulted in characteristic product anions for the ceramides containing a phytosphingosine moiety at m/z 267, 255 and 225, and for those with a sphingosine moiety at m/z 263 and 237, regardless of the length of the fatty acyl chains. The detection limit was about 0.5 pmol in selected-ion monitoring mode. Quantification using internal standards showed good linearity and a relative standard deviation of 4%. These ceramides were more sensitively detected in positive than in negative ion mode.     

A concise review of bioanalytical methods of small molecule immuno‐oncology drugs in cancer therapy

Immuno‐oncology (IO) is an emerging option to treat cancer malignancies. In the last two years, IO has accounted for more than 90% of the new active drugs in various therapeutic indications of oncology drug development. Bioanalytical methods used for the quantitation of various IO small molecule drugs have been summarized in this review. The most commonly used are HPLC and LC–MS/MS methods. Determination of IO drugs from biological matrices involves drug extraction from the biological matrix, which is mostly achieved by simple protein precipitation, liquid–liquid extraction and solid‐phase extraction. Subsequently, quantitation is usually achieved by LC–MS/MS, but HPLC–UV has also been employed. The bioanalytical methods reported for each drug are briefly discussed and tabulated for easy access. Our review indicates that LC–MS/MS is a versatile and reliable tool for the sensitive, rapid and robust quantitation of IO drugs.     

LC–MS/MS and NMR characterization of forced degradation products of mirabegron

A rapid, precise, and reliable liquid chromatography tandem mass spectrometry (LC–MS/MS) method has been developed for the characterization of stressed degradation products of mirabegron. It is used in the treatment of overactive bladder and administered to treat urinary symptoms such as urgency or frequency and incontinence. It also works by relaxing the muscles around bladder.

Mirabegron was subjected to hydrolysis (acidic, alkaline, and neutral) and peroxidation, as per ICH-specified conditions. The drug showed degradation under stress conditions. However, it was stable to neutral conditions. A total of seven degradation products were observed and the chromatographic separation of the drug and its degradation products was achieved on X-TerraRP-8 (250 mm × 4.6 mm, i.d., 5 µm) column using 0.01 M ammonium acetate as mobile phase-A and 60:40 ratio of acetonitrile (ACN):water as mobile phase-B. The degradation products were characterized by LC–MS/MS and its fragmentation pathways were proposed. Probable possible structures were drawn based on parent and daughter molecular ions. One peroxide degradant impurity was isolated using preparative LC and characterized using liquid chromatography–mass spectrometry and NMR data.     

Analysis of oxidized epigallocatechin gallate by liquid chromatography/mass spectrometry

(-)-Epigallocatechin gallate (EGCG) of catechins changes from non-colored at around neutral pH to yellow at higher pH region in aqueous solution. The pH-dependent oxidation of EGCG was analyzed by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). LC/MS/MS analysis of EGCG and its related compounds, (-)-epicatechin gallate (ECG) and (-)-epigallocatechin (EGC), successfully elucidated the structure relationship of EGCG solution involving the color change reaction at different pH conditions. The oxidation species produced at alkaline pH was detected at a different retention time from EGCG in the chromatograms of the EGCG sample. The oxidation species was found to correspond to M+14 (where M is the molecular weight of EGCG), which has two hydrogen atoms removed and addition of one oxygen atom to the gallyl moiety in the B-ring of EGCG.     

Characterization of a novel trace-level impurity in lisinopril using multi-stage mass spectrometry

An impurity in the bulk drug lisinopril was detected by simple reversed-phase high-performance liquid chromatography (HPLC). This trace-level impurity was rapidly identified as 2-(2-oxo-azocan-3-ylamino)-4-phenyl- butyric acid on the basis of the on-line multi-stage mass spectrometric evidence, and the proposed structure was further confirmed by multi-stage mass spectrometry of lisinopril and three related compounds.     

Synthesis,characterization and control of eight process-related and two genotoxic fingolimod impurities by a validated RP-UPLC method

An HPLC method has been described in the European Pharmacopoeia and United States Pharmacopeia for the determination of nine organic impurities (imp A–I) in fingolimod hydrochloride, a synthetic sphingosine-1-phosphate receptor modulator. The manufacturing process of fingolimod hydrochloride consists of multistep chemical synthesis wherein controls of precursors, intermediates and process steps should be performed to assure the final quality of the drug substance. We synthesized and isolated eight process-related impurities (FINI imp A–H) of fingolimod, which were different from the pharmacopoeial impurities. One unknown process-related impurity was found as a key intermediate (FINI) and was identified by LC–MS. Characterization of all of the impurities were done using spectroscopic techniques (¹H and ¹³C NMR, FTIR, MS), and the mechanistic pathways to the formation of these impurities were also discussed. Two of these impurities were evaluated as potential genotoxic impurities owing to their alerting structures and alkylating properties (alkyl sulfonates and alkyl halides, class 3, ICH M7). We also developed and validated an RP-UPLC method in line with ICH Q2 guidelines for control these impurities (FINI imp A–H) and to assure the pharmacopoeial quality drug substance.     

Determination of in vitro permeability of drug candidates through a caco-2 cell monolayer by liquid chromatography/tandem mass spectrometry

Studying the permeability of compounds across a Caco-2 cell monolayer is an established in vitro model to screen for oral absorption and to evaluate the mechanism of transport. This assay can also be used to evaluate compounds as potential P-glycoprotein substrates and/or inhibitors. The traditional methods of sample analysis (high-performance liquid chromatography (HPLC) with a UV or fluorescence detector) limit the throughput and sensitivity of this assay. Data are presented here describing the use of liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the analysis of samples derived from the Caco-2 cell studies. During the analysis an automatic switching valve was used to divert the flow from the HPLC column to waste for the first minute, preventing the early eluting salts from entering and contaminating the LC/MS interface. This approach allows the rapid and accurate determination of drug transport across the Caco-2 cell monolayer. The high sensitivity and specificity of LC/MS/MS make this technique an ideal candidate for the low concentration and high throughput routine analysis of Caco-2 cell solutions, especially if multiple compounds are administered and analyzed simultaneously. Thus, the use of LC/MS/MS will increase the value of the Caco-2 cell assay as an in vitro screening tool.     

Comparison of LC and LC/MS methods for quantifying <Emphasis Type="Italic">N</Emphasis>-glycosylation in recombinant IgGs

High-performance liquid chromatography (LC) and liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-MS) methods with various sample preparation schemes were compared for their ability to identify and quantify glycoforms in two different production lots of a recombinant monoclonal IgG1 antibody. IgG1s contain a conserved N-glycosylation site in the fragment crystallizable (Fc) subunit. Six methods were compared: (1) LC/ESI-MS analysis of intact IgG, (2) LC/ESI-MS analysis of the Fc fragment produced by limited proteolysis with Lys-C, (3) LC/ESI-MS analysis of the IgG heavy chain produced by reduction, (4) LC/ESI-MS analysis of Fc/2 fragment produced by limited proteolysis and reduction, (5) LC/MS analysis of the glycosylated tryptic fragment (293EEQYNSTYR301) using extracted ion chromatograms, and (6) normal phase HPLC analysis of N-glycans cleaved from the IgG using PNGase F. The results suggest that MS quantitation based on the analysis of Fc/2 (4) is accurate and gives results that are comparable to normal phase HPLC analysis of N-glycans (6).     

Analysis of urinary nucleosides. I. Optimisation of high performance liquid chromatography/electrospray mass spectrometry

In order to optimise the analysis of urinary nucleosides by high performance liquid chromatography/mass spectrometry (HPLC/MS), the HPLC separation of these compounds was performed at different 'flow rates' and 0.2mL/min was found to give both a better separation and ionisation. The ionisation conditions were optimised to give the best intensity of the molecules quasi-molecular ions. The ion distribution profile and ionisation in both positive and negative mode were examined and the detection of the protonated molecule in positive mode chosen for further analysis. The limits of detection of the method developed are reported and representative LC/MS and LC/MS/MS spectra shown. Typical urinary nucleoside chromatograms are presented.