Comparative study of drug release from electrospun nanofibers loaded with clotrimazole via two different approaches using a fully automated sequential injection system.

The development of new drug delivery platforms including the use of nanotechnology has been found of great interest in recent years. Two different loading approaches of the model antimycotic drug clotrimazole into the nanofibrous polycaprolactone and polydioxanone structures including electrospinning of a drug-polymer blend and impregnation of nanofibers with drug have been tested. The final amount of clotrimazole in the nanofibrous materials was determined by HPLC analysis and Raman spectroscopy. The electrospinning of blend approach allowed the adsorption of clotrimazole in a quantity of up to 30 % using mixtures with polymer/clotrimazole ratios from 2:1 to 8:1 (w/w). Ethanolic clotrimazole solutions with concentrations from 2.5 to 3.5 mg L-1 were used for adsorbing clotrimazole in blank nanofibers for 1-3 h with final clotrimazole content ranging from 3.0 to 5.7 %. Furthermore, a comparative liberation study including comparison with commercially available creams was carried out in low pressure flow system. The results obtained confirmed well controlled release of clotrimazole from both types of nanofibers. Compared to commercial pharmaceutical formulations containing 1 % clotrimazole where first-order release kinetics was observed, nanofibrous materials provided linear controlled release (zero-order kinetics) in the tested 3 h period.

A stationary phase with a positively charged surface allows for minimizing formic acid concentration in the mobile phase, enhancing electrospray ionization in LC-MS proteomic experiments.

The default choice of mobile phase acidifier for bottom-up LC-MS proteomic analyses is 0.10% formic acid because of its decent acidity, decent ion pairing ability, and low suppression of electrospray ionization. In recent years, state-of-the-art columns have been designed specifically to provide efficient separation even when using an MS-friendly mobile phase of low ionic strength. Despite this, no attempts have been made to improve the sensitivity of the MS-based analytical methods by reducing the amount of formic acid in the mobile phase. In this study, we evaluated the effect of reduced formic acid concentration in the mobile phase on the chromatographic behavior and MS response of peptides when separated using columns packed with a C18 stationary phase with a positively charged surface. Using 0.01% formic acid in the mobile phase maintained excellent chromatographic performance and increased MS signal response compared to the standard of 0.10%. The enhanced MS response translated to about 50% improved peptide identifications depending on the complexity and amount of sample injected. The increased retention of peptides at a reduced formic acid concentration was directly proportional to the number of acidic residues in the peptide sequence. The study was carried out by covering a spectrum of protein samples with varied complexity using analytical flow, micro-, and nanoflow regimes to expand the applicability in routine practice.

Longevity and other practical benefits of monolithic silica columns in the analysis of samples with complex matrices.

At the turn of the millennium, the monolithic columns invoked new chances in HPLC. Even more than their organic polymer-based siblings, the inorganic silica-based monoliths targeted the territory of classical fully porous particle-packed columns, promising many benefits. Based on the number of published articles, the monoliths attracted academics just in the first few years after their introduction to the market. Lately, as superficially porous particles and sub-2-micron fully porous particles dominated the market, they stayed in the focus of routine laboratories and those who really appreciated the high porosity of the monolithic bed. The monoliths' practical benefits cannot be easily traced in the literature when they gradually lose academics' interest. Nevertheless, after more than 20 years of our experience, we still favor silica monoliths for their low back pressure and longevity when analyzing samples of clinical, pharmaceutical, and environmental origin. At the same time, the high permeability of monoliths enabled the birth of sequential injection chromatography, the medium-pressure separation technique based on the flexible flow manifold. This minireview aims to check, discuss, and summarize the practical aspects of monolithic silica columns in HPLC and medium-pressure sequential injection chromatography (SIC) that may not be visible at first sight but are evident retrospectively.

Implementing reversed-phase and hydrophilic interaction liquid chromatography into the characterization of DTPA-ramucirumab conjugate before radiolabeling.

Radioimmunoconjugates represent a promising class of therapeutics and diagnostics. The characterization of intermediate chelator-antibody products, i.e., without the radionuclide, is frequently omitted, bringing significant uncertainty in the radioimmunoconjugate preparation. In the present study, we explored the utility of reversed-phase (RPLC) and hydrophilic interaction (HILIC) liquid chromatography with UV detection to characterize ramucirumab stochastically conjugated with p-SCN-Bn-CHX-A"-DTPA chelator (shortly DTPA). The conjugation was well reflected in RPLC chromatograms, while chromatograms from HILIC were significantly less informative. RPLC analyses at the intact level confirmed that the conjugation resulted in a heterogeneous mixture of modified ramucirumab. Moreover, the RPLC of DTPA-ramucirumab confirmed heterogeneous conjugation of all subunits. The peptide mapping did not reveal substantial changes after the conjugation, indicating that most parts of ramucirumab molecules remained unmodified and that the DTPA chelator was bound to various sites. Eventually, the RPLC method for analysis of intact ramucirumab was successfully applied to online monitoring of conjugation reaction in 1 h intervals for a total of 24 h synthesis, which readily reflected the structural changes of ramucirumab in the form of retention time shift by 0.21 min and increase in peak width by 0.22 min. The results were obtained in real-time, practically under 10 min per monitoring cycle. To the best of our knowledge, our study represents the first evaluation of RPLC and HILIC to assess the quality of intermediates during the on-site preparation of radioimmunoconjugates prior to radiolabeling.

Easy, Robust, and Repeatable Online Acid Cleavage of Proteins in Mobile Phase for Fast Quantitative LC-MS Bottom-Up Protein Analysis─Application for Ricin Detection.

Sample preparation involving the cleavage of proteins into peptides is the first critical step for successful bottom-up proteomics and protein analyses. Time- and labor-intensiveness are among the bottlenecks of the commonly used methods for protein sample preparation. Here, we report a fast online method for postinjection acid cleavage of proteins directly in the mobile phase typically used for LC-MS analyses in proteomics. The chemical cleavage is achieved in 0.1% formic acid within 35 s in a capillary heated to 195 °C installed upstream of the analytical column, enabling the generated peptides to be separated. The peptides generated by the optimized method covered the entire sequence except for one amino acid of trastuzumab used for the method development. The qualitative results are extraordinarily stable, even over a long period of time. Moreover, the method is also suitable for accurate and repeatable quantification. The procedure requires only one manual step, significantly decreasing sample transfer losses. To demonstrate its practical utility, we tested the method for the fast detection of ricin. Ricin can be unambiguously identified from an injection of 10 ng, and the results can be obtained within 7-8 min after receiving a suspicious sample. Because no sophisticated accessories and no additional reagents are needed, the method can be seamlessly transferred to any laboratory for high-throughput proteomic workflows.

Green Solvents in the Extraction of Bioactive Compounds from Dried Apple Cultivars.

New extraction protocols, gas-expanded liquid extraction (GXLE), and ultrasound extraction (UE) have been optimized with an emphasis on using green solvents and maximizing the extraction of 14 selected phenolic compounds, including flavonoid-based compounds and phenolic acids from dried apples. The design of the experiments' approach was applied to optimize the main extraction parameters. Fine tuning included optimization of the flow rate in GXLE and the extraction time for GXLE and UE. Optimized GXLE was carried out with CO2-ethanol-water (34/53.8/12.2; v/v/v) at a flow rate of 3 mL/min at a temperature of 75 °C and pressure of 120 bar for 30 min. UE with ethanol-water 26/74 (v/v) lasted for 10 min at 70 °C. Both methods differed in solvent consumption and sample throughput, while providing a comparable total phenolic content of 2442 µg/g with an RSD < 10% and 2226 µg/g with RSD < 6%, for GXLE and UE, respectively. Both methods were used in determining the phenolic compounds in five apple cultivars, 'Angold', 'Artiga', 'Golden Delicious', 'Meteor', and 'Topaz'. Phenolic profiles were plotted with chlorogenic acid, catechin, epicatechin, hirsutrin, phloridzin, and guaiaverin as the main components. Statistical evaluation, including pair t-test, Bland-Altman test, and linear regression did not reveal any differences between UE and GXLE results.

A simple method to quantify azo dyes in spices based on flow injection chromatography combined with chemometric tools.

Para Red (PR) and Sudan dyes have been illegally used as colorants to adulterate certain foods by enhancing their red/orange colour. In addition, they are toxic and carcinogenic. This work presents the development of a simple flow injection chromatographic method combined with chemometric tools to perform the determination of PR, Sudan I (SI) and Sudan II (SII) in food samples. The flow chromatographic system consisted of a low-pressure manifold coupled to a reverse phase monolithic column. A Partial Least Square (PLS) model was applied to resolve overlapped absorption spectra registered for each dye at the corresponding retention time. The relative errors of calibration (RMSECV, %) were 0.49, 0.85 and 0.23, and the relative errors of prediction (RMSEP, %) were 1.12, 0.75 and 0.33 for PR, SI and SII, respectively. The residual predictive deviation (RPD) values obtained were higher than 3.00 for all analytes. The method was successfully applied to quantify the dyes in six different commercial spices samples. The results were compared with the HPLC reference method concluding that there were no significant differences at the studied confidence level (α = 0.05). The proposed method can be used to rapidly determine the analytes in a simple, reliable, low-cost and environmentally-friendly manner. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-021-05299-8.

Effect of storage conditions on content of pesticide residues in sweet cherries.

Dynamics of pesticides decomposition in sweet cherry fruits at different technologies of long-term storage, ultra-low oxygen and modified atmosphere packing, and after post-harvesting application of 1-methylcyclopropen and ozone has been studied. We assumed that type of pesticide and fruit storage conditions may have a profound effect on pesticide residues content. Therefore, levels of residues after applying combinations of active ingredients including acetamiprid, boscalid, cyprodinil, fenhexamid, fenpyrazamine, fludioxonil, fluopyram, pyraclostrobin, pirimicarb, tebuconazole, thiacloprid, and trifloxystrobin were monitored. We found these contents below tolerated maximum residue limits when applied at recommended times and depended on period prior to withdrawal. Low contents of acetamiprid, boscalid, fenpyrazamine, thiacloprid, pirimicarb, and fludioxonil were found when fruit were stored in modified atmosphere packages. Ozone affected degradation of tebuconazole, pyraclostrobin, and cyprodinil. However, differences between storage regimens were not statistically significant (p ≥ 0.05). Kinetic of degradation was studied with fruits stored after treatment with 1-methylcyclopropen and ozone.

Real-time monitoring of Metridia luciferase release from cells upon interaction with model toxic substances by a fully automatic flow setup - A proof of concept.

This manuscript reports on a fully automatic sequential injection system incorporating a 3D printed module for real-time monitoring of the release of Metridia luciferase from a modified liver epithelial cell line. To this end, a simple and effective approach for the automation of flash-type chemiluminescence assays was developed. The 3D printed module comprised an apical and a basal compartment that enabled monitoring membrane processes on both sides of the cell monolayer aimed at elucidating the direction of luciferase release. A natural release was observed after transfection with the luciferase plasmid by online measurement of the elicited light from the reaction of the synthesized luciferase with the coelenterazine substrate. Model substances for acute toxicity from the group of cholic acids - chenodeoxycholic and deoxycholic acids - were applied at the 1.0 and 0.5 mmol L-1 levels. The tested cholic acids caused changes in cell membrane permeability that was accompanied by an increased luciferase release. The obtained kinetic profiles were evaluated based on the delay between the addition of the toxic substance and the increase of the chemiluminescence signal. All experiments were carried out in a fully automatic system in ca. 5 min per sample in 30 min intervals and no manual interventions were needed for a sampling period of at least 6 h.

UHPLC coupled with charged aerosol detector for rapid separation of steviol glycosides in commercial sweeteners and extract of Stevia rebaudiana.

Natural sweeteners are in high demand as a part of a healthy lifestyle. Among them, sweeteners with decreased caloric value and suitability for diabetes patients are most requested. Extension in their consumption extends the need for their quality control. A fast gradient UHPLC coupled with charged aerosol detection enabling quantitation of stevioside, rebaudioside A-D, and steviolbioside in commercial sweeteners and Stevia rebaudiana plant extracts has been developed. The method was developed to achieve high efficiency, simplicity, versatility, and low solvent consumption. All steviol glycosides were baseline-separated in less than 4 min with a total run time of 7 min. Buffer-free eluents were used in the separations and only 2.45 mL solvent were needed per analysis. The Luna Omega Polar column featuring polar modification of the C18 stationary phase was employed with mobile phases composed of water and acetonitrile for the excellent separation of polar steviol glycosides. The flow rate of the mobile phase 0.35 mL/min, column temperature 50 °C and injection volume 2 µL were used. Critical pair of glycosides, stevioside and rebaudioside A, were baseline separated with a resolution of 2.41. The universal charged aerosol detector allowed quantitation of steviol glycosides with a limit of detection and quantitation 0.15 and 0.5 µg/mL, respectively. Method intra-day precision was less than 2% (RSD), and the recovery was 89.6-105.0% and 93.8-111.4% for plant material and sweetener tablets, respectively. The quantity of steviol glycosides in three out of four commercial sweeteners was 3.0-12.3% higher than declared. The content was about 12.4% less than declared in one sample. But the difference from the labeled content corresponded to trueness and precision of the developed method together with variability of sweeteners production. The most abundant glycoside detected in sweeteners was stevioside followed by rebaudioside A. A leaf-to-stem ratio describing the dominant accumulation of steviol glycosides in leaves affected the differences in the amount of steviol glycosides among plant samples.

Sequential Injection Analysis for Automation and Evaluation of Drug Liberation Profiles: Clotrimazole Liberation Monitoring.

A fully automated sequential injection system was tested in terms of its application in liberation testing, and capabilities and limitations were discussed for clotrimazole liberation from three semisolid formulations. An evaluation based on kinetic profiles obtained in short and longer sampling intervals and steady-state flux values were applied as traditional methods. The obtained clotrimazole liberation profile was faster in the case of Delcore and slower for Clotrimazol AL and Canesten cream commercial formulations. The steady-state flux values for the tested formulations were 52 µg cm-2 h-1 for Canesten, 35 µg cm-2 h-1 for Clotrimazol AL, and 7.2 µg cm-2 h-1 for Delcore measured in 4 min sampling intervals. A simplified approach for the evaluation of the initial rate based on the gradient between the second and third sampling points was used for the first time and was found to correspond well with the results of the conventional methods. A comparison based on the ratio of the steady-state flux and the initial rate values for Canesten and Clotrimazol AL proved the similarity of the obtained results. The proposed alternative was successfully implemented for the comparison of short-term kinetic profiles. Consequently, a faster and simpler approach for dissolution/liberation testing can be used.

Benefits and Pitfalls of HPLC Coupled to Diode-Array, Charged Aerosol, and Coulometric Detections: Effect of Detection on Screening of Bioactive Compounds in Apples.

The new screening method for rapid evaluation of major phenolic compounds in apples has been developed. Suitability of coupling HPLC/UHPLC separation with the diode-array detection and universal charged aerosol detection with respect to the presence of interfering substances was tested. Characteristics of both detection techniques were compared and method linearity, limits of detection and quantitation, and selectivity of them determined. Student t-test based on slopes of calibration plots was applied for the detailed comparison. The diode-array detection provided the best results regarding sensitivity and selectivity of the developed method in terms of evaluation of phenolics profiles. The response of the charged aerosol detector was negatively affected by co-eluting substances during rapid-screening analyses. Coulometric detection was used for advanced characterization of extracts in terms of antioxidant content and strength to obtain more complex information concerning sample composition. This detection also allowed evaluation of unidentified compounds with antioxidant activity. HPLC/UHPLC separation using a combination of diode-array and coulometric detectors thus represented the best approach enabling quick, yet complex characterization of bioactive compounds in apples.

3D printed permeation module to monitor interaction of cell membrane transporters with exogenic compounds in real-time.

A new design of permeation module based on 3D printing was developed to monitor the interaction of exogenic compounds with cell membrane transporters in real-time. The fluorescent marker Rhodamine 123 (Rho123) was applied as a substrate to study the activity of the P-glycoprotein membrane transporter using the MDCKII-MDR1 genetically modified cell line. In addition, the inhibitory effect of verapamil (Ver), a prototype P-glycoprotein inhibitor, was examined in the module, demonstrating an enhanced Rho123 transfer and accumulation into cells as well as the applicability of the module for P-glycoprotein inhibitor testing. Inhibition was demonstrated for different ratios of Rho123 and Ver, and their competition in terms of interaction with the P-glycoprotein transporter was monitored in real-time. Employing the 3D-printed module, permeation testing was shortened from 8 h in the conventional module to 2 h and evaluation based on kinetic profiles in every 10 min was possible in both donor and acceptor compartments. We also show that monitoring Rho123 levels in both compartments enables calculate the amount of Rho123 accumulated inside cells without the need of cell lysis.

Lab-In-Syringe for automated double-stage sample preparation by coupling salting out liquid-liquid extraction with online solid-phase extraction and liquid chromatographic separation for sulfonamide antibiotics from urine.

A double-stage Lab-In-Syringe automated extraction procedure coupled online to HPLC for the determination of four sulfonamides in urine has been developed. Our method is based on homogeneous liquid-liquid extraction at pH 3 using water-miscible acetonitrile with induction of phase separation by the addition of a saturated solution of kosmotropic salts MgSO4 and NaCl. The procedure allowed extraction of the moderately polar model analytes and the use of a solvent that is compatible with the used separation technique. The automated sample preparation system based on the stirring-assisted Lab-In-Syringe approach was coupled on-line with HPLC-UV for the subsequent separation of the sulfonamide antibiotics. To improve both preconcentration factor and extract cleanup, the analytes were trapped at pH 10 in an anion-exchange resin cartridge integrated into the HPLC injection loop thus achieving a double-stage sample clean-up. Analytes were eluted using an acidic HPLC mobile phase in gradient elution mode. Running the analytes separation and the two-step preparation of the following sample in parallel reduced the total time of analysis to mere 13.5 min. Limits of detection ranged from 5.0 to 7.5 µg/L with linear working ranges of 50-5000 µg/L (r2 > 0.9997) and RSD ≤ 5% (n = 6) at a concentration level of 50 µg/L. Average recovery values were 102.7 ± 7.4% after spiking of urine with sulfonamides at concentrations of 2.5 and 5 mg/L followed by 5 times dilution. To the best of our knowledge, the use of Lab-In-Syringe for the automation of coupled homogeneous liquid-liquid extraction and SPE for preparation of the complex matrices suitable for separation techniques is here presented for the first time.

A flow-based platform hyphenated to on-line liquid chromatography for automatic leaching tests of chemical additives from microplastics into seawater.

An automatic flow-based system as a front end to liquid chromatography (LC) for on-line dynamic leaching of microplastic materials (polyethylene of medium density and poly(vinyl chloride)) with incurred phthalates and bisphenol A is herein presented. The microplastic particles were packed in a metal column holder, through which seawater was pumped continuously by resorting to advanced flow methodology. Each milliliter of the leachable (bioaccessible) fraction of chemical additives was preconcentrated on-line using a 10 mm-long octadecyl monolithic silica column placed in the sampling loop of the injection valve of a HPLC system that served concomitantly for analyte uptake and removal of the seawater matrix. After loading of the leachate fraction, the LC valve was switched to the inject position and the analytes were eluted and separated by a monolithic column (Onyx C18HD 100 × 4.6 mm) using an optimized acetonitrile/water gradient with UV detection at 240 nm. The automatic flow method including dynamic flow-through extraction, on-line sorptive preconcentration, and matrix clean-up was synchronized with the HPLC separation, which lasted ca. 9 min. The only two currently available multi-component certified reference materials (CRM) of microplastics (CRM-PE002 and CRM-PVC001) were used for method development and validation. Out of the eight regulated phthalates contained in the two CRMs, only the 2 most polar species, namely, dimethyl phthalate and diethyl phthalate as well as bisphenol A, were leached significantly by the seawater in less than 2 h, with bioaccessibility percentages of 51-100%. The leaching profiles were monitored and modeled with a first-order kinetic equation so as to determine the rate constants for desorption in a risk assessment scenario. Intermediate precision values of bioaccessibility data for three batches of CRMs were for the suite of targeted compounds ≤22%. This work for the first time reports a fully automatic flow method with infinite sink capacity (i.e., using a surplus of extracting solution) for the target species able to mimic the leaching of additives from plastic debris across the water body in marine settings under worst-case extraction conditions.

Fully automated method based on on-line molecularly imprinted polymer solid-phase extraction for determination of lovastatin in dietary supplements containing red yeast rice.

A fully automated method for the determination of lovastatin in dietary supplements containing red yeast rice has been developed. It uses a sequential injection analysis system combined with solid-phase extraction applying highly selective molecularly imprinted polymer sorbent. A miniaturized column for on-line extraction was prepared by packing 4.5 mg of the sorbent in a 5.0 × 2.5-mm-i.d. cartridge, which was used in the flow manifold. Sequential injection analysis manifold enabled all steps of lovastatin extraction and continuous spectrophotometric detection at 240 nm. A limit of detection of 60 µg g-1, a limit of quantitation of 200 µg g-1, and a linear calibration range of 200-2000 µg g-1 were achieved. Intra-day and inter-day precision values (RSD) were ≤ 6.7% and ≤ 4.9%, respectively, and method recovery values of spiked red yeast rice extracts at 200, 1000, and 2000 µg g-1 concentration levels were 82.9, 95.2, and 87.7%. Our method was used for determination of lovastatin lactone in four dietary supplements containing red yeast rice as a natural source of lovastatin, also known as monacolin K. The extracted samples were subsequently analyzed by the reference UHPLC-MS/MS method. Statistical comparison of results (F test, t test, α = 0.05) obtained by both methods did not reveal significant difference. A substantial advantage of the new automated approach is high sample throughput thanks to the analysis time of 7.5 min, miniaturization via down-scaling the extraction column, and smaller sample and solvent consumption, as well as reduced generation of waste. Graphical abstract ᅟ.

Determination of major phenolic compounds in apples: Part I-Optimization of high-performance liquid chromatography separation with diode array detection.

The separation of seven phenolic compounds including gallic acid, chlorogenic acid, epicatechin, quercitrin, rutin, phloridzin, and phloretin present in apple peel and pulp and differing in elution properties has been optimized using high-performance liquid chromatography with diode array detection. Several stationary phases were tested to achieve the efficient separation of phenolic compounds in fruit extracts and C18 was found to be the most efficient. Core-shell and fully porous C18 packings were assessed with respect to the complex composition of the fruit extracts. The developed high-performance liquid chromatography method comprised gradient elution in which mobile phase A was water at pH 2.8 adjusted with acetic acid and B was acetonitrile. The gradient shape was the following: 0 min 95% A/5% B, 2.5 min 85% A/15% B, 12 min 50% A/50% B, 15 min 95% A/5% B. The flow rate was 1 mL/min, injection volume 10 µL, and UV detection at 255, 280, 320, and 365 nm was applied. Our method was validated for both C18 core-shell and fully porous packings. The resolution 6.2-14.8, symmetry 0.99-1.34, peak capacity 18-60, peak area repeatability 0.45-1.00% relative standard deviation, calibration range 0.125-5 mg/mL (0.25-10 mg/mL for chlorogenic acid and rutin), correlation coefficients of calibration curve 0.9976-0.9997, and accuracy evaluated as recovery 95.56-107.54% were determined for the core-shell column.

Direct-immersion single-drop microextraction and in-drop stirring microextraction for the determination of nanomolar concentrations of lead using automated Lab-In-Syringe technique.

Two operational modes for Lab-In-Syringe automation of direct-immersion single-drop microextraction have been developed and critically compared using lead in drinking water as the model analyte. Dithizone was used in the presence of masking additives as a sensitive chromogenic complexing reagent. The analytical procedure was carried out inside the void of an automatic syringe pump. Normal pump orientation was used to study extraction in a floating drop of a toluene-hexanol mixture. Placing the syringe upside-down allowed the use of a denser-than-water drop of chloroform for the extraction. A magnetic stirring bar was placed inside the syringe for homogenous mixing of the aqueous phase and enabled in-drop stirring in the second configuration while resulting in enhanced extraction efficiency. The use of a syringe as the extraction chamber allowed drop confinement and support by gravitational differences in the syringe inlet. Keeping the stirring rates low, problems related to solvent dispersion such as droplet collection were avoided. With a drop volume of 60 µL, limits of detection of 75 nmol L-1 and 23 nmol L-1 were achieved for the floating drop extraction and the in-drop stirring approaches, respectively. Both methods were characterized by repeatability with RSD typically below 5%, quantitative analyte recoveries, and analyte selectivity achieved by interference masking. Operational differences were critically compared. The proposed methods permitted the routine determination of lead in drinking water to be achieved in less than 6 min.

Flow-batch analysis of clenbuterol based on analyte extraction on molecularly imprinted polymers coupled to an in-system chromogenic reaction. Application to human urine and milk substitute samples.

A fully automated spectrophotometric method based on flow-batch analysis has been developed for the determination of clenbuterol including an on-line solid phase extraction using a molecularly imprinted polymer (MIP) as the sorbent. The molecularly imprinted solid phase extraction (MISPE) procedure allowed analyte extraction from complex matrices at low concentration levels and with high selectivity towards the analyte. The MISPE procedure was performed using a commercial MIP cartridge that was introduced into a guard column holder and integrated in the analyzer system. Optimized parameters included the volume of the sample, the type and volume of the conditioning and washing solutions, and the type and volume of the eluent. Quantification of clenbuterol was carried out by spectrophotometry after in-system post-elution analyte derivatization based on azo-coupling using N- (1-Naphthyl) ethylenediamine as the coupling agent to yield a red-colored compound with maximum absorbance at 500nm. Both the chromogenic reaction and spectrophotometric detection were performed in a lab-made flow-batch mixing chamber that replaced the cuvette holder of the spectrophotometer. The calibration curve was linear in the 0.075-0.500mgL-1 range with a correlation coefficient of 0.998. The precision of the proposed method was evaluated in terms of the relative standard deviation obtaining 1.1% and 3.0% for intra-day precision and inter-day precision, respectively. The detection limit was 0.021mgL-1 and the sample throughput for the entire process was 3.4h-1. The proposed method was applied for the determination of CLB in human urine and milk substitute samples obtaining recoveries values within a range of 94.0-100.0%.

Online coupling of fully automatic in-syringe dispersive liquid-liquid microextraction with oxidative back-extraction to inductively coupled plasma spectrometry for sample clean-up in elemental analysis: A proof of concept.

A proof of concept of a novel automatic sample cleanup approach for metal assays in troublesome matrixes as a front-end sample pre-treatment to inductively coupled plasma optical emission spectroscopy - ICP-OES - is herein presented. Target metals, namely, copper, lead, and cadmium were complexed in-system quantitatively using ammonium pyrrolidine dithiocarbamate (APDC) and transferred into a minute volume of toluene as extractant employing lab-in-syringe magnetic stirring-assisted dispersive liquid-liquid microextraction (LIS-MSA-DLLME). After discharge of the sample, the analytes were back-extracted into nitric acid and injected on-line into ICP-OES. To promote and expedite this process in-syringe, advantage was taken from oxidative decomposition of the chelate by potassium iodate, reported in this article for the first time. Experimental conditions for LIS-MSA-DLLME were optimized by Box-Benkhen multivariate analysis using the geometric mean of analyte recoveries as the desirability function. Times of extraction and back-extraction of 300s and 100s, respectively, pH 5.5 at 30mmol/L acetate, 300µL of extraction solvent, and 600µmol/L of APDC were finally applied. Online interfacing to ICP-OES for back-extract analysis yielded average repeatabilities for Cd, Cu, and Pb of 2.9%, 3.5%, and 3.5% with limits of detections (3s) of 1.9, 1.4, and 5.6ng/mL, respectively. Oxidative back-extraction was proven reliable for the determination of metal species in coastal seawater, surrogate digestive fluids and soil leachates with recovery values for Cd, Cu, and Pb ranging from 90% to 118%, 68% to 104%, and 86% to 112%, respectively.