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.     

Evaluation of Sempervivum tectorum L. Flavonoids by LC and LC–MS

A reversed-phase high-performance liquid chromatography method with UV detection was developed for determination of [(N-morpholine)methylene]daunorubicin hydrochloride (DD-M) during studies of its stability. In this LC method the following were used: an RP-column, the mobile phase—acetonitrile:methanol:solution A (9:1:10 v/v/v) [solution A contains 2.88 g of sodium laurilsulfate and 1.6 mL of phosphoric acid(V)] with a flow rate of 1.4 mol L⁻¹ and quinine hydrochloride as an internal standard. The detection wavelength was 254 nm. The method was validated with regard to linearity, limit of detection, limit of quantitation, selectivity and precision. Hydrolysis of the DD-M catalyzed by hydrogen ions in hydrochloric acid and a spontaneous reaction of the DD-M degradation under the influence of the water in sodium hydroxide took place. The thermodynamic parameters of these reactions—energy, enthalpy and entropy of activation—were calculated. It was observed that a positive salt effect occurred in hydrochloric acid.

     

Determination of multiclass pesticides in food commodities by pressurized liquid extraction using GC–MS/MS and LC–MS/MS

Pressurized liquid extraction (PLE) was applied to the simultaneous extraction of a wide range of pesticides from food commodities. Extractions were performed by mixing 4 g of sample with 4 g of Hydromatrix and (after optimization) a mixture of ethyl acetate:acetone (3:1, v/v) as extraction solvent, a temperature of 100°C, a pressure of 1000 psi and a static extraction time of 5 min. After extraction, the more polar compounds were analyzed by liquid chromatography (LC), and the apolar and semipolar pesticides by gas chromatography (GC); in both cases LC and GC were coupled with mass spectrometry in tandem (MS/MS) mode. The overall method (including the PLE step) was validated in GC and LC according to the criteria of the SANCO Document of the European Commission. The average extraction recoveries (at two concentration levels) for most of the analytes were in the range 70–80%, with precision values usually lower than 15%. Limits of quantification (LOQ) were low enough to determine the pesticide residues at concentrations below or equal to the maximum residue levels (MRL) specified by legislation. In order to assess its applicability to the analysis of real samples, aliquots of 15 vegetable samples were processed using a conventional extraction method with dichloromethane, and the results obtained were compared with the proposed PLE method; differences lower than 0.01 mg kg⁻¹ were found.     

The identity confirmation of 99gTc-DMSA complexes by using NMR and HPLC–MS/MS methods

The analyses of ^99gTc-DMSA complexes prepared under alkali and acidic reactions were reported. Modern analytical, separation and spectral methods such as NMR (¹H-NMR, ¹³C-NMR, APT, COSY and HSQC) and Q-TOF HPLC–MS/MS system with ESI were employed to determine the identity and characterization of the products. The structure of ^99gTc(V)DMSA was clearly confirmed and its fragmentation path in negative and positive ionisation mode was suggested. The effect of ascorbic acid and new alternative labelling with the use of NH ₄ ^99g TcOCl₄ was examined. Surprisingly, ^99gTc(III)DMSA complex was not formed under acidic reaction conditions. ^99gTc(V)DMSA complex was the main reaction product under both experimental conditions. This result suggests the key role of ^99g/99mTc concentration during the process of radiopharmaceuticals preparation.     

Quantitative determination of cationic modified polysaccharides on hair using LC–MS and LC–MS–MS

Cationic polysaccharides containing N-hydroxypropyl-N,N,N-trimethylammonium substituents are widely used as conditioning agents for hair-care products. A sensitive method has been developed for the quantitation of these polymers. After acidic extraction from hair the polysaccharides are hydrolyzed using trifluoroacetic acid. The cationic monoglycosides are determined using liquid chromatography–tandem mass spectrometry (LC–MS–MS). The developed method is independent of hair treatment. Even hair cut from test persons after customary hair wash can be analyzed. After treatment of natural and bleached hair tresses using a real-life treatment procedure 180 g and 300 g of polymer per gram hair were quantified, respectively. Additionally the fragmentation mechanism of the cationic N-hydroxypropyl-N,N,N-trimethylammonium group during electrospray ionization was investigated. A mass loss of 60 Da in combination with loss of a single charge is observed and associated with cleavage of trimethylamine and a proton. It is assumed that this process is promoted by the anionic counter-ion which might be hydroxide in an aqueous environment.     

Determination of Fenofibric Acid in Human Plasma by LC–MS/MS and LC–UV

A sensitive, high-throughput and economic liquid chromatographic method for determination of fenofibric acid in human plasma was developed and validated by ultraviolet detection and tandem mass spectrometry, then applied in pharmacokinetic study to investigate Lipanthyl™ 200 mg MC bioavailability under food and fasting conditions. Fenofibric acid with 2-chloro fenofibric acid-d6 (internal standard) was extracted from 100 µL of human plasma by acetonitrile in a single extraction step. 25 and 2 µL from supernatant were injected onto ACE column, 50 mm, 5 micron with 4.6 mm inner diameter for LC–UV and 2.1 mm for LC–MS/MS, and both systems were eluted isocratically by water:methanol:formic acid (35:65:0.1, v/v/v), with a constant flow rate of 1 mL min⁻¹. The established calibration curve was linear between 0.05–20 µg mL⁻¹, and the within- and between-day precisions were all below 13 % in both LC–MS/MS and LC–UV systems during validation, and accuracies ranged between 91 and 112 %. Twenty-eight healthy adult subjects participated in this clinical study, and the pharmacokinetic parameters including coefficient of variation were calculated and discussed. A dramatic decrease in C _max and AUC0-72 (3.63- and 1.85-fold, respectively) were observed for Lipanthyl™ MC under fasting conditions with more variable inter subject measurements comparing to the fed state.

     

Analysis of 10 urinary BTEX metabolites using LC–MS/MS

Benzene, toluene, ethylbenzene, and xylene (BTEX) are a group of volatile organic compounds that are ubiquitous in the environment due to numerous anthropogenic sources. Exposure to BTEX poses a health hazard by increasing the risk for damage to multiple organs, neurocognitive impairment and birth defects. Urinary BTEX metabolites are useful biomarkers for the evaluation of BTEX exposure, because of the ease of sampling and their longer physiological half-lives compared with parent compounds. A method that utilizes LC–MS/MS was developed and validated for simultaneously monitoring of 10 urinary BTEX metabolites. During the sample preparation an aliquot of urine was diluted with an equal volume of 1% formic acid; internal standard solution was added, and then the sample was centrifuged and analyzed. The analytes were separated on the Kinetex-F5 column by applying a linear gradient, consisting of 0.1% formic acid and methanol. The method was validated according to the FDA Bioanalytical Method Validation Guidance for Industry. The mean method's accuracies of the spiked matrix were 81–122%; the inter-day precision ranged from 4 to 20%; the limits of quantitation were 0.5–2 μg/L. The method was used for the evaluation of baseline levels of urinary BTEX metabolites in 87 firefighters.     

Separation and identification of selenotrisulfides in epithelial cell homogenates by LC–ICP–MS and LC–ESI-MS after incubation with selenite

To elucidate how selenite is metabolised in the intestine after oral intake, it was incubated with homogenized epithelial cells from pigs. When the metabolites were analysed by LC–ICP–MS, two major selenium metabolites were separated in the supernatant from the homogenate. These metabolites were formed instantly but disappeared within 15 min. No other selenium-containing compounds appeared during this time. Hence, the secondary reaction products were either volatilised or precipitated. To verify the identity of the compounds, a larger amount of selenite was incubated with epithelial cells. The presence of Cys-Se-SG and GS-Se-SG was verified by LC–ESI-MS. Selenotrisulfides were synthesized by reaction of L-cysteine and L-glutathione with sodium selenite. The reaction mixture contained three main products: selenodicysteine (Cys-Se-Cys), selenocysteine glutathione (Cys-Se-SG), and selenodiglutathione (GS-Se-SG). The two transient selenium compounds in the epithelial cell incubation mixture co-eluted with the synthesized Cys-Se-SG and GS-Se-SG, respectively. The identities of these compounds were verified by LC–ESI-MS. Hence, these selenium metabolites have now been identified by ESI-MS after isolation from epithelial cells.     

Detection, quantification, and identification of dermorphin in equine plasma and urine by LC–MS/MS for doping control

Dermorphin is a unique opioid peptide that is 30–40 times more potent than morphine. It was misused and went undetected in horse racing until 2011 when intelligence obtained from a few North American race tracks suggested its use. To prevent such misuse, a reliable analytical method became necessary for detection and identification of dermorphin in post-race horse samples. This paper describes the first liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for such a purpose. Equine plasma and urine samples were pre-treated with ethylenediamine tetra-acetic acid and urea prior to solid-phase extraction (SPE) on Oasis MCX cartridges. Resulting eluates were dried under vacuum and analyzed by LC–MS/MS for dermorphin. The matrix effect, SPE efficiency, intra-day and inter-day accuracy and precision, and stability of the analyte were assessed. The limit of detection was 10 pg/mL in plasma and 20 pg/mL in urine, and the limit of confirmation was 20 pg/mL in plasma and 50 pg/mL in urine. Dermorphin in plasma is stable at ambient temperature, but its diastereomer is unstable. With isotopically labeled dermorphin as an internal standard, the quantification range was 20–10,000 pg/mL in plasma and 50–20,000 pg/mL in urine. The intra-day and inter-day accuracy was from 91 % to 100 % for the low, intermediate, and high concentrations. The intra-day and inter-day coefficients of variation were less than 12 %. The method differentiates dermorphin from its diastereomer. This method is very specific for identification of dermorphin in equine plasma and urine, as assessed by BLAST search and targeted SEQUEST search, and by MS/MS spectrum library search. The method has been successfully applied to analysis of samples collected following dermorphin administration to research horses and of official post-race samples.

Characterization of peptide–protein interactions using photoaffinity labeling and LC/MS

The combination of photoaffinity labeling (PAL) with modern mass spectrometric techniques is a powerful approach for the characterization of peptide–protein interactions. Depending on the analytical strategy applied, a PAL experiment can provide different levels of information ranging from the identification of interaction partners to the structural characterization of ligand-binding sites. On the basis of LC/MS data generated in the framework of the identification of the binding site of the neuropeptide corticotropin-releasing factor (CRF) on its binding protein (CRFBP), the key role of LC/MS in the characterization of photoadducts on different structural levels was demonstrated. Covalent photoadducts of rat CRFBP (rCRFBP) were obtained by PAL with different mono- and bifunctional benzophenone photoprobes designed on the basis of the sequence of the synthetic CRF fragment human/rat CRF^6–33 which binds to CRFBP with high affinity. In view of the stoichiometry, LC/MS analysis revealed that the photoadducts consisted of one molecule of photoprobe and one molecule of rCRFBP. For a further characterization of the photoadducts on the oligopeptide level, enzymatic digests of unlabeled rCRFBP and of the respective photoadduct were compared by peptide mapping monitored with LC/MS. Thereby, it was found that the photoprobe that contained the photophore at its N-terminus labeled the amino acid sequence rCRFBP(34–38), whereas the photoprobe that contained the photophore at its C-terminus labeled rCRFBP(12–26). On the basis of the characterization of the photoadduct formed by rCRFBP and the bifunctional photoprobe that contained photophores on both termini, semiquantitative comparison of different enzymatic digests was accomplished by application of the mass-selective multiple ion chromatogram strategy.     

Investigation of the biotransformation of melarsoprol by electrochemistry coupled to complementary LC/ESI–MS and LC/ICP–MS analysis

Melarsoprol is the only currently available drug for treatment of the late stage of African trypanosomiasis (sleeping sickness). Unfortunately, the arsenic-containing drug causes serious side effects, for which the mechanisms have not been elucidated so far. This investigation describes the study of the melarsoprol biotransformation processes by electrochemical (EC) techniques. Based on EC, potential oxidation reactions of melarsoprol are examined. Moreover, the reactivity of melarsoprol, its metabolite melarsen oxide, and their oxidation products toward the tripeptide glutathione and the proteins hemoglobin and human serum albumin is evaluated. The combination of different analytical techniques allows the identification as well as the quantification of the biotransformation products. The hyphenation of liquid chromatography (LC) and electrospray ionization mass spectrometry (ESI–MS) is applied for identification and structure elucidation, which implies the determination of exact masses and fragmentation patterns. For the selective detection of arsenic containing metabolites, LC coupled to inductively coupled plasma mass spectrometry is utilized. Based on the obtained data, the oxidative biotransformation of melarsoprol can be predicted, revealing novel species which have been suspected, but not been identified up to now. The results of the protein studies prove that melarsen oxide, the active derivative of melarsoprol, strongly binds to human hemoglobin and forms different adducts via the free cysteinyl groups of the hemoglobin α- and β-chain.     

Stress Studies and Identification of Degradation Products of Cephalexin Using LC–PDA and LC–MS/MS

A stability indicating RP-HPLC method for cephalexin has been developed and validated to identify and characterize potential degradation products. Drug was subjected to hydrolytic (acidic, basic, and neutral), oxidative, thermal, and photolytic stresses as per ICH guidelines Q1A (R2) and Q1B. Chromatographic separation was achieved on C8 column with mixture of ammonium acetate buffer pH 4.5 and acetonitrile in gradient mode as a mobile phase with PDA detection. Specificity of the method was established by peak purity studies. Method was validated as per ICH guideline Q2 (R1) for accuracy, precision, linearity, sensitivity, and robustness. Kinetics for each degradation condition was studied with respect to order of reaction and rate constant. Method was found to comply with acceptance criteria of validation parameters with respect to specificity (peak purity greater than 0.999) linearity (r ² greater than 0.99), accuracy (% recovery in the range of 98–102%), and precision (% RSD not more than 2). A total of six degradation products were generated in different stress conditions; these were identified and structures were proposed using LC–MS/MS. Cephalexin undergoes degradation in almost all the conditions. The developed stability indicating method is suitable for analysis of stability samples as it adequately separates all degradation products. Degradation products generated in photolytic and oxidative conditions are reported for the first time.     

Determination of cannabinoids in plasma using salting-out-assisted liquid–liquid extraction followed by LC–MS/MS analysis

The detection of the markers of Cannabis consumption in biological specimens is an important task for drug testing laboratories in varous contexts. A simple assay combining salting-out assisted liquid–liquid extraction sample preparation and LC–MS/MS analysis was applied to the measurement of Δ⁹-tetrahydrocannabinol, 11-nor-9-carboxy-Δ⁹-tetrahydrocannabinol (THC-COOH), 11-hydroxy-Δ⁹-tetrahydrocannabinol, cannabinol and cannabidiol concentrations in 100 μl plasma specimens. The assay had linearity of 1–100 ng ml⁻¹ for THC-COOH and 0.5–50 ng ml⁻¹ for the other tested cannabinoids. Assay validation criteria were fulfilled. Extraction yields (88.7–97.3%) and internal-standard correct matrix effects (−9.6 to +5.4%) were acceptable. The assay was applied to 238 clinical specimens from trauma patients, with 19 samples presenting quantifiable concentrations of at least one of the target compounds. The developed assay is a simple and efficient strategy for simultaneous measurement of Δ⁹-tetrahydrocannabinol, THC-COOH, 11-hydroxy-Δ⁹-tetrahydrocannabinol, cannabinol and cannabidiol concentrations in plasma specimens.     

Analysis of Linarin and Its Metabolites in Rat Urine by LC–MS/MS

Liquid chromatography–ion trap mass spectrometry was employed to investigate the metabolism of linarin in rats. Identification and structural elucidation of the metabolites were performed by comparing the differences in molecular masses, retention times, and full scan MS ⁿ spectra between linarin and its metabolites. Six metabolites (acacetin, apigenin, acacetin glucuronide, apigenin glucuronide, acacetin sulfate, apigenin sulfate) were detected in rat urine after oral administration of linarin at the dose of 50 mg kg^?1. Furthermore, a selective and sensitive liquid chromatography–triple quadruple mass spectrometry assay was developed and validated for the simultaneous determination of linarin and acacetin (the major metabolite of linarin) in rat urine. Chromatographic separation was carried out on a C₁₈ column, and mass spectrometric detection was performed using a triple-quadrupole mass spectrometer coupled with an electrospray ionization source in the positive ion mode. Quantitation of linarin and acacetin was performed using selected reaction monitoring of precursor–product ion transitions at m/z 593 → 285 for linarin, 285 → 242 for acacetin, and 303 → 153 for hesperitin (internal standard), respectively. The assay exhibited good linearity (r > 0.9900) for both linarin and acacetin. The intra- and inter-day precisions were <13.4 % and the accuracy was between ?8.1 and 3.1 %. The method was successfully applied to the urinary excretion study of linarin in rats after oral administration of linarin.     

Determination of Eleutherosides in Dietary Supplements by LC–MS/MS

A simple and specific method for the simultaneous determination of eleutherosides B and E in powdered rhizomes of Eleutherococcus senticosus extract and in solid and liquid dietary supplements was developed and validated. E. senticosus extracts, often mixed with other plants or herbal extracts, are widely used in food supplements because of the tonic and adaptogenic activities referred to the eleutherosides B and E. In this study, samples were analyzed by a liquid chromatography-electrospray tandem mass spectrometry (LC–ESI-MS/MS) method operated in single reaction monitoring (SRM). Validation was carried out in terms of limit of detection (LOD), limit of quantitation (LOQ), linearity, precision and trueness. LOD and LOQ values were fixed at 3 μg L^?1 and 10 μg L^?1, respectively, whereas linearity was established within 10–1,000 μg L^?1 range for both compounds. Good precision was obtained for both eleutherosides in terms of intra-day precision (RSD % lower than 4 %) and inter-day precision (RSD % lower than 6 %). Good percentage recoveries were obtained for both eleutherosides (91.5–103.6 %). Finally, the developed method was successfully applied to analyze a number of solid and liquid commercial dietary supplements containing E. senticosus extracts, also mixed with other herbal extracts.     

Determination of chlormequat in pig serum and sow milk by LC–MS/MS

Chlormequat is a plant growth regulator widely used on cereals, and there is general concern that it may impair human fertility. A LC–MS/MS method for the analysis of chlormequat in milk and serum was developed and validated in connection with an investigation on the effect of chlormequat on pig reproduction. Validation of the method was based on recovery tests at three spiking levels, determined as double determinations and repeated at least four times. Samples were extracted with methanol–water–acetic acid, centrifuged, filtrated and determined by LC–MS/MS. The mean recoveries were in the range 80–110%, and the LOD was 0.2 ng/g for serum and 0.3 ng/g for milk. The values for repeatability and reproducibility were within 2/3 of the limits given by the Horwitz equation. Samples of pig serum (59) and sow milk (27) were analyzed using the method. Chlormequat was determined in four milk samples in the range of 0.4 ng/g to 1.2 ng/g and in all serum samples in the range of 0.2 ng/g−4.0 ng/g.     

Marker peptide combination for source identification of gelatins obtained from Equidae hides by LC–MS/MS detection

Gelatin, which is a soluble, natural polymer produced by partial hydrolysis of collagens, has been extensively used in food and pharmaceuticals. Donkey-hide gelatin is a well-known traditional Chinese medicine. The certification of donkey-hide gelatin is one of the most concerned issues for consumers. Currently, the source identification of the homologous family species, including donkey, horse and their hybrids, remains a major challenge. Here, three peptides (SGQPGTVGPAGVR, GASGPAGVR and GATGPAGVR) of the equine family species were screened using high-resolution LC-MS. The combination of these three peptides has been used as a high-performance marker in LC-MS/MS to identify the animal source (donkey, horse, and their hybrids) of donkey-hide-gelatin-containing products. Horse-hide gelatin and hybrid-hide gelatin can be detected in gelatin-based products even at very low percentages (0.05% and 0.10%, respectively). The marker peptide combination method is useful for source identification of homologous family species and is not only highly sensitive but also rapid.