Modeling the retention mechanism for high‐performance liquid chromatography with a chiral ligand mobile phase and enantioseparation of mandelic acid derivatives

The chromatographic retention mechanism describing relationship between retention factor and concentration of Cu²⁺(l ‐phenylalanine)₂ using chiral ligand mobile phase was investigated and eight mandelic acid derivatives were enantioseparated by chiral ligand exchange chromatography. The relationship between retention factor and concentration of the Cu²⁺(l ‐phenylalanine)₂ complex was proven to be in conformity with chromatographic retention mechanism in which chiral discrimination occurred both in mobile and stationary phase. Different copper(II) salts, chiral ligands, organic modifier, pH of aqueous phase, and conventional temperature on retention behavior were optimized. Eight racemates were successfully enantioseparated on a common reversed‐phase column with an optimized mobile phase composed of 6 mmol/L of l ‐phenylalanine or N,N‐dimethyl‐l ‐phenylalanine and 3 mmol/Lof copper(II) acetate or copper(II) sulfate aqueous solution and methanol.     

Isotopic variants of light and heavy l-pyroglutamic acid succinimidyl esters as the derivatization reagents for dl-amino acid chiral metabolomics identification by liquid chromatography and electrospray ionization mass spectrometry

l-Pyroglutamic acid succinimidyl ester (l-PGA-OSu) and its isotopic variant (l-PGA[d₅]-OSu) were newly synthesized and evaluated as the chiral labeling reagents for the enantioseparation of amino acids, in terms of separation efficiency by reversed-phase chromatography and detection sensitivity by ESI-MS/MS. The enantiomers of amino acids were easily labeled with the reagents at 60 °C within 10 min in an alkaline medium containing triethylamine. Although all the diastereomers derived from 18 proteolytic amino acids could not be satisfactorily separated, the pairs of 9 amino acids were completely separated by reversed-phase chromatography using the small particle (1.7 μm) ODS column (Rs = 1.95–8.05). The characteristic daughter ions, i.e., m/z 84.04 and m/z 89.04, were detected from all the derivatives by the collision induced dissociation of the protonated molecular ions. A highly sensitive detection at a low-fmol level (0.5–3.2 fmol) was also obtained from the selected reaction monitoring (SRM) chromatograms. An isotope labeling strategy using light and heavy l-PGA-OSu for the differential analysis of the dl-amino acids in different sample groups is also presented in this paper. The differential analysis of biological sample (i.e., human serum) and food product (i.e., yogurt) were tried to demonstrate the efficiency of the proposed method. The ratios of the dl-amino acids in human serum samples, spiked with the different concentrations of d-amino acids, were determined by the procedures using l-PGA-OSu and l-PGA[d₅]-OSu. The d/l ratios in the two sample groups at different concentrations of amino acids were similar to the theoretical values. Furthermore, the ratios of d/l-alanine values in different yogurt products were comparable to the ratios obtained from the d/l values using only light reagent (i.e., l-PGA-OSu). Consequently, the proposed strategy is useful for the differential analysis not only in biological samples but also in food products.     

Purity determination and uncertainty evaluation of theophylline by mass balance method, high performance liquid chromatography and differential scanning calorimetry

Purity determination of pure organic substance is essential for the establishment of traceability to SI units. A mass balance method was employed to determine the purity of theophylline certified reference materials (CRM), compared with high performance liquid chromatography (HPLC) and differential scanning calorimetry (DSC). In the approach of the mass balance, the impurities were identified by ion trap time-of-flight mass spectrometer (IT-TOF-MS) and quantified by HPLC. The purity of theophylline CRM determined by mass balance method was 99.82% with an extended uncertainty of 0.1% (k = 2). The uncertainty evaluation of purity demonstrated that the accuracy of the mass balance method is better than that of HPLC and DSC. It indicated that the mass balance is suitable for the CRM and pharmaceutical standards.     

Validation of a confirmatory method for the determination of melamine in egg by gas chromatography-mass spectrometry and ultra-performance liquid chromatography-tandem mass spectrometry

A sensitive and reliable method was developed and validated for detection and confirmation of melamine in egg based on gas chromatography-mass spectrometry (GC-MS) and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Trichloroacetic acid solution was used for sample extraction and precipitation of proteins. The aqueous extracts were subjected to solid-phase extraction by mixed-mode reversed-phase/strong cation-exchange cartridges. Using ultra-performance liquid chromatography and electrospray ionization in the positive ion mode, melamine was determined by LC-MS/MS, which was completed in 5 min for each injection. For the GC-MS analysis, extracted melamine was derivatized with N,O-bis(trimethylsilyl)trifluoracetamide prior to selected ion monitoring detection in electron impact mode. The average recovery of melamine from fortified samples ranged from 85.2% to 103.2%, with coefficients of variation lower than 12%. The limit of detection obtained by GC-MS and UPLC-MS/MS was 10 and 5 μg kg⁻¹, respectively. This validated method was successfully applied to the determination of melamine in real samples from market.     

Development and validation of a turbidimetry method for hydrogen sulphide evaluation in effluents of phosphoric acid production unit

The aim of this study was to develop and validate a simple, new and efficient method for the analysis of hydrogen sulphide (H₂S) released from the effluents of phosphoric acid production units. H₂S sampling was performed by absorption of the industrial gas into a cadmium acetate absorbing solution. The formed cadmium sulphide (CdS), as a result of a chemical reaction, was analysed by turbidimetry. A methodical validation study of the proposed method was performed according to the requirements of ISO 17025 standards. The proposed method was demonstrated to be precise, linear and accurate over a concentration range of 6.91–92.16 mg/l. Detection and quantification limits were equal to 5.09 mg/l and 6.91 mg/l, respectively. Sample analysis had to be performed within 48 h of the sampling step. The turbidimetry method was applied successfully to the industrial gaseous effluents and can be considered as an economical alternative to the iodometric method.     

Development and validation of a gas chromatography-mass spectrometric method for the determination of sildenafil and desmethyl-sildenafil in whole blood

Sildenafil (SDL) is a phosphodiesterase type 5 inhibitor and it is approved for the treatment of erectile dysfunction and pulmonary hypertension. SDL is extensively metabolized to its pharmacologically active metabolite, desmethyl‐sildenafil (DSDL). A sensitive and specific GC/MS method for the determination of SDL and DSDL in whole blood was developed and validated to support therapeutic drug monitoring of SDL patients. The combination of solid‐phase extraction with derivatization using BSTFA with 1% TMCS in acetonitrile efficiently reduced matrix effect and improved sensitivity of the method. In this assay, protriptyline was used as internal standard for both analytes. The LODs were 1.50 and 5.00 ng/mL for SDL and DSDL, respectively, whereas the respective LOQs were 5.00 and 15.0 ng/mL. The calibration curves were linear up to 500.0 ng/mL (SDL: R² 0.992, DSDL: R² 0.990). Absolute recovery values for both analytes ranged from 83.1 to 93.2%. Within‐ and between‐batch accuracy was less than 11.8 and 10.2%, respectively, whereas within‐ and between‐batch precision was less than 8.1 and 10.8%, correspondingly. The developed method is suitable for the determination of SDL and DSDL concentrations in blood samples obtained from patients under Viagra^® treatment, for pharmacokinetic studies or for the investigation of related forensic cases.     

Development and validation of an assay method for the determination of trifluoroacetic acid in a cyclosporin-like drug

An improved method for the assay of trifluoroacetic acid (TFA) in a cyclosporin-like drug substance is presented, based on ion chromatography with suppressed conductivity detection. Column fouling by the drug molecule is avoided by use of a sample preparation method in which the drug substance is precipitated at alkaline pH whilst the TFA remains in solution. The new method requires a smaller sample mass than a previous method based on headspace-GC-FID whilst achieving an improvement in sensitivity. During validation, the method's performance was found to be consistent with usual acceptance criteria, and the method was found to be robust in routine use.     

Preparation and evaluation of a macroporous molecularly imprinted hybrid silica monolithic column for recognition of proteins by high performance liquid chromatography

A novel type of macroporous molecularly imprinted hybrid silica monolithic column was first developed for recognition of proteins. The macroporous silica-based monolithic skeleton was synthesized in a 4.6 mm i.d. stainless steel column by a mild sol–gel process with methyltrimethoxysilane (MTMS) as a sole precursor, and then vinyl groups were introduced onto the surface of the silica skeleton by chemical modification of γ-methacryloxypropyltrimethoxysilane (γ-MAPS). Subsequently, the molecularly imprinted polymer (MIP) coating was copolymerized and anchored onto the surface of the silica monolith. Bovine serum albumin (BSA) and lysozyme (Lyz), which differ greatly in molecular size, isoelectric point, and charge, were representatively selected for imprinted templates to evaluate recognition property of the hybrid silica-based MIP monolith. Some important factors, such as template–monomer molar ratio, total monomer concentration and crosslinking density, were systematically investigated. Under the optimum conditions, the obtained hybrid silica-based MIP monolith showed higher binding affinity for template than its corresponding non-imprinted (NIP) monolith. The imprinted factor (IF) for BSA and Lyz reached 9.07 and 6.52, respectively. Moreover, the hybrid silica-based MIP monolith displayed favorable binding characteristics for template over competitive protein. Compared with the imprinted silica beads for stationary phase and in situ organic polymer-based hydrogel MIP monolith, the hybrid silica MIP monolith exhibited higher recognition, stability and lifetime.     

Development and evaluation of a generic evolutionary method for protein-ligand docking

We have developed a generic evolutionary method with an empirical scoring function for the protein-ligand docking, which is a problem of paramount importance in structure-based drug design. This approach, referred to as the GEMDOCK (Generic Evolutionary Method for molecular DOCKing), combines both continuous and discrete search mechanisms. We tested our approach on seven protein-ligand complexes, and the docked lowest energy structures have root-mean-square derivations ranging from 0.32 to 0.99 A with respect to the corresponding crystal ligand structures. In addition, we evaluated GEMDOCK on crossdocking experiments, in which some complexes with an identical protein used for docking all crystallized ligands of these complexes. GEMDOCK yielded 98% docked structures with RMSD below 2.0 A when the ligands were docked into foreign protein structures. We have reported the validation and analysis of our approach on various search spaces and scoring functions. Experimental results show that our approach is robust, and the empirical scoring function is simple and fast to recognize compounds. We found that if GEMDOCK used the RMSD scoring function, then the prediction accuracy was 100% and the docked structures had RMSD below 0.1 A for each test system. These results suggest that GEMDOCK is a useful tool, and may systematically improve the forms and parameters of a scoring function, which is one of major bottlenecks for molecular recognition.     

Optimization of a liquid chromatography ion mobility-mass spectrometry method for untargeted metabolomics using experimental design and multivariate data analysis

High-resolution mass spectrometry coupled with pattern recognition techniques is an established tool to perform comprehensive metabolite profiling of biological datasets. This paves the way for new, powerful and innovative diagnostic approaches in the post-genomic era and molecular medicine. However, interpreting untargeted metabolomic data requires robust, reproducible and reliable analytical methods to translate results into biologically relevant and actionable knowledge. The analyses of biological samples were developed based on ultra-high performance liquid chromatography (UHPLC) coupled to ion mobility - mass spectrometry (IM-MS). A strategy for optimizing the analytical conditions for untargeted UHPLC-IM-MS methods is proposed using an experimental design approach. Optimization experiments were conducted through a screening process designed to identify the factors that have significant effects on the selected responses (total number of peaks and number of reliable peaks). For this purpose, full and fractional factorial designs were used while partial least squares regression was used for experimental design modeling and optimization of parameter values. The total number of peaks yielded the best predictive model and is used for optimization of parameters setting.     

The generalized hybrid orbital method for combined quantum mechanical/molecular mechanical calculations: formulation and tests of the analytical derivatives

 Hybrid quantum mechanical (QM) and molecular mechanical (MM) potentials are becoming increasingly important for studying condensed-phase systems but one of the outstanding problems in the field has been how to treat covalent bonds between atoms of the QM and MM regions. Recently, we presented a generalized hybrid orbital (GHO) method that was designed to tackle this problem for hybrid potentials using semiempirical QM methods [Gao et al. (1998) J Phys Chem A 102: 4714–4721]. We tested the method on some small molecules and showed that it performed well when compared to the purely QM or MM potentials. In this article, we describe the formalism for the determination of the GHO energy derivatives and then present the results of more tests aimed at validating the model. These tests, involving the calculation of the proton affinities of some model compounds and a molecular dynamics simulation of a protein, indicate that the GHO method will prove useful for the application of hybrid potentials to solution-phase macromolecular systems. Received: 4 October 1999 / Accepted: 18 December 1999 / Published online: 5 June 2000     

Development and optimization of a method for the analysis of phenols and chlorophenols from aqueous samples by gas chromatography-mass spectrometry, after solid-phase extraction and trimethylsilylation

A 3-step analytical procedure was developed and optimized for the simultaneous determination of 6 phenols (phenol, o-, m-, p-cresol, catechol and resorcinol) and 19 chlorophenols (all mono-, di-, tri-, and tetrachlorophenol isomers and pentachlorophenol) from environmental water samples. The analytical scheme consists of (1) solid-phase extraction (SPE) carried out on hypercrosslinked styrene-divinylbenzene (Isolute ENV+) cartridge; (2) derivatization with trimethylsilyl-N,N-dimethylcarbamate (TMSDMC); (3) analysis of the derivatives with capillary gas chromatography-mass spectrometry, in the selective ion monitoring mode. Ethyl acetate, ethyl acetate/acetic acid (5 v/v%) mixture, dichloromethane and acetonitrile were compared as to their ability to elute the phenols and chlorophenols from the ENV + sorbent in the smallest solvent volume possible. The optimized extraction step uses a minimal amount of organic solvent (4 mL ethyl acetate). Derivatization of the phenols and chlorophenols with TMSDMC was studied with respect to conversion, reagent excess, medium, temperature and the stability of the trimethylsilyl derivatives. If the reagent is applied in sufficient excess, the reaction takes place instantaneously at room temperature, and the derivatives remain stable for 24 h, making the procedure simple, fast and convenient. The overall method gave detection limits of 0.05-100 ng/L for all compounds except resorcinol which could not be retained on the SPE cartridge. The complete optimized analytical scheme was applied to ground water and river water samples collected in Hungary.     

Evaluation of the combination of a dispersive liquid–liquid microextraction method with micellar electrokinetic chromatography coupled to mass spectrometry for the determination of estrogenic compounds in milk and yogurt

In this work, the suitability of a methodology based on dispersive liquid–liquid microextraction (DLLME) has been evaluated for the extraction of four endoestrogens (estriol, 17α‐estradiol, 17β‐estradiol, and estrone), an exoestrogen (17α‐etynylestradiol), and a mycotoxin (zearalenone), together with some of their major metabolites (2‐methoxyestradiol, α‐zearalanol, β‐zearalanol, α‐zearalenol, and β‐zearalenol) from different types of milk (whole and skimmed cow milk and semiskimmed goat milk) and whole natural yogurt. The methodology includes a previous protein precipitation with acidified ACN and a defatting step with n‐hexane. Separation of the analytes, determination, and quantification were developed by MEKC coupled to ESI‐MS using a BGE containing an aqueous solution of ammonium perfluorooctanoate as MS friendly surfactant. Calibration, precision, and accuracy studies of the described DLLME‐MEKC‐MS/MS method were evaluated obtaining a good linearity and LODs in the low micrograms per liter range.     

LLE for the extraction of alcohol from aqueous solutions with diethyl ether and dichloromethane at 293.15 K, parameter estimation using a hybrid genetic based approach

Experimental liquid-liquid equilibrium (LLE) data for the extraction of methanol, ethanol and 1-propanol from water by diethyl ether and dichloromethane at 293.15 K and at ambient pressure were investigated. Data for the binodal curves have been determined by cloud-point titration method and conjugate points on tie-line were obtained by correlating the refractive index of the binodal curves as a function of composition. The experimental ternary (liquid + liquid) equilibrium data have been estimated using the NRTL and UNIQUAC activity coefficient models to obtain the binary interaction parameters of these components by a combination of Levenberg-Marquardt method and the genetic algorithm based method. The distribution coefficients and the selectivity factor of the solvent used were calculated and presented. From our experimental and calculated results, we conclude that for the extraction of alcohol from aqueous solutions with dichloromethane solvent has a higher selectivity factor than the diethyl ether solvent.     

Preparation and evaluation of o‐phenanthroline immobilized on a hybrid silica monolith modified with ionic liquids for reversed‐phase pressurized capillary electrochromatography

A novel o‐phenanthroline‐immobilized ionic‐liquid‐modified hybrid monolith for capillary electrochromatography was synthesized based on chloropropyl‐silica, which was prepared by the in situ polymerization of tetramethoxysilane and 3‐chloropropyltrimethoxysilane via a sol–gel process. The morphology of the hybrid monolith was characterized by scanning electron microscopy, and relatively stable anodic electroosmotic flow was observed under a broad pH ranged from pH 3.0 to 9.0. The separation mechanism was investigated by separating four neutral molecules (toluene, dimethylformamide, formamide, and thiourea). The obtained hybrid monolith possessed an obviously reversed‐phase retention mechanism, but when the acetonitrile content in the mobile phase was >90% v/v, a weak hydrophilic mechanism was observed on the resultant o‐phenanthroline‐modified chloropropyl‐silica hybrid monolith. The reproducibility of the column was also investigated by measuring relative standard deviations of the migration time for four neutral molecules. Relative standard deviations of run to run (n = 3), day to day (n = 3), and column to column (n = 3) were in the range of 0.4–0.7, 0.9–2.1, and 1.4–3.3%, respectively. Basic separations of various polar analytes including phenols and aromatic amines were successfully achieved.     

Development and validation of a direct plasma injection LC-MS method for YM087 and YM440 and metabolites in human plasma

Summary A fully automated, direct plasma-injection technique using an LC coupled to a quadrupole mass spectrometric detector with an on-line, sample clean-up procedure for two new pharmaceutical products, YM087 and YM440, has been developed. Plasma samples containing YM440 were mixed with internal standard or plasma containing YM087 and were injected into a chromatographic system based on two coupled columns. An extraction column packed with alkyl-diol-silica (ADS) was used for online sample cleanup. Using a back-flush technique analytes were subsequently transferred to an analytical column, for separation. Detection was based on tandem mass spectrometry either in electronspray or in APCI mode. Despite the relatively small volumes of plasma injected, reasonably low limits of quantitation were achieved. Validation of both assays was performed using guidelines concerning method validation. All parameters studied were within acceptance criteria. The methods were successfully applied to clinical pharmacokinetic studies.     

Evaluation of a sensitive HPLC method for the determination of Malondialdehyde, and application of the method to different biological materials

Summary Reactive oxygen species (ROS), important mediators of cell and tissue injury during inflammation, are produced by several types of inflammatory cells. The formation of ROS can be monitored by detection of lipid peroxidation products. The extremely broad spectrum of biological effects of aldehydic lipid peroxidation products has necessitated the development of a technique that enables the sensitive routine quantitation of aldehydes formed in biological materials. Malondialdehyde (MDA) is a by-product of enzymatic eicosanoid formation and an end-product of nonenzymatic peroxidation of polyunsaturated fatty acids with three or more bisallylic double bonds. The determination of the thiobarbituric acid derivative of MDA (TBA-MDA) is a widely used method for estimating overall lipid peroxidation. We describe a rapid, isocratic, simple, and sensitive high-performance liquid chromatographic (HPLC) method with spectrofluorimetric detection for measurement of MDA-TBA in human biological samples such as plasma, urine, wound secretions, amniotic fluid, sputum and tissue samples. By use of this method, picomole quantities of MDA can be readily and specifically detected in different biological materials. Coefficients of variation of repeated MDA-TBA assays were 4.4% within run and 6.9% from run to run. Reference values are given for a variety of human body fluids and for rat tissues.     

Development of validated LC-MS/MS method for imidacloprid and acetamiprid in parsley and rocket and evaluation of their dissipation dynamics

Fast and efficient method based on quick, easy, cheap, effective, rugged and safe followed by liquid chromatography–tandem mass spectrometry for acetamiprid and imidacloprid residues determination in parsley and rocket was developed and validated. Linearity (R²) ranged from 0.996 to 0.999. Accuracy ranged from 95.9 to 99.1%. Precision was <11%. Limit of quantification was 2.8–10.3?µg/kg. Matrix effect was evaluated. Dissipation of acetamiprid and imidacloprid in parsley and rocket was investigated under open field conditions. The dissipation behavior of both compounds followed first-order kinetics. Half-lives (t_1/2) were 2.68 and 4.24 days with dissipation rate (k) values of 0.25 and 0.165 days^?1 for acetamiprid; 0.24 (in parsley) and 0.25 (in rocket) days^?1 with k values of 0.636 (in parsley) and 0.718 (in rocket) days for imidacloprid. Preharvest interval values were 5.53 and 1.42 days for acetamiprid; 2.5 and 0.49 days for imidacloprid in parsley and rocket, respectively. Residues of both compounds were searched in real samples. None of the real samples violated maximum residues limits of the European Commission regulations.     

A highly sensitive LC–MS/MS method for simultaneous determination of glycyrrhizin and its active metabolite glycyrrhetinic acid: Application to a human pharmacokinetic study after oral administration

A highly sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method for simultaneous determination of glycyrrhizin (GL) and its active metabolite, glycyrrhetinic acid (GA), from human plasma was validated and applied to a human pharmacokinetic study. The analytes were extracted from human plasma using an Oasis MAX cartridge and chromatographic separation was performed on an Inertsil ODS‐3 column. The detection was performed using an API 4000 mass spectrometer operating in the positive electrospray ionization mode. Selected ion monitoring transitions of m /z 823 → 453 for GL and m /z 471 → 149 for GA were obtained. The response was a linear function of concentration over the ranges of 0.5–200 ng/mL for GL and 2–800 ng/mL for GA (both R ² > 0.998). Using this method, the pharmacokinetics of GL after single oral administration of a clinical dose (75 mg) to six healthy male Japanese volunteers were evaluated. GL was detected in the plasma of all subjects and the average peak concentration was 24.8 ± 12.0 ng/mL. In contrast, peak concentration of GA was 200.3 ± 60.3 ng/mL, i.e. ~8‐fold higher than that of GL. This is the first report clarifying pharmacokinetic profiles of GL and GA simultaneously at a therapeutic oral dose of a GL preparation.     

Risk assessment,development and validation of a GC-ECD-based method for the quantification of cypermethrin from green pea

The present work describes the persistence, dissipation behaviour, half-life, risk assessment and novel gas chromatography method for the residue estimation of cypermethrin in green pea by spraying cypermethrin 10EC at 50 g a.i. ha⁻¹ at fruiting stage followed by another application at a 10 day interval. The sample extraction and cleanup was followed bya modified quick, easy, cheap, effective, rugged, and safe method, and the residues of cypermethrin were determined using a validated gas chromatography method. The initial deposits were found to be 1.21 mg kg⁻¹ following the application of insecticide at 50 g a.i. ha⁻¹. Cypermethrin residues declined to below the detection limit of 0.05 mg kg⁻¹ after 15 days at the recommended dosage. The half-life of cypermethrin was 2.66 days at 50 g a.i. ha⁻¹. For risk assessment studies, the waiting period of 15 days is recommended as safe for consumption for the insecticide. The GC-ECD method was validated according to the SANTE guidelines by various analytical parameters including linearity, accuracy, detection and quantification limits. The developed method is simple, selective and repeatable, and can be used for the standardization of pesticides on fruits and vegetables.