Characterization of impurities in dirithromycin by liquid chromatography/ion trap mass spectrometry

With a recently developed liquid chromatographic (LC) method, using a phosphate buffer, several unknown impurities present in dirithromycin samples were separated. In this paper, a reversed-phase liquid chromatography-tandem mass spectrometry method is described for the investigation of dirithromycin and related substances. The method employed uses a Zorbax Extend C18 column (250 mm x 4.6 mm I.D.), 5 microm, and a mobile phase consisting of acetonitrile, 2-propanol, water and ammonium acetate solution pH 8.5. Mass spectral data are acquired on an LCQ ion trap mass spectrometer equipped with an electrospray ion (ESI) source operated in the positive ion mode. The LCQ is ideally suited for the identification of related substances because it provides on-line LC/MS(n) capability, which allows efficient identification without time-consuming isolation and purification procedures. Using this method, the fragmentation behavior of dirithromycin and its related substances was studied and the unknown impurities occurring in commercial samples were investigated. In total the structures of nine impurities were elucidated, among which three were different analogues with a modification in the side chain on the oxazine ring. Two impurities showed a different alkyl group in position C13. In two impurities the desosamine sugar was involved with changes in the degrees of methylation of the amino group. One unknown impurity was identified as dirithromycin F and another unknown was characterized as dirithromycin N-oxide.     

Characterization of impurities in spiramycin by liquid chromatography/ion trap mass spectrometry

A reversed-phase liquid chromatography/tandem mass spectrometry method is described for the investigation of spiramycin and related substances. The method uses an XTerra C18 column (250 x 4.6 mm i.d.), 5 microm, and a mobile phase consisting of acetonitrile, methanol, water and ammonium acetate solution, pH 6.5. Mass spectral data were acquired on an LCQ ion trap mass spectrometer equipped with atmospheric pressure chemical ionization (APCI) operated in the positive ion mode. Using this method, the fragmentation behavior of spiramycin and its related substances was studied and the unknown impurities occurring in commercial samples were investigated. In total 17 compounds were identified, among which three reported as specified impurities in the European Pharmacopoeia. The other impurities showed mainly a modification in the forosamine sugar or in the substituent at C-3 and C-6 positions. In one impurity, the mycarose sugar is absent.     

Characterization of amiodarone metabolites and impurities using liquid chromatography/atmospheric pressure chemical ionization mass spectrometry

Using the high performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry (HPLC/APCI-MS/MS) technique, together with established trends from the literature, the structures of metabolites and impurities of amiodarone, an anti-arrhythmic drug, have been assigned. By comparing analyses of products of incubation with rat liver microsomes with controls in which glucose 6-phosphate dehydrogenase was omitted, metabolites could be distinguished from impurities. Structures for the two proposed metabolites and four impurities are proposed.     

Characterization of explosives by liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry using electrospray ionization and parent-ion scanning techniques

Analytical techniques for the detection of small amounts of explosives (in the picogram range) are now involved in various application. Some of them concern soil, water and air monitoring in order to face environmental problems related to improper handling procedures either in stocking or in wasting of the explosive products. Other areas are strictly related to forensic analysis of samples coming either from explosion areas where the matrix is various (metal, glass, wood, scraps), or from explosives transportation related to international terrorism. Generally speaking, for these applications the bulk of the matrix seriously interferes in the detection of the explosive analyte, which is usually present at trace levels. Unfortunately, despite some improvements, analytical techniques developed up today in this domain are still faced to two main constraints: the introduction of new products with unanticipated chemico-physical properties and the requirement of a routine and fast analytical method which can handle any matrix with a minimal clean-up and performing a sensitivity compatible either with the ever-decreasing demanded detection limit and with the ever-decreasing available specimen amount. These requirements can be fulfilled now by the new LC-MS and LC-MSMS techniques: mass spectrometry (MS) is likely an universal detector but even specific, especially when implemented in tandem MS (MSMS); LC is by far the most suitable technique to handle such a kind of compounds. Moreover, of a particular concern are some explosives which are reported to be thermally stable but difficult to dissolve. Some of the experiments on characterization of explosives [Octagen (HMX), Ethyleneglycol dinitrate (EGDN), Exogen (RDX), Propanetriol trinitrate (NG), Trinitrotoluene (TNT), N-Methyl-N-tetranitrobenzenamine (TETRYL), Dintrotoluene (DNT), Bis-(nitrooxy-methyl) propanediol dinitrate (PETN), Hexanitrostilbene (HNS), Triazido-trinitrobenzene (TNTAB), Tetranitro-acridone (TENAC), Hexa-nitrodiphenylamine (HEXYL), Nitroguanidine (NQ)] by LC-MS and LC-MSMS with the API-IonSpray source and using the Parent-Scan technique are presented.     

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.     

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.     

Characterization of boldione and its metabolites in human urine by liquid chromatography/electrospray ionization mass spectrometry and gas chromatography/mass spectrometry

Boldione (1,4-androstadiene-3,17-dione) is a direct precursor (prohormone) to the anabolic steroid boldenone (1,4-androstadiene-17beta-ol-3-one). It is advertised as a highly anabolic/androgenic compound promoting muscularity, enhancing strength and overall physical performance, and is available on the Internet and in health stores. This work was undertaken to determine and characterize boldione and its metabolites in human urine, using both liquid chromatography with electrospray ionization mass spectrometry and gas chromatography with mass spectrometry and derivatization. Boldione and its three metabolites were detected in dosed human urine after dosing a healthy volunteer with 100 mg boldione. The excretion studies showed that boldione and its metabolites were detectable in urine for 48 h after oral administration, with maximum excretion rates after 1.8 and 3.6 h (boldenone case). The amounts of boldione and boldenone excreted in urine from this 100 mg dose were 34.45 and 15.95 mg, respectively.     

Characterization of dehydroascorbic acid solutions by liquid chromatography/mass spectrometry.

The composition of a commercial dehydroascorbic acid (DA) solution at pH 2 was investigated by liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) to establish the nature of its different forms and its decomposition products. In freshly prepared solutions, dimeric forms of DA and the hydrated bicyclic hemiketal of DA are the species mainly present in solution. In the presence of light, the initial dimeric species disappears over time to give other dehydrated dimers some of which decompose to the monomer. The comparison of these data with similar data obtained for ascorbic acid (AA) solutions under the same experimental conditions revealed that, in the presence of light, the aging of such AA solutions gives rise to only the hemiketal form of DA, and that no dimeric species of DA were formed. The presence of the hemiketal form of DA was not revealed by analysis of the same AA solutions using the conventional LC/UV technique. The natural form of DA from the oxidation of AA is the hydrated bicyclic form.     

Characterization of metabolites of Celecoxib in rabbits by liquid chromatography/tandem mass spectrometry

The metabolism of the anti-inflammatory drug Celecoxib in rabbits was characterized using liquid chromatography (LC)/tandem mass spectrometry (MS/MS) with precursor ion and constant neutral loss scans followed by product ion scans. After separation by on-line liquid chromatography, the crude urine samples and plasma and fecal extracts were analyzed with turbo-ionspray ionization in negative ion mode using a precursor ion scan of m/z 69 (CF(3)) and a neutral loss scan of 176 (dehydroglucuronic acid). The subsequent product ion scans of the [M - H] ions of these metabolites yielded the identification of three phase I and four phase II metabolites. The phase I metabolites had hydroxylations at the methyl group or on the phenyl ring of Celecoxib, and the subsequent oxidation product of the hydroxymethyl metabolite formed the carboxylic acid metabolite. The phase II metabolites included four positional isomers of acyl glucuronide conjugates of the carboxylic acid metabolite. These positional isomers were caused by the alkaline pH of the rabbit urine and were not found in rabbit plasma. The chemical structures of the metabolites were characterized by interpretation of their product ion spectra and comparison of their LC retention times and the product ion spectra with those of the authentic synthesized standards.     

Characterization of glutathione conjugates of chloroacetanilide pesticides using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry and liquid chromatography/ion trap mass spectrometry

Glutathione S-transferases (GSTs) isolated from maize were used to catalyze the conjugation of glutathione (GSH) with chloroacetanilide herbicides, producing stable conjugates that were structurally characterized using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/QqToF-MS) and liquid chromatography/ion trap mass spectrometry (LC/IT-MS). Enzyme-mediated dechlorination of alachlor, metolachlor, and propachlor resulted during GSH conjugation as revealed by the mass spectra of the conjugates, which was confirmed by the loss of the chlorine isotopic signature and from high accurate mass measurements. Several fragmentation patterns in the mass spectra of the chloroacetanilide-GSH conjugates can be used to verify the identities of the enzyme reaction products, such as characteristic ions corresponding to the neutral loss of glutamic acid residue (129 Da) and water (18 Da) observed in the product ion spectrum. For the first time, data are presented showing detection of chloroacetanilides that are conjugated with two GSH molecules, in addition to the known single GSH conjugates.     

Monitoring stevioside in soju by high-performance liquid chromatography and liquid chromatography/mass spectrometry

A method using high-performance liquid chromatography (HPLC) with UV absorption detection was developed to monitor stevioside in soju, a distilled spirits product that is commercially available. The method uses a single-step dilution for sample preparation. It completely eliminates the time-consuming process of solid-phase extraction. A method using HPLC/mass spectrometry was optimized to confirm the identities of stevioside and other related impurities, including rebaudioside A, rebaudioside C, and dulcoside. The method was validated. The validation parameters included range (10.1-1007.3 ppm), precision, linearity, accuracy, robustness, system suitability, and intermediate precision. Stevioside standard solutions at 6 concentration levels were prepared for the validation work, including the tests for precision, linearity, and accuracy. The solutions were prepared in triplicate for each concentration. The relative standard deviation for the precision test was <3% for all 6 concentration levels. The correlation coefficient for the linearity within the concentration range was determined to be > 0.999. The average recovery ranged from 95.7 to 101.1% for the soju samples spiked with stevioside standard. The detection limit for stevioside was estimated at 75 ppb. The method was used to screen several soju samples; no detectable stevioside was found in the samples.     

Applicability of mass spectrometry to detect coeluting impurities in high-performance liquid chromatography

This paper describes the results of selectivity optimization and internal standard prediction for the quantitation of estradiol and levonorgestrel in transdermal patches by reversed-phase liquid chromatography (RPLC) based on the linear solvation energy relationships (LSERs). The patch samples are prepared by swelling with acetonitrile (ACN) and the separation is performed by Zorbax Eclipse XDB ODS columns. A proper retention range is first determined with a binary mobile phase of ACN and water based on the general resolution equation. The interference to estradiol from a levonorgestrel impurity is then eliminated by a ternary mobile phase of acetonitrile-methanol-water with a composition predicted by LSERs. When the resolution is optimized and the "open window" in the chromatogram for an internal standard is selected, LSERs are used to predict the candidate compounds to be evaluated as the internal standard. The approach described in this study can be used, in general, to considerably improve the efficiency of RPLC method development, particularly for neutral samples. Finally, the LSER approach for the selectivity optimization is compared with a statistical response surface methodology (RSM) based on a central composite design (CCD) in terms of the effectiveness and number of experiments. It is concluded that, although the predicted mobile phase composition to achieve the desired selectivity is about the same, the LSER approach is more efficient and fewer experiments are required.     

Determination of steroids by liquid chromatography/mass spectrometry

On-line atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) liquid chromatography/mass spectrometry (LC/MS) were evaluated for the analysis of a variety of steroids. Steroids were classified into three major groups based on the spectra and the sensitivities observed: (I) those containing a 3-one, 4-ene functional group, (II) those containing at least one ketone group without conjugation, and (III) those containing hydroxy group(s) only. In the APCI mode, the best sensitivity and the lowest detection limit for all three groups were obtained by using a mobile phase consisting of methanol and 1%–2% acetic acid in water. The APCI spectra were characterized by MH⁺, MH⁺-H₂O, MH⁺-2H₂O, etc., with the degree of H₂O loss being compound dependent: group I steroids produced stable MH⁺ and group III steroids showed extensive water loss. In the electrospray mode the best sensitivity and the lowest detection limit for the first two groups were obtained when pure methanol and water were used as the mobile phase. This condition produced abundant stable MNa⁺ due to ubiquitous sodium. Detection limits in the 5–15 pg range can be easily achieved using ESI LC/MS. Addition of ammonium acetate or use of acetonitrile in the mobile phase, common in the LC/MS analysis of steroids, decreased the sensitivity for the group I and II steroids and thus should be avoided. For group III steroids, the detection limit can be improved by the addition of acetic acid to the mobile phase.     

Characterization of metabolites of tanshinone IIA in rats by liquid chromatography/tandem mass spectrometry

The metabolism of tanshinone IIA was studied in rats after a single-dose intravenous administration. In the present study, 12 metabolites of tanshinone IIA were identified in rat bile, urine and feces with two LC gradients using LC-MS/MS. Seven phase I metabolites and five phase II metabolites of tanshinone IIA were characterized and their molecular structures proposed on the basis of the characteristics of their precursor ions, product ions and chromatographic retention time. The seven phase I metabolites were formed, through two main metabolic routes, which were hydroxylation and dehydrogenation metabolism. M1, M4, M5 and M6 were supposedly tanshinone IIB, hydroxytanshinone IIA, przewaquinone A and dehydrotanshinone IIA, respectively, by comparing their HPLC retention times and mass spectral patterns with those of the standard compounds. The five phase II metabolites identified in this research were all glucuronide conjugates, all of which showed a neutral loss of 176 Da. M9 and M12 were more abundant than other identified metabolites in the bile, which was the main excretion path of tanshinone IIA and the metabolites. M12 was the main metabolite of tanshinone IIA. M9 and M12 were proposed to be the glucuronide conjugates of two different semiquinones and these semiquinones were the hydrogenation products of dehydrotanshinone IIA and tanshinone IIA, respectively. This hydrogenized reaction may be catalyzed by the NAD(P)H: quinone acceptor oxidoreductase (NQO). The biotransformation pathways of tanshinone IIA were proposed on the basis of this research.     

Characterization of carbofuran photodegradation by-products by liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry

The structural elucidation of by-products arising from carbofuran photodegradation using a high-pressure UV lamp has been investigated by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) employing a quadrupole time-of-flight mass spectrometer. Exact mass measurements of the [M + H]+ ions of the by-products and of product ions allowed the elemental formulae and related structures of seven photodegradation by-products (resulting, respectively, from photo-Fries rearrangement, hydroxylation of the benzene ring, oxidation of the 2,3-dihydrobenzofuran ring, cleavage of the carbamate group, hydrolysis of the ether group and the newly observed radical coupling and decarboxylation processes) to be determined confidently. Accurate mass measurements of product ions allowed ambiguities to be removed concerning neutral losses having the same nominal mass, namely CO and C2H4, allowing the fragmentation patterns to be rationalized.     

Determination of chlormequat and mepiquat in foods by liquid chromatography/mass spectrometry or liquid chromatography/tandem mass spectrometry: interlaboratory study

Interlaboratory validation studies have been performed on 2 methods for the determination of chlormequat (CLQ) and mepiquat (MPQ). Both methods used identical extraction procedures and stable isotope internal standardization but differed in the use of liquid chromatography/mass spectrometry (LC/MS) or LC/tandem mass spectrometry (LC/MS/MS) for the determination, the amount of internal standard used, and the expected limit of detection. After addition of deuterated internal standards, CLQ and MPQ were extracted with methanol-water and determined by LC//MS or LC/MS/MS with positive electrospray ionization. Eight European laboratories participated in the LC/MS method study, analyzing mushroom, pear, wheat flour, and fruit puree with residues of CLQ in the range 0.040-1.19 mg/kg and of MPQ in the range 0.041-0.39 mg/kg. For CLQ, the Horwitz ratio (HoRat) values for individual test materials/levels were in the range 0.85-1.13 with a mean of 1.00, showing good method performance. For MPQ, the Ho values for mushroom, pear (both levels), and wheat flour were in the range 0.83-0.94, again indicating good method performance. For the determination of MPQ in infant food (fruit puree) at 0.041 mg/kg, the Ho was 1.7 when a value of 0 reported by one participant was excluded. In the LC/MS/MS study, in which 11 laboratories participated, a separate sample set was analyzed with residues of CLQ in the range 0.007-1.03 mg/kg and of MPQ in the range 0.008-0.72 mg/kg. Ho values for CLQ were in the range 0.27-1.36 and for MPQ in the range 0.51-2.10, all corresponding to acceptable method performance.     

Separation and characterization of clindamycin phosphate and related impurities in injection by liquid chromatography/electrospray ionization mass spectrometry

A simple high‐performance liquid chromatography/electrospray ionization tandem mass spectrometric (HPLC/ESI‐MS/MS) method has been developed for the rapid identification of clindamycin phosphate and its degradation products or related impurities in clindamycin phosphate injection. Detection was performed by quadrupole time‐of‐flight mass spectrometry (Q‐TOFMS) via an ESI source in positive mode. Clindamycin phosphate and its related substances lincomycin, 7‐epilincomycin‐2‐phosphate, lincomycin‐2‐phosphate, clindamycin B, clindamycin B‐2‐phosphate, and clindamycin were identified simultaneously by HPLC/ESI‐MS/MS results. Based on the MS/MS spectra of their quasi‐molecular ions, the fragmentation pathways of clindamycin phosphate and its related substances were compared and proposed, which are specific and useful for the identification of the lincosamide antibiotics and related impurities. The method was rapid, sensitive and specific and can be used to identify clindamycin phosphate and its related impurities in clindamycin phosphate injection without control compounds. Copyright © 2009 John Wiley & Sons, Ltd.     

Detection of sulfur-containing impurities in pharmaceutical samples by high performance liquid chromatography/chemical reaction interface mass spectrometry

This paper describes the use of chemical reaction interface mass spectrometry (CRIMS) combined with liquid chromatography for the detection of trace level sulfur-containing impurities in pharmaceutical materials. A mixture of sulfur- and non-sulfur-containing compounds were analyzed initially to test the system. Then the determination of trace level impurities in a cimetidine drug substance was carried out. Detection of sulfur-containing impurities at less than 0.1% of the major component was obtained with good linearity. The results obtained are consistent with the expected results for this sample and illustrate the applicability of the technique.     

Characterization of low-molecular weight peptides in champagne wine by liquid chromatography/tandem mass spectrometry

This paper reports the use of an on-line LC–ESI–MS/MS method for the identification and quantification of di- and tripeptides in champagne wine without laborious sample pretreatment. The identification of these compounds, in their underivatised form, is based on identical retention times and ESI–MS spectra to those of reference standards. The presence of nine dipeptides (Arg–Ile, Ile–Arg, Ile–Val, Lys–Phe, Lys–Tyr, Phe–Lys, Tyr–Gln, Tyr–Lys, Val–Ile) and the absence of two tripeptides (Phe–Arg–Arg and Lys–Met–Asn) have been evidenced in the matrix. Calibration curves for each analyte were established using Phe–Arg as internal standard. The calibration curves were linear in the concentration range 0.1–10 mg L⁻¹ with a determination coefficient, r², better than 0.992. The accuracy for the calibration standard was estimated at between 92 and 102%. This method allows high recovery and satisfies the necessary requirements with respect to accuracy, repeatability and sensitivity. The first application of this analytical method to the measurement of di- and tri-peptides in different vintages of champagne wine is reported. Compositional changes in the peptides occurred depending on the vintage.