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.     

Identification of major metabolites in rat urine and plasma of N(6) -(4-hydroxybenzyl) adenine riboside by LC/MS/MS

N(6) -(4-hydroxybenzyl) adenine riboside, a novel neuroprotective compound found in Gastrodia elata at trace level, is regarded as a potential drug for the treatment of neural degenerative disease. To understand the metabolism of this compound, the metabolites in rat urine and plasma of N(6) -(4-hydroxybenzyl) adenine riboside were analyzed by HPLC-ESI-MS/MS after oral administration of this compound. Beside the parent compound, six phase I metabolites and four phase II metabolites in urine were detected by scanning all possible metabolites in extracted ion chromatograms mode. By comparing their product ion spectra and retention times with those of parent compound, these metabolites were identified and proved to be mainly formed via hydrolysis or hydroxylation in phase I, N-sulfation or N-glucuronidation in phase II or their combinations. Similarly, the parent compound, one phase I metabolite and two phase II metabolites were also identified in rat plasma. Therefore, the in vivo metabolic pathways of N(6) -(4-hydroxybenzyl) adenine riboside in rat were proposed.     

Enantiomer identification in the flavour and fragrance fields by "interactive" combination of linear retention indices from enantioselective gas chromatography and mass spectrometry

This study describes the development of a gas chromatography-mass spectrometry (GC-MS) library to identify optically active compounds in the flavour and fragrance field using enantioselective GC with cyclodextrin derivatives (CDs) as chiral selectors in combination with MS. The library operates on the "interactive" combination of linear retention indices (I(T) values) in parallel to MS spectra, so as to identify enantiomers reliably and to measure EE and/or ER unequivocally. Since MS is not a selective probe to discriminate optical isomers, mass spectra (or diagnostic ions in SIM mode) are used to locate the enantiomer(s) in the chromatogram, and I(T) values to identify it(them) safely and reliably in particular in complex mixtures. The library has been built up through the following steps: Some applications of the library are also reported. (a) Selection of CD derivatives able to cover a wide range of racemate separations. Four cyclodextrin derivatives were used: 2,6-di-O-methyl-3-O-pentyl-beta-CD, 2,3-di-O-methyl-6-O-tert-butyldimethylsilyl-beta-CD, 2,3-di-O-ethyl-6-AO-tert-butyldimethylsilyl-beta-CD, and 2,3-di-O-acetyl-6-O-tert-butyldimethylsilyl-beta-CD. (b) Determination of the analytes' I(T) values and evaluation of their stability and reliability at both intra- and inter-laboratory level. (c) Determination of the range within which the I(T) of an enantiomer has to be correctly identified, i.e. determination of a common retention index allowance (RIA). (d) Construction of the library, at the moment comprising the enantiomers of 134 racemates. A record has been attributed to each enantiomer including I(T) values determined on the four CD coated columns, mass spectrum, IUPAC chemical names, CAS numbers, molecular weight, and, when separated, racemate enantiomer resolution on the CD investigated. Some applications of the library are also reported.     

Confirmatory analysis of sulfonamide antibacterials in bovine liver and kidney: extraction with hot water and liquid chromatography coupled to a single- or triple-quadrupole mass spectrometer

A simple, specific, and rapid confirmatory method for determining 12 sulfonamide (SAs) antibacterials in bovine liver and kidney is presented. This method is based on the matrix solid-phase dispersion technique with hot water as extractant followed by liquid chromatography/mass spectrometry (LC/MS) with an electrospray ion source. The method was tailored for use with both single-quadrupole MS (I) and triple-quadrupole MS (II) instruments. After acidification and filtration of the aqueous extract, a 250-microL aliquot was injected into instrument I while only 25 microL was analyzed by instrument II. With instrument I MS data acquisition was performed in the selected ion monitoring (SIM) mode, selecting at least three ions for each target compound. With instrument II the selected reaction monitoring (SRM) mode with three fragmentation reactions for each compound was chosen. With the exception of sulfaquinoxaline (SQX), recovery of the analytes at the 50 ppb level in both liver and kidney was 72-96% with relative standard deviations (RSDs) ranging between 3 and 11%. The very poor recovery of SQX was due to its rapid enzymatic oxidation when in contact with the two tissues. With instrument I, limits of quantification (LOQs, S/N = 10) were 5-14 ppb of SAs. Even lower LOQs (1-8 ppb) were estimated by using instrument II, even though the extract volume analyzed was ten times lower than that with instrument I. With both matrices and using instrument I, severe ion signal suppression was experienced for the early-eluted SAs when trying to fractionate analytes by using a short chromatographic run time. This effect was traced to polar endogenous co-extractives eluted in the first part of the chromatographic run that interfered with gas-phase ion formation for SAs. Adopting more selective chromatographic conditions minimized this effect.     

Mass spectrometric and gas and high-performance liquid chromatographic behaviour of an impurity in 2,5-hexanedione

The analysis of 2,5-hexanedione, a metabolic compound of several industrial solvents, is normally carried out using gas chromatographic (GC) or GC-mass spectrometric (MS) techniques. This work, with the aim of verifying the possibility of determining the diketone by means of a high-performance liquid chromatographic (HPLC) method with UV detection, illustrates the importance of the choice of a 2,5-hexanedione standard for the determination of the diketone response factor. In some commercial diketone samples the presence of an impurity, which may interfere with the analysis of the target analyte, was ascertained. This impurity showed GC and HPLC behaviour similar to that of 2,5-hexanedione, but gave a very different UV response. This impurity was identified as 3-methylcyclopent-2-enone by means of MS, GC-MS, HPLC-photodiode-array detection, IR and UV spectrometry. The structure was confirmed by comparing the chromatographic, mass and ultraviolet data of the unknown compound with those of a synthesized reference sample. The well known difficulty in determining 2,5-hexanedione by HPLC with UV detection was reconfirmed owing to its low molar absorptivity.     

Determination of midazolam and its major hydroxy metabolite in plasma by gas liquid chromatography. Application to a pharmacokinetic study

A sensitive gas liquid chromatographic (GLC) assay was developed for plasma determinations of 8-chloro-6-(2′ -fluorophenyl)-1-methyl-4H-imidazo[1,5 α][1,4]benzodiazepine (compound I) and its hydroxymethylimidazo metabolite (compound II). The internal standards used were 8-chloro-6-(2′ -chlorophenyl)-1 -methyl-4H-imidazo[1,5 α][1,4]benzodiazepine (compound VI) and 7-chloro-5-(2′ -fluorophenyl)-1, 3-dihydro-1-(hydroxyethyl)-2H-1,4-benzodiazepin-2-one (compound VII) for compounds I and II, respectively. Following extraction, and silylation for compound II, compounds I and II were analyzed by GLC using a glass column packed with 5% OV-101 on Gas-Chrom Q, and a ⁶³Ni electron-capture detector. The GLC method was validated by a CI-GC/MS technique. The detection limit of the assay is approximately 4–5 ng/ml for compound I and 3 ng/ml for compound II. The method was used in comparative pharmacokinetic studies of the distribution of the two compounds in arterial and venous blood.     

Photostabilization of commercial polypropylene by hindered piperidine compounds: An ESR and luminescence study

The photostabilization of commercial polypropylene by a hindered piperidine stabilizer, bis [2,2,6,6-tetramethyl-4-piperidinyl] sebacate (I) and by a model N-oxy radical compound, 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxy (II) is examined using ESR and luminescence spectroscopy. ESR spectroscopy shows that I operates through the formation of a stable nitroxyl radical in the polymer. On the other hand, II disappears rapidly during the early stages of photo-oxidation but continues to act as an effective stabilizer. A low steady-state equilibrium concentration of nitroxyl radicals is believed to be responsible for the high photostabilizing efficiency of I. Both compounds also inhibit the photolysis of the luminescent α,β-unsaturated impurity groups present in the polymer; possible mechanisms are discussed.     

Identification and quantitation of cis-ketoconazole impurity by capillary zone electrophoresis-mass spectrometry

trans-Ketoconazole was identified and quantified as impurity of cis-ketoconazole, an antifungal compound, by capillary zone electrophoresis-electrospray-mass spectrometry (CZE-ESI-MS). The chirality of this impurity was demonstrated separating their enantiomers by adding heptakis-(2,3,6-tri-O-methyl)-beta-cyclodextrin to the separation buffer in capillary electrophoresis (CE) with UV detection. However, MS detection was hyphenated to the CE instrument for its identification. As both compounds are diastereomers, they have the same m/z values and are needed to be separated prior to the MS identification. A 0.4M ammonium formate separation buffer at pH 3.0 enabled the separation of the impurity from cis-ketoconazole. Under these conditions, the optimization of ESI-MS parameters (composition and flow of the sheath-liquid, drying temperature, drying gas flow, and capillary potential) was carried out to obtain the best MS sensitivity. CZE-ESI-MS optimized conditions enabled the identification of trans-ketoconazole as impurity of cis-ketoconazole. In addition, the quantitation of this impurity was achieved in different samples: cis-ketoconazole standard and three different pharmaceutical formulations (two tablets and one syrup) containing this standard. In all cases, percentages higher than 2.0 were determined for the impurity. According to ICH guidelines, these values required the identification and quantitation of any impurity in drug substances and products.     

Correlation between isoniazid resistance and superoxide reactivity in mycobacterium tuberculosis KatG

Isoniazid is an antituberculosis prodrug that requires activation by the catalase-peroxidase (KatG) of Mycobacterium tuberculosis. The activated species, presumed to be an isonicotinoyl radical, couples to NADH forming an isoniazid-NADH adduct that ultimately confers antitubercular activity. We have compared the catalytic properties of three KatGs associated with isoniazid resistance (resistance mutation KatGs, (RM)KatGs: R104L, H108Q, S315T) to wild-type enzyme and two additional lab mutations (wild-type phenotype KatGs, (WTP)KatGs: WT KatG, Y229F, R418L). Neither catalase nor peroxidase activities, nor the presence/absence of the Met-Tyr-Trp cross-link (as probed by LC/MS on tryptic digests of the protein), exhibited any correlation with isoniazid resistance. The yields of isoniazid-NADH adduct formed were determined to be 1-5, 4-12, and 20-70-fold greater for the (WTP)KatGs than the (RM)KatGs for the compound I, II, and III pathways, respectively, strongly suggesting a role for oxyferrous KatG (supported by superoxide consumption measurements) that correlates with drug resistance. Stopped-flow UV-visible spectroscopic studies revealed that all KatGs were capable of forming both compound II and III intermediates. Rates of compound II decay were accelerated 4-12-fold in the presence of isoniazid (vs absence) for the (WTP)KatGs but were unaffected by the drug for the (RM)KatGs. A mechanism for isoniazid resistance which accounts for the observed reactivity for each of the compound I, II, and III intermediates is proposed and suggests that the compound III pathway may be the primary factor in determining overall isoniazid resistance by specific KatG mutants, with secondary contributions arising from the compound I and II pathways.     

Assessing the suitability of capillary electrophoresis‐mass spectrometry for biomarker discovery in plasma‐based metabolomics

The actual utility of capillary electrophoresis‐mass spectrometry (CE‐MS) for biomarker discovery using metabolomics still needs to be assessed. Therefore, a simulated comparative metabolic profiling study for biomarker discovery by CE‐MS was performed, using pooled human plasma samples with spiked biomarkers. Two studies have been carried out in this work. Focus of study I was on comparing two sets of plasma samples, in which one set (class I) was spiked with five isotope‐labeled compounds, whereas another set (class II) was spiked with six different isotope‐labeled compounds. In study II, focus was also on comparing two sets of plasma samples, however, the isotope‐labeled compounds were spiked to both class I and class II samples but with concentrations which differ by a factor two between both classes (with one compound absent in each class). The aim was to determine whether CEMS‐based metabolomics could reveal the spiked biomarkers as the main classifiers, applying two different data analysis software tools (MetaboAnalyst and Matlab). Unsupervised analysis of the recorded metabolic profiles revealed a clear distinction between class I and class II plasma samples in both studies. This classification was mainly attributed to the spiked isotope‐labeled compounds, thereby emphasizing the utility of CE‐MS for biomarker discovery.     

Identification of a bioactive impurity in a commercial sample of 6-methyl-2-p-tolylaminobenzo[d][1,3]oxazin-4-one (URB754)

The compound URB754 was recently identified as a potent inhibitor of the endocannabinoid-deactivating enzyme monoacylglycerol lipase (MGL) by screening of a commercial chemical library. Based on HPLC/MS, NMR and EI/MS analyses, the present paper shows that the MGL-inhibitory activity attributed to URB754 is in fact due to a chemical impurity present in the commercial sample, identified as bis(methylthio)mercurane. Although this organomercurial compound is highly potent at inhibiting MGL (IC50 = 11.9 +/- 1.1 nM), its biological use is prohibited by its toxicity and target promiscuity.     

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.     

2,2'-二乙炔基-1,1'-联萘为模板构筑的新的环芳化合物的合成及其光学性质

基于2,2'-二取代的联萘衍生物在手性构型上高度稳定的特点,分别以光学活性的(R)-和(S)-2,2'-二乙炔基-1,1'-联萘为模板,设计了2个有趣的拓扑环芳分子四联萘笼状对映异构体(R,R,R,R)-2和(S,S,S,S)-2.其合成路线涉及保护基的控制导入、苯连接桥的链接、保护基的脱去以及偶合成环反应4个步骤.用MS,IR,UV-Vis,1HNMR,13CNMR和元素分析等技术对其进行了表征,并比较了其光学性质.研究结果表明,采用这种模板合成方法能够有效地获得具有单一手性的目标化合物.镜像特征的圆二色(CD)谱和比旋光度[α]D的测定结果清楚地反映了它们的对映异构关系.     

Gas chromatography/negative ion chemical ionization mass spectrometry and liquid chromatography/electrospray ionization tandem mass spectrometry quantitative profiling of N-acetylcysteine conjugates of valproic acid in urine: application in drug metabolism studies in humans

We report a GC/NICI-MS assay and a LC/ESI-MS/MS assay for the analysis of N-acetylcysteine (NAC) conjugates of (E)-2,4-diene VPA (NAC I and NAC II) identified in humans. The assay also includes the analysis of the NAC conjugate of 4,5-epoxy VPA (NAC III), an identified metabolite in rats treated with 4-ene VPA for its use in metabolic studies in animals. The highly sensitive GC/MS assay was designed to monitor selectively the diagnostic and most abundant [M - 181](-) fragment anion of the di-PFB derivatives of NAC I, NAC II, and NAC IV, the internal standard (IS) and the PFB derivative of NAC III. The higher selectivity of LC/MS/MS methodology was the basis for an assay which could identify and quantitate the underivatized conjugates simultaneously using MRM of the diagnostic ions m/z 130 and 123 arising from the CID of their protonated molecular ions [MH](+). The GC/MS assay employed liquid-liquid extraction whereas the LC/MS/MS assay used a solid-phase extraction procedure. Linearity ranges of the calibration curves were 0.10-5.0microg ml(-1) by GC/MS and 0.10-1.0microg ml(-1) by LC/MS/MS for NAC I, NAC II and NAC III (r(2) = 0.999 or better). Both assays were validated for NAC I and NAC II and provided good inter- and intra-assay precision and accuracy for NAC I and NAC II. The LOQ by LC/MS/MS was 0.1microg ml(-1), representing 1 ng of NAC I and NAC II. The same LOQ (0.1microg ml(-1)) was observed by GC/MS and was equivalent to 100 pg of each metabolite. NAC III was detected at concentrations as low as 0.01 microg ml(-1) by both methods. The total urinary excretion of the NAC conjugates in four patients on VPA therapy was determined to be 0.004-0.088% of a VPA dose by GC/MS and 0.004-0. 109% of a VPA dose by LC/MS/MS.     

Über die Enantiomerentrennung durch Verteilung zwischen flüssigen Phasen. 4. Mitteilung. Enantioselektivität der diastereoisomeren Weinsäure-dimenthylester gegenüber α-Aminoalkohol-Salzen

Separation of Enantiomers by Partition between Liquid Phases. Enantioselectivity of Diastereoisomeric Dimenthyl Tartarates towards α-Aminoalcohol Salts The enantioselectivity of the easily obtainable diastereoisomeric dimenthyl tartarates I and II towards the salts of a series of α-aminoalcohols 1 - 10 was investigated by partition between aqueous and lipophilic phase. The measured enantiomer distribution constants Q (=K_A/K_B) confirm the previously observed configurational relationships between the lipophilic tartarates and the preferentially extracted enantiomer of the hydrophilic ammonium slat. However, the (R,R)-ester I is appreciably more selective than its (S,S)-diastereoisomer II or other previously investigated esters. The tartarates I - III transport norephedrin salts through bulk lipophilic membranes with enantioselectivity comparable to that observed in partition experiments. The most enantioselective ester I can be used as an efficient mobile phase for preparative separation of norephedrine enantiomers by flash partition chromatography.     

Thorough evaluation of the validity of conventional enantio-gas chromatography in the analysis of volatile chiral compounds in mandarin essential oil: A comparative investigation with multidimensional gas chromatography

The present research is focused on the determination of the enantiomeric distribution of chiral compounds, contained in mandarin essential oils, by means of conventional chiral gas chromatography with flame ionization detection (enantio-GC-FID); the results attained were compared with those derived from heart-cutting multidimensional GC-mass spectrometry (MDGC/MS), to evaluate the reliability of the monodimensional technique as a tool for quality control. The Deans-switch MDGC system was equipped with two GC ovens, which were connected via a heated transfer line, a flame ionization detector (FID1) in the first dimension and a quadrupole MS as second-dimension detector. The a priori knowledge of potential co-elutions concerning target compounds (an enantiomer and an interfering compound), when using enantio-GC-FID, could enable the use of corrected enantiomer excess values. Correction factors could be calculated through a preliminary GC-FID analysis (using an apolar column), considering the peak areas of the known interferences. The method used for the calculation of a so-called “coelution correction factor” is described, along with some examples.     

Simultaneous determination of metformin and glipizide in human plasma by liquid chromatography-tandem mass spectrometry

A method has been developed for the simultaneous quantification of metformin (I) and glipizide (II) in human plasma. It is based on high-performance liquid chromatography with electrospray ionization tandem mass (LC-ESI-MS/MS) spectrometric detection in positive ionization mode. Phenformin (III) and gliclazide (IV) were used as internal standards for I and II, respectively. The MS/MS detection was performed in multiple reaction monitoring (MRM) mode. The precursor-product ion combinations of m/z 130 --> 71, 446 --> 321, 206 --> 60 and 324 --> 127 were used to quantify I, II, III and IV, respectively. This method was validated in the concentration ranges of 0.02-4 microg/mL for I and 0.004-0.8 microg/mL for II. It was utilized to support a clinical pharmacokinetic study after single dose oral administration of a combination of I and II.     

Impurity-profiling UPLC methods for quantitative analysis of some antiemetics formulated with pyridoxine

Cyclizine hydrochloride (CYC) and meclozine hydrochloride (MEC) are antihistaminic drugs generally co-formulated with pyridoxine hydrochloride (PYR) to treat nausea and vomiting in pregnancy. Several analytical techniques have been applied for the determination of CYC or MEC with PYR, but determination of CYC impurity; benzhydrol (BEH) or MEC impurity; or 4-chlorobenzophenone (BEP) has not been paid attention to. Therefore, micellar UPLC method is introduced for analysis of ternary mixtures containing PYR together with both CYC and BEH (mixture I) or MEC and BEP (mixture II). Chromatographic separation was achieved using a Hypersil gold C₈ column (50 × 2.1 mm, 1.9 μm) using 0.01 M sodium dodecyl sulfate modified to pH 3.5 using phosphoric acid:acetonitrile (45:55 by volume) for mixture I and 0.1% sodium dodecyl sulfate, 0.1% sodium bicarbonate adjusted to pH 2.6 by phosphoric acid:acetonitrile (47:53 by volume) for mixture II as mobile phases. The separated peaks were detected at 230 and 245 nm for mixtures I and II, respectively. The adopted methods were validated in conformance with the International Conference on Harmonization (ICH) recommendations and were properly applied in commercial pharmaceutical formulation analysis. Comprehensive ecological comparison was achieved, confirming a higher ecological value of the presented methods compared to the earlier reported methods.     

Identification and structural characterization of febuxostat metabolites in rat serum and urine samples using UHPLC–QTOF/MS

Febuxostat is a novel nonpurine type of highly selective xanthine oxidoreductase inhibitor. A rapid and sensitive ultra‐high‐performance liquid chromatography–quadrupole time‐of‐flight mass spectrometry method for simultaneous separation and determination of febuxostat and its metabolites in rat serum and urine was developed at various time points after oral administration to the rats. The febuxostat metabolites were predicted by biotransformation software and transformed to a personal compound database to quickly determine the possible metabolites from the MS1 data. The possibility of the MS/MS fragmentation was calculated by the Molecular Structure Correlator software. As a result, five phase I and two phase II metabolites in rat serum, and seven phase I and three phase II metabolites in rat urine were identified, of which four metabolites (M2, M5, M6, M7) have not been reported before. The metabolite toxicities are predicted, and the results are helpful for the design of new xanthine oxidoreductase inhibitors.