A liquid chromatography-mass spectrometry method for the enantioselective multiresidue determination of nine chiral agrochemicals in urine using an enrichment procedure based on graphitized carbon black.

Many agrochemicals are chiral molecules, and most of them are marketed as racemates or diastereomeric mixtures. Stereoisomers that are not the active enantiomer have little or no pesticidal activity and can exert serious toxic effects towards non-target organisms. Thus, investigating the possible exposure to different isomers of chiral pesticides is an urgent need. The present work was aimed at developing a new enantioselective high-performance liquid chromatography-mass spectrometry method for the simultaneous determination of nine chiral pesticides in urine. Two solid-phase extraction (SPE) procedures, based on different carbon-based sorbents (graphitized carbon black (GCB) and buckypaper (BP)), were developed and compared. By using GCB, all analytes were recovered with yields ranging from 60 to 97%, while BP allowed recoveries greater than 54% for all pesticides except those with acid characteristics. Baseline separation was achieved for the enantiomers of all target agrochemicals on a Lux Cellulose-2 column within 24 min under reversed-phase mode. The developed method was then validated according to the FDA guidelines for bioanalytical methods. Besides recovery, the other evaluated parameters were precision (7-15%), limits of detection (0.26-2.21 µg/L), lower limits of quantitation (0.43-3.68 µg/L), linear dynamic range, and sensitivity. Finally, the validated method was applied to verify the occurrence of the pesticide enantiomers in urine samples from occupationally exposed workers.

Enantiomeric analysis of drugs in water samples by using liquid-liquid microextraction and nano-liquid chromatography.

The nano-LC technique is increasingly used for both fast studies on enantiomeric analysis and test beds of novel stationary phases due to the small volumes involved and the short conditioning and analysis times. In this study, the enantioseparation of 10 drugs from different families was carried out by nano-LC, utilizing silica with immobilized amylose tris(3-chloro-5-methylphenylcarbamate) column. The effect on chiral separation caused by the addition of different salts to the mobile phase was evaluated. To simultaneously separate as many enantiomers as possible, the effect of buffer concentration in the mobile phase was studied, and, to increase the sensitivity, a liquid-liquid microextraction based on the use of isoamyl acetate as sustainable extraction solvent was applied to pre-concentrate four chiral drugs from tap and environmental waters, achieving satisfactory recoveries (>70%).

Carbon nanomaterial-based membranes in solid-phase extraction.

Carbon nanomaterials (CNMs) have some excellent properties that make them ideal candidates as sorbents for solid-phase extraction (SPE). However, practical difficulties related to their handling (dispersion in the atmosphere, bundling phenomena, reduced adsorption capability, sorbent loss in cartridge/column format, etc.) have hindered their direct use for conventional SPE modes. Therefore, researchers working in the field of extraction science have looked for new solutions to avoid the above-mentioned problems. One of these is the design of CNM-based membranes. These devices can be of two different types: membranes that are exclusively composed of CNMs (i.e. buckypaper and graphene oxide paper) and polysaccharide membranes containing dispersed CNMs. A membrane can be used either as a filter, operating under flow-through mode, or as a rotating device, operating under the action of magnetic stirring. In both cases, the main advantages arising from the use of membranes are excellent results in terms of transport rates, adsorption capability, high throughput, and ease of employment. This review covers the preparation/synthesis procedures of such membranes and their potential in SPE applications, highlighting benefits and shortcomings in comparison with conventional SPE materials (especially, microparticles carbonaceous sorbents) and devices. Further challenges and expected improvements are addressed too.

Potentiality of miniaturized techniques for the analysis of drugs of abuse.

Capillary electromigration (CE) and liquid chromatographic techniques (CLC/nano-LC) are miniaturized techniques offering distinct advantages over conventional ones in the field of separation science. Among these, high efficiency, high chromatographic resolution, and use of minute volumes of both mobile phase and sample volumes are the most important. CE and CLC/nano-LC have been applied to the analysis of many compounds including peptides, proteins, drugs, enantiomers, ions, etc. Over the years, the methods described here have also been used for the analysis of compounds of clinical, forensic, and toxicological interest. In this review article, the main features of the mentioned techniques are summarized. Their potentiality for the analysis of drugs of abuse are discussed. Some selected applications in this field in the period of 2015-present are also reported.

Some thoughts about enantioseparations in capillary electrophoresis.

In this overview the goal of the authors was to analyze from the historical perspective the reasons of success and failure of chiral capillary electrophoresis. In addition, the current trends are analyzed, unique advantages of capillary electrophoresis are highlighted and some future directions are discussed.

Enantioseparation of tryptophan and its unnatural derivatives by nano-LC on CSP-teicoplanin silica based.

This work deals with the potentiality of nano liquid chromatography (Nano-LC) for the chiral separation of racemic mixture of tryptophan and some selected derivatives by using 100 µm i.d. fused silica capillary packed with teicoplanin bonded to 5 µm diol silica stationary phase. The experiments were carried out by using a cheap and laboratory-assembled nano-LC-UV system. Elution was done in an isocratic mode using a polar organic mobile phase. In order to find the optimum chiral separation of the studied enantiomers, some chromatographic experimental parameters were systematically studied and optimized. Among them, mobile phase composition, namely organic modifier type and concentration, buffer type and pH and aqueous content and sample solvent dilution on retention time, retention factor and enantioresolution factor were studied. Baseline enantioresolution and good peak shape was achieved utilizing the mobile phase containing 40 mM ammonium formate at pH pH 2.5 in ACN/water/acetone (60:30:10, v/v/v) at 520 nL/min in less than 8 min analysis time.

Analysis of Enantiomers in Products of Food Interest.

The separation of enantiomers has been started in the past and continues to be a topic of great interest in various fields of research, mainly because these compounds could be involved in biological processes such as, for example, those related to human health. Great attention has been devoted to studies for the analysis of enantiomers present in food products in order to assess authenticity and safety. The separation of these compounds can be carried out utilizing analytical techniques such as gas chromatography, high-performance liquid chromatography, supercritical fluid chromatography, and other methods. The separation is performed mainly employing chromatographic columns containing particles modified with chiral selectors (CS). Among the CS used, modified polysaccharides, glycopeptide antibiotics, and cyclodextrins are currently applied.

Oxidized Buckypaper for Stir-Disc Solid Phase Extraction: Evaluation of Several Classes of Environmental Pollutants Recovered from Surface Water Samples.

This paper describes, for the first time, the use of oxidized buckypaper (BP) as a sorbent membrane of a stir-disc solid phase extraction module. The original device, consisting of a BP disc ( d = 34 mm) enveloped in a polypropylene mesh pouch, was designed to extract organic micropollutants (OMPs) from environmental water samples in dynamic mode. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was used to analyze the extracts. Several classes of pesticides and pharmaceuticals were chosen as model compounds to evaluate key parameters affecting the recovery rates. To this end, the effects of adsorption time, desorption time, stirring speed, type and volume of solvent, and sample volume were thoroughly examined. After optimization, a novel and in-depth study was conducted to find a correlation between physicochemical properties of the analytes and extraction yields. Recoveries were mainly governed by a combination of log P and p Ka values. As indicated, hydrophilic compounds with log P < 1 showed poor affinity for the oxidized BP, compounds having log P > 1 exhibited recoveries ranging between 50% and 100% depending on their p Ka, while compounds with p Ka between 6 and 7.5 gave low yields irrespective of their log P. The analytical method was also validated and tested as large scale screening method of OMPs in surface waters. The analysis of real samples revealed the presence of some nonsteroidal anti-inflammatory drugs, sulfonamides, and pesticides at low ng L-1 concentration levels with relative standard deviations lower than 8%.

An overview to nano-scale analytical techniques: Nano-liquid chromatography and capillary electrochromatography.

Nano-liquid chromatography (nano-LC) and CEC are microfluidic techniques mainly used for analytical purposes. They have been applied to the separation and analysis of a large number of compounds, e.g., peptides, proteins, drugs, enantiomers, antibiotics, pesticides, nutraceutical, etc. Analytes separation is carried out into capillaries containing selected stationary phase. The mobile phase is moved either by a pump (nano-LC) or by an EOF, respectively. The two tools can offer some advantages over conventional techniques, e.g., high selectivity, separation efficiency, resolution, short analysis time and consumption of low volumes of mobile phase. Flow rates in the range 50-800 nL/min are usually applied. The low flow rate reduces the chromatographic dilution increasing the mass sensitivity. Special attention must be paid in avoiding peak dispersion selecting the appropriate detector, injector and tube connection. Finally due to the low flow rate these microfluidic techniques can be easily coupled with mass spectrometry.

An attempt for fast separation of enantiomers in nano-liquid chromatography and capillary electrochromatography.

In the present study, an attempt was made to achieve separation of enantiomers within a minute in nano-LC and CEC. In order to achieve this goal several parameters were optimized from the viewpoint of the property of chiral analytes, concentration of the chiral selector in the packing material, capillary dimensions, and separation mode. The enantiomers of several of the applied chiral sulfoxides could be resolved with the analysis time <1 min. Some instrumental obstacles hindering further reduction of analysis time are also highlighted.

Online sample concentration and analysis of drugs of abuse in human urine by micelle to solvent stacking in capillary zone electrophoresis.

A sensitive and rapid CZE-UV method was developed to determine drugs and their metabolites' presence in human urine. Ten drugs of abuse were analyzed including four amphetamines, cocaine, cocaethylene, heroin, morphine, 6-monoacetylmorphine, and 4-methylmethcathinone. An MSS (micelle to solvent stacking) approach was evaluated to enhance method sensitivity. This method considers composition of the micellar sample solution matrix and the injection time. Several analytical conditions influencing the resolution of the drugs mixture as pH and buffer concentration, organic solvent content, were also investigated. The base-line separation of all studied analytes in the same run was achieved within 18 min in an uncoated fused silica capillary (50 µm id × 60 cm) using a background solution containing 50 mM phosphate buffer pH 2.5 and 30% ACN v/v. Other experimental parameters such as applied voltage and capillary temperature were set up at 20 kV and 20°C, respectively. LOD values ranging between 15 and 75 ng/mL for all studied compounds were obtained. From a comparison with conventional CZE, the proposed method provides an increase of sensitivity (39- to 55-fold enhancement factor). Under optimal MSS-CZE conditions, good linearity was achieved (R2 ≤ 0.9998). The method was finally applied to the analysis of urine samples spiked with a standard mixture after a sample pretreatment, reaching satisfactory recovery values.

Rapid determination of nucleotides in infant formula by means of nano-liquid chromatography.

A rapid method for the quantification of five ribonucleotides 5'- monophophates (adenosine, cytidine, guanosine, inosine, uridine, 5'-monophosphate), in infant formula, has been proposed using nano-LC. To separate the studied compounds, capillary columns packed with different C18-based stationary phases were investigated. All the columns tested were laboratory prepared. The experiments were performed in ion-pairing RP chromatographic mode using tetrabutylammonium hydroxide as ion-pairing reagent. The method was developed using a core-shell XB-C18 capillary column with a mobile phase consisting of 5% v/v methanol and 95% v/v 100 mM ammonium formate, pH 8, containing 20 mM tetrabutylammonium hydroxide. All compounds were baseline resolved in less than 5 min with a flow rate of 500 nL/min in isocratic elution mode. Nucleotides were detected at 260 nm. Analytical validation parameters were evaluated. The RSD values for intraday and interday repeatability for retention time and peak area were <2.4 and 4.2%, respectively. The method linearity was good (R(2) < 0.9995) for the studied compounds. LOD and limit of quantitation were 0.25 and 0.50 µg/mL, respectively. The method was applied to the determination of nucleotides in infant formula, subjected to a centrifugal ultrafiltration process, prior their analysis. The amounts found were in agreement to the labeled contents.

Capillary electrochromatography and nano-liquid chromatography coupled to nano-electrospray ionization interface for the separation and identification of estrogenic compounds.

Nano-LC and CEC were coupled to MS through a nanospray or a pressurized liquid-junction interface for the simultaneous separation and determination of 11 estrogenic compounds. Different stationary phases, that is, phenyl, C18, and C18 bidentate silica hydrate, were studied. For both techniques, the phenyl stationary phase was the best option, considering separation efficiency, selectivity, and resolution. Under the optimized conditions, the baseline separation of the target compounds (including estradiol and zearalanol epimers) was achieved in less than 20 min in nano-LC-MS and less than 13 min in CEC-MS. Molecular imprinted polymer SPE was used for extracting the target compounds from mineral water samples with the analysis of nano-LC-MS. The whole molecular imprinted polymer SPE nano-LC-MS method was validated through a recovery study at two levels of concentration. Sensitivity was improved by on-column focusing technique obtaining LODs in the range 1.4-55.4 ng/L.

Determination of estrogenic compounds in milk and yogurt samples by hollow-fibre liquid-phase microextraction-gas chromatography-triple quadrupole mass spectrometry.

An environmentally friendly method based on hollow-fibre liquid-phase microextraction (HF-LPME) was developed for the extraction of selected estrogenic compounds (i.e. four natural sexual hormones: estrone, 17ß-estradiol, 17α-estradiol and estriol; two exoestrogens: 17α-ethynylestradiol and 2-methoxyestradiol; two synthetic stilbenes: dienestrol and hexestrol; and five resorcylic acid lactones: zearalenone, α-zearalanol, ß-zearalanol, α-zearalenol and ß-zearalenol), from whole cow and semi-skimmed goat milk and whole natural yogurt. After the optimization of the sample preparation procedure, spiked extracts were derivatized to their trimethylsilyl products using N,O-bis(trimethylsilyl)trifluoroacetamide reagent and then analyzed by gas chromatography-tandem mass spectrometry (GC-MS/MS). Once optimum extraction conditions were established (protein precipitation with acetonitrile, extraction and the back-extraction in acetonitrile following the HF-LPME procedure), the method was validated and the calibration range, precision and accuracy were studied. The RSD values for the intra- and inter-day precision of the peak areas were in the range 0.65-9.69 and 1.00-11.47 %, respectively. The determination coefficients were higher than 0.991 for method calibration curves while LOD and LOQ values were between 0.06-2.55 and 0.16-6.11 µg/L for whole cow milk, 0.04-1.70 and 0.11-4.86 µg/L for semi-skimmed goat milk and 0.07-3.73 and 0.23-9.81 µg/L for natural yogurt, respectively. Finally, the accuracy and precision of the method were evaluated, obtaining a value in the range 84 81-119 % and RSD values lower than 20 % in all cases.

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, 17ß-estradiol, and estrone), an exoestrogen (17α-etynylestradiol), and a mycotoxin (zearalenone), together with some of their major metabolites (2-methoxyestradiol, α-zearalanol, ß-zearalanol, α-zearalenol, and ß-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.

Capillary electrochromatography-mass spectrometry for the determination of 5-nitroimidazole antibiotics in urine samples.

The separation of eight antibiotics belonging to 5-nitroimidazole family was carried out by means of CEC coupled with MS. Preliminary experiments were carried out with ultraviolet detection in order to select the proper stationary and mobile phase. Among the different stationary phases studied (namely Lichrospher C18, 5 µm particle size; Cogent(TM) Bidentate C18, 4.2 µm; Pinnacle II™ Phenyl, 3 µm; Pinnacle II™ Cyano, 3 µm), Cogent™ Bidentate C18 (4.2 µm) gave the best performance. For CEC-MS coupling, a laboratory assembled liquid-junction-nano-spray interface was used. In order to achieve a good sensitivity, special attention was paid to both optimization of the sheath liquid composition as well as selection of the injection mode. Under optimized CEC-ESI-MS conditions, the separation was accomplished within 22 min by using a column packed with a mixture of Bidentate C18:Lichrospher Silica-60 (5 µm) 3:1 w/w, an inlet pressure of 11 bar, a voltage of 15 kV, and a mobile phase composed by 45:10:45 v/v/v ACN/MeOH/water containing ammonium acetate (5 mM pH 5). A combined hydrodynamic and electrokinetic injection of 8 bar, 15 kV, and 96 s was adopted. The method was validated in terms of repeatability and intermediate precision of retention times and peak areas, linearity, and LODs and LOQs. RSDs values were <2.9% for retention times and <16.1% for peak areas in both intraday and interday experiments. LOQ values were between 0.09 and 0.42 µg/mL for all compounds. Finally, the method was applied to the determination of three most employed 5-nitroimidazole antibiotics (metronidazole, secnidazole, and ternidazole) in spiked urine samples, subjected to a SPE procedure. Recovery values in the 67-103% range were obtained. Furthermore, for the selected antibiotics, CEC-MS(2) spectra were obtained providing the unambiguous confirmation of these drugs in urine samples.

Use of a Novel Sub-2 µm Silica Hydride Vancomycin Stationary Phase in Nano-Liquid Chromatography. II. Separation of Derivatized Amino Acid Enantiomers.

A novel vancomycin silica hydride stationary phase was synthesized and the particles of 1.8 µm were packed into fused silica capillaries of 75 µm internal diameter (I.D.). The chiral stationary phase (CSP) was tested for the separation of some derivatized amino acid enantiomers by using nano-liquid chromatography (nano-LC). Some experimental parameters such as the type and the content of organic modifier, the pH, and the concentration of the buffer added to the mobile phase were modified and the effect on enantioselectivity, retention time, and enantioresolution factor was studied. The separation of selected dansyl amino acids (Dns-AAs), e.g., Asp, Glu, Leu, and Phe in their enantiomers was initially achieved utilizing a mobile phase containing 85% (v/v) methanol (MeOH) and formate buffer measuring the enantioresolution factor and enantioselectivity in the range 1.74-4.17 and 1.39-1.59, respectively. Better results were obtained employing a more polar organic solvent as acetonitrile (ACN) in the mobile phase. Optimum results (Rs 1.41-6.09 and α 1.28-2.36) were obtained using a mobile phase containing formate buffer pH 2.5/water/MeOH/ACN 6:19:12.5:62.5 (v/v/v/v) in isocratic elution mode at flow rate of 130 nL/min.

Enantiomeric separation of new cathinone derivatives designer drugs by capillary electrochromatography using a chiral stationary phase, based on amylose tris(5-chloro-2-methylphenylcarbamate).

In this study, a chiral CEC method for the enantiomeric separation of ten cathinone derivatives, by means of a polysaccharide-based chiral stationary phase, has been developed. Capillary columns of 100 µm id packed with amylose tris(5-chloro-2-methylphenylcarbamate) coated on silica, also called Sepapak 3 or Lux Amylose-2, were used to achieve the enantioseparation of the studied designer drugs. Enantioresolution, chromatographic retention, and separation efficiency were evaluated in dependence of mobile-phase composition in terms of the content of the organic modifier, nature, and pH buffer. To obtain a sensitivity improvement, a field-amplified sample injection was evaluated optimizing the sample solvent composition and injection time. The LODs and LOQs values were in the range 25-100 and 50-150 ng/mL, respectively, for all the racemic compounds. Good results in terms of resolution (Rs ), separation efficiency (N/m), and short analysis times were obtained using a mixture of ACN/methanol/sodium acetate pH 9 (89/10/1, v/v/v). Applying a voltage of 10 kV and a temperature of 20°C, the analyzed cathinone derivatives were separated in their enantiomers in less than 10 min. A study, concerning the method precision, in terms of intra- and interday repeatability and column-to-column reproducibility was carried out in accordance with the analytical procedures for method validation. Intra- and interday repeatability provided RSD values in the ranges 1.1-1.7, 1.3-2.3% for retention time and 1.3-2.6, 2.1-3.4% for peak area, respectively.

Use of novel phenyl-hexyl core-shell particles in nano-LC.

This paper reports the use of novel phenyl-hexyl core-shell particles packed into fused silica capillaries in nano-LC. Capillary columns of different id of 25, 50, 75, 100, and 150 µm were packed employing the slurry packing method. The columns were used for the separation of a model mixture containing five aromatic hydrocarbons. Benzene, toluene, ethylbenzene, n-propylbenzene, and n-butylbenzene were separated utilizing an isocratic elution mode. Mixtures of water/ACN at different ratio were studied to find optimal experimental conditions for baseline separation of all sample components. As expected with this novel stationary phase, an RP chromatographic mechanism was observed. A mixture of water/ACN, 30:70, v/v allowed the complete resolution of the studied analytes. Efficiency increased by decreasing the capillary id recording the highest number of plates per meter with capillaries of 25 µm id. The decrease of the column id also resulted in a flatter dependence of the plate numbers on the linear flow rate of the mobile phase allowing the increase of the flow rate of the mobile phase without significant decrease of efficiency.

Enantiomeric separation of amlodipine and its two chiral impurities by nano-liquid chromatography and capillary electrochromatography using a chiral stationary phase based on cellulose tris(4-chloro-3-methylphenylcarbamate).

In this work, a novel polysaccharide-based chiral stationary phase, cellulose tris(4-chloro-3-methylphenylcarbamate), also called Sepapak 4 has been evaluated for the chiral separation of amlodipine (AML) and its two impurities. AML is a powerful vasodilatator drug used for the treatment of hypertension. Capillary columns of 100 µm id packed with the chiral stationary phase were used for both nano-LC and CEC experiments. The optimization of the mobile phase composed of ACN/water, (90:10, v/v) containing 15 mM ammonium borate pH 10.0 in nano-LC allowed the chiral separation of AML and the two impurities, but not in a single run. With the purpose to obtain the separation of the three pairs of enantiomers simultaneously, CEC analyses were performed in the same conditions achieving better enantioresolution and higher separation efficiencies for each compound. To fully resolve the mixture of six enantiomers, parameters such as buffer pH and concentration sample injection have been then investigated. A mixture of ACN/water (90:10, v/v) containing 5 mM ammonium borate buffer pH 9.0 enabled the complete separation of the three couples of enantiomers in less than 30 min. The optimized CEC method was therefore validated and applied to the analysis of pharmaceutical formulation declared to contain only AML racemate.