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.     

Inclusion Complexes of Manidipine with γ-Cyclodextrin and Identification of Photodegradation Products

The paper presents results of a study of photochemical decomposition of manidipine and its inclusion complexes with γ-cyclodextrin and identification of photodegradation products. The process was qualitatively assessed by the UV spectrophotometry and by HPLC-MS. The quantitative assessment of its efficiency was performed on the basis of kinetic parameters and quantum yields. The main product of photodegradation was nitrophenylpyridine derivative, while the concentration of nitrozophenylpyridine derivative being the other product of this process, was about 20 times lower. The inclusion complexes of manidipine with γ-cyclodextrin were obtained in the liquid phase. The stoichiometry of the complexes was determined from the Benesi–Hildebrand equation. The photochemical stability of manidipine in inclusion complexes was compared with that of manidipine in non-complexed form.This revised version was published online in July 2005 with a corrected issue number.     

Comparison of LC and UPLC Coupled to MS–MS for the Determination of Sulfonamides in Egg and Honey

Determination of ten sulfonamides (SAs) in egg and honey has been compared using column liquid chromatography (LC) and ultra-performance liquid chromatography (UPLC) coupled to tandem mass spectrometry (MS–MS). A liquid–liquid extraction with acetonitrile followed by solid-phase extraction on a Strata-X cartridge was developed for sample preparation. The analytical performance of both methods was compared applying the alternative matrix-comprehensive in-house validation approach using specially designed software InterVal?. Using UPLC the separation time was shortened about 30% reducing the run time by 8 min and a better resolution was achieved compared to LC. Due to higher peak efficiency achieved with UPLC, the decision limit values obtained by both techniques were almost equal (6.61–9.43 μg kg^?1 and 7.25–11.9 μg kg^?1 for UPLC and LC, respectively), despite the fact that in UPLC twice lower sample volumes were injected. Satisfactory and comparable recoveries (80–110%) were obtained by UPLC and LC for all the SAs, except for sulfacetamide by LC and sulfabenzamide by both methods. For a majority of the spiked compounds, UPLC gave significantly better precision.     

Fast Determination of Fumonisin B1 and B2 in Corn Using a Modified QuEChERS Method and LC–MS–MS

A fast analytical method for the simultaneous determination of fumonisin B₁ (FB₁) and fumonisin B₂ (FB₂) in corn using a novel QuEChERS method and LC–MS–MS was developed and validated. Samples were extracted with methanol–water (3:1 v/v) by means of ultrasonic extraction. The extract was purified with a novel modified QuEChERS method. Firstly, FB₁ and FB₂ in the extract were retained with primary secondary amine (PSA). Then, FB₁ and FB₂ were released from PSA with 1.0 % formic acid in methanol. The final eluate was diluted with water, and analyzed by LC–MS–MS on a Waters Acquity BEH C₁₈ column with 0.1 % formic acid in water/methanol as mobile phase with gradient elution. Mean recoveries of 83.5–102.4 % with CVs of 3.6–10.5 % were obtained at fortification levels of 2, 50 and 1,000 μg kg⁻¹. The limit of quantification was 2.0 μg kg⁻¹.

     

Advantages of LC–MS–MS compared to LC–MS for the determination of nitrofuran residues in honey

In the framework of developing analyses for exogenous contaminants in food matrices such as honey, we have compared data obtained by high-performance liquid chromatography coupled with mass spectrometry (LC–MS) to those provided by high-performance liquid chromatography and tandem mass spectrometry (LC–MS–MS). Initial results obtained with LC–MS showed that the technique lacked selectivity, which is why the method was validated by LC–MS–MS. This method involves a solid-phase extraction (SPE) of nitrofuran metabolites and nitrofuran parent drugs, a derivatization by 2-nitrobenzaldehyde for 17 h, and finally a clean-up by SPE. The data obtained show that the limits of detection varied between 0.2 and 0.6 μg kg⁻¹ for the metabolites and between 1 and 2 μg kg⁻¹ for nitrofuran parent drugs. The method was applied to different flower honeys. The results showed that nitrofurans (used as antibiotics) are consistently present in this matrix, the predominant compound being furazolidone. Figure Working bees     

LC–DAD and LC–ESI–MS Chromatographic Fingerprinting and Quantitative Analysis for Evaluation of the Quality of Huang-Lian-Jie-Du-Tang

LC–DAD coupled with electrospray tandem mass spectrometry (LC–ESI–MS–MS) has been used to evaluate the quality of the traditional Chinese medicine Huang-Lian-Jie-Du-Tang (HLJDT). Twenty-five chromatographic peaks were obtained from a C₁₈ analytical column by gradient elution with acetonitrile and formate buffer (containing 0.5% formic acid) at a flow rate of 1.0 mL min^?1. The linearity, precision, and accuracy of the data obtained were acceptable. Thirteen components were identified by ESI–MS, and seven of these were quantitatively analyzed by LC–DAD. The method was used to analyze ten batches of HLJDT, and both chromatographic fingerprints and quantitative data were used to evaluate the quality of the HLJDT. It was concluded that this LC–DAD–ESI–MS method enables more fully validated and complete evaluation and monitoring of the quality of HLJDT.     

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.     

LC–MS–MS Assay for Simultaneous Quantification of Fexofenadine and Pseudoephedrine in Human Plasma

A rapid, sensitive, and simple HPLC–MS–MS method, with electro-spray ionization and cetirizine as internal standard (IS), has been developed and validated for simultaneous quantification of fexofenadine and pseudoephedrine in human plasma. The analytes were isolated from plasma by solid-phase extraction (SPE) on Oasis HLB cartridges. The compounds were chromatographed on an RP 18 column with a mixture of ammonium acetate (10 mm, pH 6.4) and methanol as mobile phase. Quantification of the analytes was based on multiple reaction monitoring (MRM) of precursor-to-product ion pairs m/z 502 → 466 for fexofenadine, m/z 166 → 148 for pseudoephedrine, and m/z 389 → 201 for cetirizine. The linear calibration range for both analytes was 2–1,700 ng mL⁻¹ (r = 0.995), based on analysis of 0.1 mL plasma. Extraction recovery was 91.5 and 80.88% for fexofenadine and pseudoephedrine, respectively. The method was suitable for analysis of human plasma samples obtained 72 h after administration of a drug containing both fexofenadine and pseudoephedrine.     

LC–MS–MS Analysis of Mirtazapine in Plasma, and Determination of Pharmacokinetic Data for Rats

A sensitive LC–MS–MS method with electrospray ionization has been developed for analysis of mirtazapine in rat plasma. After addition of diazepam as internal standard, liquid–liquid extraction was used to produce a protein-free extract. Chromatographic separation was achieved on a 150 × 4.6 mm, 5 μm particle, ODS column with 84:16 (v/v) methanol–water containing 0.1% ammonium acetate and 0.01% glacial acetic acid as mobile phase. LC–MS–MS was performed in selected-ion-monitoring (SIM) mode using target fragment ions m/z 195.09 for mirtazapine and m/z 192.80 for the IS. Calibration plots were linear over the range of 0.516–618.8 ng mL^?1. The lower limit of quantification was 0.516 ng mL^?1. Intra-day and inter-day precision were better than 12.6 and 8.8%, respectively. Mean recovery of mirtazapine from plasma was in the range 87.41–90.06%; average recovery was 88.40% (RSD 3.95%). Significant gender differences between mirtazapine pharmacokinetic data were observed in this study.     

Comparison of LC–UV, LC–ELSD and LC–MS Methods for the Determination of Sesquiterpenoids in Various Species of Artemisia

Simple and specific analytical methods for the quantitative determination of sesquiterpenoids from various species of Artemisia plant samples were developed. By LC–UV, LC–ELSD, the separation was achieved by reversed-phase chromatography on a C18 column with water and acetonitrile both containing 0.025% trifluoroacetic acid as the mobile phase. In the LC–MS system, trifluoroacetic acid was replaced by 0.1% formic acid. The wavelength used for quantification of sesquiterpenoids with a diode array detector was 205 nm. The limits of detection by LC–MS was found to be 5, 10, 25, 50, 50 ng mL^?1. The limits of detection by LC–UV and LC–ELSD were found to be 5.0, 3.0, 100, 100, 7.5 μg mL^?1, by LC–UV and 50, 25, 30, 100 and 75 μg mL^?1 by LC–ELSD. LC–mass spectrometry coupled with electrospray ionization (ESI) interface is described for the identification and quantification of sesquiterpenoids in various plant samples. This method involved the use of the [M + H]⁺ ions of sesquiterpenoids in the positive ion mode with extractive ion monitoring.     

LC–ESI–MS Determination of Phospholipids and Lysophospholipids

Phospholipids are major constituents of biological membranes in plants and animals. Phospholipid-containing substances, such as lecithin, have also received increasing attention as emulsifiers in pharmaceutical drug delivery systems. The phospholipids phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, their lysophosphatidyl hydrolysis products and sphingomyelin, were quantified in lecithin and lecithin-containing drug-delivery emulsions. Lysophospholipids are constituents of lecithin, but their formation during thermal sterilization of the investigated drug delivery systems might contribute to the concentrations found in the emulsions.     

AFB1–AP Conjugate for Enzyme Immunoassay of Aflatoxin B1 in Corn Samples

Abstract

This article describes the conjugation between aflatoxin B₁ (AFB₁), one of the major mycotoxins, and alkaline phosphatase (AP), one of the most used enzymes for immunoassays. In addition, an application of the ELISA method for aflatoxin B₁ determination in corn is presented. Three AFB₁–AP conjugates in different toxin–enzyme ratios were prepared and tested. The ELISA results, developed with the most effective conjugate obtained, showed a satisfactory working range between 2.4 and 4000 ng of toxin/g of corn. The detection limit was 2 ng/g in corn samples, and recoveries ranged from 105 to 120%.     

Identification of the absorptive constituents and their metabolites in vivo of Ziziphi Spinosae Semen by UPLC–ESI–Q-TOF–MS/MS

In this research, ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC–ESI–Q-TOF–MS/MS) was used for detection and identification of the absorptive constituents and their metabolites in rat plasma, urine and feces following oral administration of Ziziphi Spinosae Semen alcohol extract. After structure elucidation, a total of 12 compounds in rat plasma, comprising seven prototypes and five metabolites, 28 compounds in urine, comprising 17 prototypes and 11 metabolites, and 23 compounds in feces, comrpising 17 prototypes and six metabolites, have been tentatively identified by comparison with standard compounds and reference literature information. To the best of our knowledge, this is the first comprehensive and systematical metabolic study on the seed. Mostly importantly, we propose that gastric acid could convert jujubosides into an absorbable form of ebelin lactone oligosaccharides, which may be responsible for the low bioavailability and specific bioactivities of these compounds. Additionally, we deduced that the absorption site of ebelin lactone oligosaccharides is located in the stomach, and that the ebelin lactone form of jujubosides may be more suitable for absorption than its hydrolysis product. Our investigation will be helpful to narrow the scope for potentially active ingredients of the seed, and pave the way for determination of the pharmacological mechanism of the seed.     

Identification of Metabolites of Epimedin A in Rats Using UPLC/Q–TOF–MS

Herba Epimedii (Epimedium) is a kind of tonic herb, widely used in China. Epimedin A is a major component of Herba Epimedii with bioactivities. Analysis of the metabolic profile in vivo plays a pivotal role in understanding how traditional Chinese medicine works. And the metabolites of epimedin A might influence the effects of Herba Epimedii. Moreover, the metabolic routes of epimedin A provide an important basis for safety evaluation. Until now, little has been known about the metabolism of epimedin A. The current study was designed to characterize the metabolic pathways of epimedin A in vivo. The metabolites in rat plasma, bile, feces, and urine were identified by UPLC/Q–TOF–MS analysis. A total of 27 metabolites from epimedin A were detected or tentatively identified. The major metabolic processes were hydrolysis, hydrogenation, hydroxylation, dehydrogenation, demethylation, and conjugation with glucuronic acid and different sugars. The present study revealed the metabolic pathways of epimedin A in rat for the first time, and epimedin A could undergo extensive phase I and phase II metabolism in rat. These findings would provide an important basis for the further study and clinical application of epimedin A. In addition, the results of this work have shown the feasibility of the UPLC/Q–TOF–MS approach for rapid and reliable characterization of metabolites.

     

Identification of Acepromazine and Its Metabolites in Horse Plasma and Urine by LC–MS/MS and Accurate Mass Measurement

Acepromazine maleate (Sedalin^?) was administered orally to six thoroughbred horses at a dose of 0.15?mg?kg^?1. Urine and blood samples were collected up to 412?h post-administration. Plasma and urine were hydrolysed; plasma samples were then processed using liquid–liquid extraction and urine samples using solid-phase extraction. A sensitive tandem mass spectrometric method was developed in this study, achieving a lower limit of quantification for acepromazine of 10?pg?mL^?1 in plasma and 100?pg?mL^?1 in urine. Acepromazine, hydroxyethylpromazine, hydroxyacepromazine, hydroxyethylpromazine sulphoxide, hydroxyethylhydroxypromazine, dihydroxyacepromazine and dihydroxyhydroxyethylpromazine were detected in the post-administration samples. The parent drug and its metabolites were identified using a combination of UPLC–MS/MS and accurate mass measurement. Separation of the structural isomers hydroxyethylpromazine sulphoxide and hydroxyethylhydroxypromazine was another significant outcome of this work and demonstrated the advantages to be gained from investing in chromatographic method development.     

Analysis of Phlebodium decumanum Fronds by High-Performance Liquid Chromatography by Ultraviolet-Visible and Quadrupole Time-of-Flight Tandem Mass Spectrometry (HPLC–UV–VIS–QTOF–MS/MS)

Phlebodium decumanum (P. decumanum), together with other tropical ferns commonly known as Calaguala, have been empirically applied since ancient times to ameliorate inflammatory disorders, skins diseases and even cancer. There are evidences of antineoplastic potential and anti-inflammatory, immunomodulatory and antioxidant properties of the extract. Preliminary studies have also shown direct antitumor activity of the extracts. In the present article, the phytochemical composition of a hydro-ethanolic extract of P. decumanum is investigated using high-performance liquid chromatography (HPLC) with ultraviolet–visible (UV–vis) diode array detection (DAD) and electrospray ionization (ESI) quadrupole time-of-flight tandem mass spectrometry (QTOF–MS/MS) detection. First, the chromatographic profile is established as a representative fingerprint of the compounds extracted from the fern. Then, a total of 122 chemicals, including 23 flavonoids, 47 phenolic acids (34 hydroxycinnamic acids and 13 hydroxybenzoic acids), 9 amino and amino-sugar derivatives, 24 organic acids and their derivatives, and other metabolites, have been characterized. Moreover, 12 unknown compounds were also detected, providing the first comprehensive characterization available on the phytochemical composition of the leaves of P. decumanum.     

Identification of Metabolites of Epimedin A in Rats Using UPLC/Q–TOF–MS

Herba Epimedii (Epimedium) is a kind of tonic herb, widely used in China. Epimedin A is a major component of Herba Epimedii with bioactivities. Analysis of the metabolic profile in vivo plays a pivotal role in understanding how traditional Chinese medicine works. And the metabolites of epimedin A might influence the effects of Herba Epimedii. Moreover, the metabolic routes of epimedin A provide an important basis for safety evaluation. Until now, little has been known about the metabolism of epimedin A. The current study was designed to characterize the metabolic pathways of epimedin A in vivo. The metabolites in rat plasma, bile, feces, and urine were identified by UPLC/Q–TOF–MS analysis. A total of 27 metabolites from epimedin A were detected or tentatively identified. The major metabolic processes were hydrolysis, hydrogenation, hydroxylation, dehydrogenation, demethylation, and conjugation with glucuronic acid and different sugars. The present study revealed the metabolic pathways of epimedin A in rat for the first time, and epimedin A could undergo extensive phase I and phase II metabolism in rat. These findings would provide an important basis for the further study and clinical application of epimedin A. In addition, the results of this work have shown the feasibility of the UPLC/Q–TOF–MS approach for rapid and reliable characterization of metabolites.