Characterization of impurities in cefpodoxime proxetil using LC–MSn

Reversed-phase liquid chromatography coupled with electrospray ionization tandem mass spectrometry (ESI-MS/MS) was used to characterize impurities in cefpodoxime proxetil, an ester-modified prodrug. Based on the mechanisms by which cephalosporins are degraded, stress tests were designed and performed. The bulk material and capsule were eluted through a C18 column with formic acid–methanol–water as the mobile phase. In total, 15 impurities were characterized in commercial samples, including 7 known impurities and 8 new impurities. The structures of these unknown compounds were deduced via comparison with the fragmentation patterns of cefpodoxime proxetil. Data from this systematic study will help improve the safety and quality of cefpodoxime proxetil.Key words: Cefpodoxime proxetil, Cephalosporins, Impurities, LC–MS, Structure identification     

Effect of hydroalcoholic extract of leaves of Colocasia esculenta on marble-burying behavior in mice: Implications for obsessive–compulsive disorder

Abstract

Context: Over the past decades, the inhibition of spontaneous burying of glass marbles by mice has been used as an index of anxiolytic drug action in the so-called marble-burying test. Although Colocasia esculenta Linn. (Araceae), commonly known as elephant ear (English), possesses several medicinal properties, little is known for its use in neurological activity.

Objective: The current research evaluated the anti-obsessive–compulsive disorder (anti-compulsive) activity of the hydroalcoholic extract of leaves of Colocasia esculenta (HECE) for the first time using the marble-burying behavior test in mice.

Materials and methods: In the present study, the effect of HECE (25 and 50?mg/kg) intraperitoneally (i.p.) was examined using the marble-burying behavior test, which is an animal model of obsessive compulsive disorder (OCD), using Swiss albino mice.

Results and discussion: The acute toxicity studies showed that the LD₅₀ value of the HECE in mice was 1000?mg/kg by i.p. route. The effect of HECE (25 and 50?mg/kg, i.p.) was characterized by significant reduction in the number of buried marbles as compared with the control group (p?<?0.001). The effect of HECE was comparable with that of fluoxetine (5?mg/kg, i.p.) – a reference standard drug used in the treatment of obsessive–compulsive disorder (p?<?0.001). Fluoxetine and HECE do not produce any overt motor dysfunction.

Conclusions: The results of the study for the first time show that the plant possesses anti-compulsive activity, confirming the traditional claims. Future research should focus on the identification and the anti-compulsive activity of the constituents from this plant.     

A generic approach for the determination of trace hydrazine in drug substances using in situ derivatization-headspace GC–MS

In situ derivatization-headspace GC–MS methodology has been developed for the determination of hydrazine in drug substance at low ppm levels. This general method uses acetone or acetone-d₆ as the derivatization reagent. The resulting acetone azine or acetone azine-d₁₂ can then be analyzed by headspace GC–MS. The method gives excellent sensitivity with a limit of quantitation (LOQ) as low as 0.1 ppm when the API (active pharmaceutical ingredient) samples are prepared at 10 mg per headspace injection vial. The spike recoveries of hydrazine at the 1 ppm level were between 79% and 117% in various APIs tested. The precisions (%RSD) of six preparations of the hydrazine standards at the concentration of 1 ppm level were typically between 2.7 and 5.6%. A linear range of concentrations from 0.1 to 10 ppm has been demonstrated with R² ≥ 0.999. This general method has been tested in a number of API matrices and successfully applied to the determination of hydrazine in support of API batch releases and process chemistry at GlaxoSmithKline.     

Effects of triptolide on pharmacokinetics of amlodipine in rats by using LC–MS/MS

Context: Triptolide and amlodipine are often simultaneously used for reducing urine protein excretion after renal transplantation in China clinics.

Objective: This study investigated the effects of triptolide on the pharmacokinetics of amlodipine in male Sprague–Dawley rats.

Materials and methods: The pharmacokinetics of amlodipine (1?mg/kg) with or without triptolide pre-treatment (2?mg/kg/day for seven?days) were investigated using a sensitive and reliable LC–MS/MS method. Additionally, the inhibitory effects of triptolide on the metabolic stability of amlodipine were investigated using rat liver microsome incubation systems.

Results: The results indicated that when the rats were pre-treated with triptolide, the C_max of amlodipine increased from 13.78?±?3.57 to 19.96?±?4.56?ng/mL (p?T_max increased from 4.04?±?1.15 to 5.89?±?1.64?h (p?AUC_0–t increased by approximately 104% (p?p?Conclusions: In conclusion, these results indicated that triptolide could affect the pharmacokinetics of amlodipine, possibly by inhibiting the metabolism of amlodipine in rat liver when they are co-administered.     

Characterization of odor-active compounds in cooked meat of farmed obscure puffer (Takifugu obscurus) using gas chromatography–mass spectrometry–olfactometry

The volatile and odor-active compounds in cooked meat of farmed obscure puffer (Takifugu obscurus) were analyzed by gas chromatography–mass spectrometry–olfactometry (GC–MS–O). The volatile compounds were extracted by the simultaneous distillation–extraction (SDE) method, then separated and identified by GC–MS. Odor-active compounds in the SDE extract were characterized by GC–MS–O. A total of 68 volatile compounds were found, including 23 aldehydes, 10 alcohols, nine ketones, 17 N- or S-containing compounds and aromatics, three acids, three alkanes, and three esters. Of these, 31 odor-active compounds were detected and identified. Trimethylamine (fishy), octanal (grassy, leafy, green), (E)-2-octenal (roast, fatty), 1-octen-3-ol (fishy, fatty, mushroom, grassy), 2-ethyl-1-hexanethiol (cooked fish), (E,E)-2,4-octadienal (cooked meat, sweet), 2-acetylthiazole (meaty, roast, nutty, sulfur), 2-acetylpyrrole (nutty, walnut, bread) were identified as the key odorants in the cooked meat of farmed obscure puffer based on posterior intensity and time-intensity methods.     

Quantification of the major metabolites of bromhexine in human plasma using RRLC–MS/MS and its application to pharmacokinetics

(E)-4-hydroxydemethylbromhexine (E-4-HDMB) and (E)-3-hydroxydemethylbromhexine (E-3-HDMB) were found as major metabolites, while (Z)-4-hydroxydemethylbromhexine and (Z)-3-hydroxydemethylbromhexine as minor metabolites of bromhexine in human plasma. These compounds were identified in comparison with synthetic authentic samples. A sensitive and selective rapid resolution liquid chromatography tandem mass spectrometry (RRLC–MS/MS) method was developed to quantify the concentration of bromhexine and its two major metabolites (E-4-HDMB and E-3-HDMB) in human plasma. Following solid phase extraction, the analytes were separated on a Zorbax 1.8 μm particle size reversed-phase C₁₈ column, using a gradient elution program with solvents consisting of 0.1% formic acid in acetonitrile and 0.1% formic acid in 5 mM ammonium acetate at a flow rate of 0.7 mL/min. Detection was carried out with an Agilent 6460 triple-quadrupole mass spectrometer operated with an electrospray ionization source mode operated in the positive ion mode. The recovery of bromhexine, E-4-HDMB, E-3-HDMB, and internal standard (IS) was 63.1–70.9%, 60.5–68.4%, 57.0–63.5%, and 87.8%, respectively. The matrix factors of bromhexine, E-4-HDMB, E-3-HDMB, and IS were 89.9–96.7%, 89.6–94.8%, 90.4–91.4%, and 103%, respectively. After an oral administration of 8.0 mg bromhexine to five healthy male subjects, AUC_0–24 h values of bromhexine, E-4-HDMB, and E-3-HDMB were found to be 93.5 ± 31.9, 34.0 ± 14.5, and 15.8 ± 6.89 ng h/mL, respectively; while C_max values were 24.6 ± 5.16, 3.11 ± 1.13, and 5.36 ± 2.55 ng/mL, respectively. Plasma concentration of bromhexine, E-4-HDMB, and E-3-HDMB declined with t_1/2 which gave 3.6 ± 0.5, 8.4 ± 2.7, and 6.4 ± 2.5 h, respectively.     

LC–MS/MS studies of ritonavir and its forced degradation products

Forced degradation of ritonavir (RTV), under the conditions of hydrolysis (acidic, basic and neutral), oxidation, photolysis and thermal stress as prescribed by ICH was studied using LC–MS/MS. Eight degradation products were formed and their separation was accomplished on Waters XTerra^® C₁₈ column (250 mm × 4.6 mm i.d., 5 μm) using water:methanol:acetonitrile as (40:20:40, v/v/v) mobile phase in an isocratic elution mode by LC. The method was extended to LC–MS/MS for characterization of the degradation products and the pathways of decomposition were proposed. No previous reports were found in the literature regarding the characterization of degradation products of ritonavir.     

Non-destructive profiling of volatile organic compounds using HS-SPME/GC–MS and its application for the geographical discrimination of white rice

The authenticity determination of white rice is crucial to prevent deceptive origin labeling and dishonest trading. However, a non-destructive and comprehensive method for rapidly discriminating the geographical origins of white rice between countries is still lacking. In the current study, we developed a volatile organic compound based geographical discrimination method using headspace solid-phase microextraction coupled to gas chromatography–mass spectrometry (HS-SPME/GC–MS) to discriminate rice samples from Korea and China. A partial least squares discriminant analysis (PLS-DA) model exhibited a good classification of white rice between Korea and China (accuracy = 0.958, goodness of fit = 0.937, goodness of prediction = 0.831, and permutation test p-value = 0.043). Combining the PLS-DA based feature selection with the differentially expressed features from the unpaired t-test and significance analysis of microarrays, 12 discriminatory biomarkers were found. Among them, hexanal and 1-hexanol have been previously known to be associated with the cultivation environment and storage conditions. Other hydrocarbon biomarkers are novel, and their impact on rice production and storage remains to be elucidated. In conclusion, our findings highlight the ability to rapidly discriminate white rice from Korea and China. The developed method maybe useful for the authenticity and quality control of white rice.     

Identification and characterization of degradation products of irbesartan using LC–MS/TOF,MS, on-line H/D exchange and LC–NMR

Irbesartan was subjected to hydrolytic, oxidative, photolytic and thermal stress, according to ICH guideline Q1A (R2). The drug showed degradation only in acidic, basic and photoacidic conditions, while it was stable to other stress conditions. A total of three degradation products were formed, which were separated on a C-8 column employing a gradient HPLC method. Initially, a complete mass fragmentation pathway of the drug was established with the help of MS/TOF, MSⁿ and H/D exchange studies. Subsequently, the degradation products were subjected to LC–MS/TOF and on-line H/D exchange mass studies to obtain their accurate mass, fragment pattern and number of labile hydrogens. The MS results helped to assign tentative structures to degradation products, which were verified through ¹H and 2D COSY LC–NMR experiments. The products were identified as (2′-(1H-tetrazol-5-yl)biphenyl-4-yl)methanamine, 1-(1-((2′-(1H-tetrazol-5-yl)biphenyl-4-yl)methylamino)pentylideneamino)cyclopentane carboxylic acid and 2-butyl-3-(tetrazolo[1,5-f]phenanthridin-6-ylmethyl)-1,3-diazaspiro[4.4]non-1-en-4-one. The structures were justified by mechanisms of their formation.     

Separation and characterization of forced degradation products of abacavir sulphate by LC–MS/MS

Abacavir sulphate was subjected to forced degradation under the conditions of hydrolysis (acid, alkali and neutral), oxidation, photolysis and thermal stress as prescribed by ICH. Eight degradation products were formed and their separation was accomplished on Waters XTerra C₁₈ (250 mm × 4.6 mm, 5 μm) column using 20 mM ammonium acetate:acetonitrile as a mobile phase in gradient elution mode by LC. The degradation products were characterized by LC–MS/MS and its fragmentation pathways were proposed. No previous reports were found in the literature regarding the degradation behavior of abacavir sulphate.     

Characterization of metabolites of worenine in rat biological samples using liquid chromatography–tandem mass spectrometry

The in vivo and in vitro metabolites of worenine in rat were identified or characterized using a specific and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method. In vivo samples including rat urine, feces, and plasma samples were collected after ingestion of 25 mg/kg worenine to healthy rats. The in vivo and in vitro samples were cleaned up by a solid-phase extraction procedure (C18 cartridges) and a liquid–liquid extraction procedure, respectively. Then these pretreated samples were injected into a reversed-phase C18 column with mobile phase of methanol–ammonium acetate (2 mM, adjusted to pH 3.5 with formic acid) (60:40, v/v) and detected by an on-line MS/MS system. As a result, at least twenty-seven metabolites and the parent medicine were found in rat urine after ingestion of worenine. Seven metabolites and the parent medicine were identified or characterized in rat feces. Three metabolites and the parent medicine were detected in rat plasma. One metabolite was found in the rat intestinal flora incubation mixture, and three metabolites were characterized in the homogenized liver incubation mixture. The main phase I metabolism of worenine in rat was dehydrogenization, hydrogenation, hydroxylation, and demethylene reactions, and that of phase II was sulfation and glucuronidation.     

GC–MS analysis of incenses for possible presence of allergenic nitromusks

A Gas chromatographic method with mass detector was developed to identify and determine nitromusks in incense sticks of different origin (India, China, Tibet). The proposed method was found useful to correlate dermatological allergic reactions with the use and composition of commercial incense sticks. The incense sticks were powdered, extracted with methanol and after the addition of 1-eicosanol as internal standard, injected into the GC–MS, using 25 m bonded phase fused capillary column methyl, 5% phenyl silicone (0.32 mm I.D., 0.25 μm film thickness). Musk ambrette was identified and determined in one kind of chinese incense together with musk ketone and musk xylene. This latter compound was also found alone in another kind of chinese incense.     

Phytochemical analysis of an antiviral fraction of Radix astragali using HPLC–DAD–ESI–MS/MS

Radix astragali (Huangqi in Chinese) is a well-known traditional Chinese medicinal herb that has been used clinically in China for centuries to cure various diseases. To profile the antiviral constituents of this herb, a high-performance liquid chromatography–diode array detector–electrospray ionization–tandem mass spectrometry (HPLC–DAD–ESI–MS/MS) analytical method was developed to separate and determinate the active part of the extract of Radix astragali, which showed potent inhibition of several viruses. By comparing their retention time and MS data with those obtained from the authentic compounds and the published data, a total of 18 compounds, comprising 11 flavonoids and 7 saponins, were identified. This study provides an approach to rapidly characterize bioactive constituents in traditional Chinese medicines (TCMs).     

LC–MS analysis of saponins of Achyranthes root in the Japanese market

Journal of Natural Medicines - LC–MS analyses of saponin fractions of Achyranthes roots in the Japanese market revealed that there were three patterns for the saponin fraction of their water...     

Quantification of phenolic antioxidants in rat cerebrospinal fluid by GC–MS after oral administration of compounds

A gas chromatographic-mass spectrometric (GC–MS) method for qualitative and subsequent quantitative analysis of phenolic antioxidants compounds, presents in olive oil, in rat cerebrospinal fluid (CSF) after oral administration of compounds is proposed. The procedure involves the extraction of compounds from the samples by a traditional microliquid–liquid extraction method, followed by a silylation step before the GC–MS analysis. The chromatographic separation was performed by using a low bleed DB5-MS fused-silica capillary column. The presence of 21 phenolic compounds was tested in CSF extracts and only free tyrosol, hydroxytyrosol and ferulic acid were detected. Those compounds were then quantitatively determined using the proposed methology. The molecular ion for silylated compounds appears at 370 m/z for hydroxytyrosol, 282 m/z for tyrosol and 338 m/z for ferulic acid respectively, while the base peak appears at 267 m/z, 179 m/z and 338 m/z. α-Naphthol was used as a surrogate (216 and 201 m/z). The detection capabilities obtained were 74, 92 and 79 ng/mL respectively. The method was applied to the determination of trace amounts of compounds in rat cerebrospinal fluid after oral administration. The animals were fed with a standard chow diet (free of phenolic antioxidants) in order to avoid the influence of any other component of the diet on the CSF of the animals.     

Determination of volatile nitrosamines in various meat products using comprehensive gas chromatography–nitrogen chemiluminescence detection

An optimized method was developed for the extraction, pre-concentration and analysis of nitrosamines (NAs) in various meat products. Values of reproducibility, linearity, limit of detection (LOD) and limit of quantitation (LOQ) for six NA standards (N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosodi-n-propylamine, N-nitrosopyrrolidine, N-nitrosopiperidine, N-nitrosodi-n-butylamine) were determined. The LODs using this method were between 1.66–3.86 and LOQs between 6.96–16.71 μg L⁻¹. The screening of four different types of meat samples (sausage, salami, sucuk and doner kebab) showed that all samples contained levels of various NAs, identified with high confidence using comprehensive gas chromatography (GCxGC) and a fast responding element specific nitrogen chemiluminescence detector (NCD). The sum of the six NAs were highest in the doner kebab samples, being between 0.51–16.63 μg kg⁻¹ and were lowest in the sausage samples at 0.45–2.93 μg kg⁻¹. The described method is simple, rapid, selective and sensitive.