Elution Of Analytes Research Articles

An automatic chemiluminescence method based on the multi-pumping flow system coupled with the fluidized reactor and direct-injection detector: Determination of uric acid in saliva samples

A novel approach for the automatic chemiluminescence (CL) analysis of the complex samples is proposed. A multi-pumping flow system was successfully combined with fluidized reactor and direct-injection CL detector. The possibility of the approach was demonstrated on the determination of uric acid in saliva samples. Uric acid is clinically important analyte and its determination in biological fluids is related to human organism dysfunctions, such as gout. For the first time, the fast luminol – N-bromosuccinimide (NBS) reaction in an alkaline medium was used for the CL determination of uric acid in saliva samples. The CL intensity is greatly quenched in the presence of the analyte.The method includes on-line separation of uric acid from the saliva samples based on fluidized beds strategy using anion-exchange resin Dowex® 2×8 followed by the elution and CL determination using a direct-injection CL detector. The stroke pulsations of the solenoid micro-pumps provided the floating of the anion exchange resin in a sample phase and uric acid separation from the sample matrix into a sample pre-treatment block of flow system. To obtain efficient elution of analyte an eluent circulation was applied. Under the optimal conditions, the detector response for uric acid was linear in the logarithmic concentration ranges from 6·10−6 to 1·10−3molL−1. The limit of detection, calculated from a blank test based on 3σ, was 2·10−6molL−1. Fluidized bed strategy allows us to exclude saliva matrices influence on the luminol–NBS CL reaction, which results in improved selectivity. The applicability of the method developed is demonstrated with the help of real sample analysis. The obtained results are confirmed by reference HPLC-UV method.

Enantioseparation of Chiral Sulfoxides on Amylose-Based Columns: Comparison of Normal Phase Liquid Chromatography and Supercritical Fluid Chromatography

We present enantioseparation of a series of racemic sulfoxides on three different amylose-based polysaccharide columns. Two of them have the amylose units modified with dimethylphenyl carbamoyl groups (Chiralpak AD-H and Chiralpak IA), while the third one possesses a carbamoyl moiety with an additional chiral centre (Chiralpak AS-H). The enantioseparation of selected analytes was achieved in high-performance liquid chromatography (HPLC) and the full analyte set was enantiomerically resolved using supercritical fluid chromatography (SFC). Comparing the results obtained in both modes, we show that enantioseparation under SFC conditions is superior to HPLC mode in terms of speed, while retaining excellent enantioselectivity and resolution. Faster elution of analytes was observed on increasing the polarity of the co-solvent (modifier) in the mobile phase. This trend is apparent in both chromatographic modes. Documenting the important role of the additional chiral centre, Chiralpak AS-H provided the best chromatographic parameters resulting in the enantioseparation of all analytes.

Multicore Magnetic Nanoparticles Coated with Oligomeric Micelles: Characterization and Potential for the Extraction of Contaminants over a Wide Polarity Range.

Oligomeric micelles from sodium undecylenate (oSUD) were chemisorbed to magnetic iron oxide nanoparticles (MNPs) through a single-step synthetic route involving the simultaneous nanoparticle formation and functionalization in an aqueous medium. The resulting spherical nanoparticles (MNPs-oSUD) consisted of a concatenation of iron oxide cores, with an average size of 7.7 nm, bound by oSUD micelles (particle average diameter of ca. 200 nm). Micellar coverage was ∼50% of the MNP-oSUD (by weight) and offered multiple retention mechanisms (e.g., dispersion, hydrogen bonding, polar, and ionic) for solute solubilization while keeping it intact during analyte elution. The high density of micelles and variety of interactions provided by this sorbent rendered it highly efficient for the extraction of aromatic amines in a wide polarity range (log Kow values from -0.80 to 4.05) from textiles, urine, and wastewater. Extraction took 5 min, no cleanup or evaporation of the extracts was needed and the method, based on LC-MS/MS quantitation, proved matrix-independent. Recoveries for 17 aromatic amines in samples were in the range of 93%-123% while those with negative log Kow values were in the range of 69%-87%. Detection limits for aromatic amines in textiles (0.007-2 mg kg-1) were well below the limits legislated by the European Union (EU) (30 mg kg-1) and those in urine and wastewater (0.004-1.5 μg L-1) were at the level usually found in real-world applications. All the analyzed samples were positive in aromatic amines. The easy synthesis and excellent extraction properties of MNPs-oSUD anticipate their high potential not only for multiresidue analysis but also in other fields such as water remediation.

A highly selective nanocomposite based on MIP for curcumin trace levels quantification in food samples and human plasma following optimization by central composite design.

A selective and rapid method was developed for quantification of curcumin in human plasma and food samples using molecularly imprinted magnetic multiwalled carbon nanotubes (MMWCNTs) which was characterized with EDX and FESEM. The role of sorbent mass, volume of eluent and sonication time on response in solid phase microextraction procedure were optimized by central composite design (CCD) combined with response surface methodology (RSM) using Statistica. Preliminary experiments reveal that among different solvents, methanol:dimethyl sulfoxide (4:1V/V) led to efficient and quantitative elution of analyte. A reversed-phase high performance liquid chromatographic technique with UV detection (HPLC-UV) was applied for detection of curcumin content. The assay procedure involves chromatographic separation on analytical Nucleosil C18 column (250×4.6mm I.D., 5μm particle size) at ambient temperature with acetonitrile-water adjusted at pH=4.0 (20:80, v/v) as mobile phase at flow rate of 1.0mLmin-1, while UV detector was set at 420nm. Under optimized conditions, the method demonstrated linear calibration curve with good detection limit (0.028ngmL-1) and R2=0.9983. The proposed method was successfully applied to biological fluid and food samples including ginger powder, curry powder, and turmeric powder.

Rapid ultrasensitive chemometrics-fluorescence methodology to quantify fluoroquinolones antibiotics residues in surface water

A sensitive method for the determination of fluoroquinolones in surface waters at trace concentration level is presented. The proposed two-step methodology consists in a solid-phase extraction using C-18 membranes followed measurement of the emission molecular fluorescence spectra over extracted membrane without elution of the analytes. Membrane background signal was removed by the used of chemometrics calculations, in addition chemometrics was as well used for the direct and simultaneous determination of the studied compounds. The method was optimized for the analysis of three fluoroquinolones: enoxacin (ENO), norfloxacin (NOR) and ofloxacin (OFLO). The fluorescence of these compounds increase drastically when they are in the membrane, thus with this method low concentrations are possible to be determined, as the concentration in which these compounds appear in surface water. Limits of detection at the ng•L–1 level were estimated for ENO, NOR and OFLO.

Determination of haloacetic acids in water using layered double hydroxides as a sorbent in dispersive solid‐phase extraction followed by liquid chromatography with tandem mass spectrometry

In the present study, highly efficient and simple dispersive solid-phase extraction procedure for the determination of haloacetic acids in water samples has been established. Three different types of layered double hydroxides were synthesized and used as a sorbent in dispersive solid-phase extraction. Due to the interesting behavior of layered double hydroxides in an acidic medium (pH˂4), the analyte elution step was not needed; the layered double hydroxides are simply dissolved in acid immediately after extraction to release the analytes which are then directly introduced into a liquid chromatography with tandem mass spectrometry system for analysis. Several dispersive solid-phase extraction parameters were optimized to increase the extraction efficiency of haloacetic acids such as temperature, extraction time and pH. Under optimum conditions, good linearity was achieved over the concentration range of 0.05-100 μg/L with detection limits in the range of 0.006-0.05 μg/L. The relative standard deviations were 0.33-3.64% (n = 6). The proposed method was applied to different water samples collected from a drinking water plant to determine the concentrations of haloacetic acids.

Threads for tear film collection and support in quantitative amino acid analysis.

The collection of tears for chemical composition analysis is complicated by both the difficulty in sampling the tear film and the relatively low microliter volumes available for analysis. The experiments in this study are focused on the demonstration of a method for determining amino acids from tear samples. Phenol red thread was used to absorptively collect tear fluid for qualitative and quantitative analyses of amino acids in basal, reflex, and emotional tears. The thread is also used as a support for sample preparation followed by elution with a buffer. The phenol red indicator on the thread turns from yellow to red with 15-s tear absorption and allows accurate volume measurement from 100nL to over 1μL. Derivatization of amino acids was performed directly on the thread with primary amine reactive fluorescamine for fluorescence detection. Analyte elution was performed via centrifugation with the thread in a pipet tip suspended in a centrifuge tube. Collected tear eluate was analyzed via capillary electrophoresis with LED-induced fluorescence. Glycine, glutamate, and aspartate were baseline resolved and used for method characterization. Recoveries were at 50% for a single derivation and elution step but average recoveries near 90% were found with two-step processing. Glutamate and aspartate are shown to be stable stored on thread for 3days. Basal, reflex, and emotional tears were analyzed from three subjects showing distinct amino acid profiles for each tear type. The demonstration of this method may facilitate the development of routine tear compositional analysis to assess ocular health. Graphical Abstract Schematic drawing of thread-based tear collection and quantitative analysis.

In-syringe extraction using dissolvable layered double hydroxide-polymer sponges templated from hierarchically porous coordination polymers

Herein we report the use of cobalt porous coordination polymers (PCP) as intermediates to prepare advanced extraction media based on layered double hydroxides (LDH) supported on melamine polymer foam. The obtained dissolvable Ni–Co LDH composite sponges can be molded and used as sorbent for the in-syringe solid-phase extraction (SPE) of phenolic acids from fruit juices. The proposed sorbent is obtained due to the surfactant-assisted self-assembly of Co(II)/imidazolate PCPs on commercially available melamine foam, followed by the in situ conversion of the PCP into the final dissolvable LDH coating. Advantageous features for SPE are obtained by using PCPs with hierarchical porosity (HPCPs). The LDH-sponge prepared using intermediate HPCPs (HLDH-sponge) is placed in the headspace of a glass syringe, enabling flow-through extraction followed by analyte elution by the dissolution of the LDH coating in acidic conditions. Three phenolic acids (gallic acid, p-hydroxybenzoic acid and caffeic acid) were extracted and quantified using high performance liquid chromatography. Using a 5mL sample volume, the obtained detection limits were 0.15–0.35μgL−1. The proposed method for the preparation of HLDH-sponges showed a good reproducibility as observed from the intra- and inter-day RSD’s, which were <10% for all analytes. The batch-to-batch reproducibility for three different batches of HLDH-sponges was 10.6–11.2%. Enrichment factors of 15–21 were obtained. The HLDH-sponges were applied satisfactorily to the determination of phenolic acids in natural and commercial fruit juices, obtaining relative recoveries among 89.7–95.3%.

Determination of cocaine and its metabolites in plasma by porous membrane-protected molecularly imprinted polymer micro-solid-phase extraction and liquid chromatography—tandem mass spectrometry

A selective molecularly imprinted polymer synthesized for the selective retention of cocaine (COC) and its metabolites [benzoylecgonine (BZE), ecgonine methyl ester (EME), and cocaethylene (CE)] was used as a solid adsorbent for assessing cocaine abuse by plasma analysis. The MIP beads (50mg) were loaded inside a cone shaped device made of a polypropylene (PP) membrane for micro-solid-phase extraction (μ-SPE). High performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) was used for quantifying the analytes after MIP-μ-SPE. The best retention capabilities were reached when loading plasma samples (within the 0.1–5.0mL range), previously adjusted to pH 5.5 by orbital-horizontal shaking (150rpm, 50°C) for 10min. Analyte elution was achieved by subjecting the MIP-μ-SPE device to ultrasound (37kHz, 325W) with 10mL of dichloromethane/2-propanol/ammonium hydroxide (76:20:4) for 8min. After eluate evaporation to dryness and re-dissolution in 100μL of mobile phase, the MIP-μ-SPE method yielded a pre-concentration factor of 50. Precision was assessed by intra-day and inter-day assays, and accuracy (intraday and inter-day analytical recovery, as well as the analysis of a BTMF 1/11-B control serum sample) show that the developed method is highly precise and accurate. In addition, the limits of detection, ranging from 0.061ngmL−1 for COC to 0.87ngmL−1 for BZE, were low enough for confirmative conclusions regarding cocaine abuse. The method was used for screening/quantifying cocaine and metabolites in plasma samples from poly-drug abusers.

Determination of Rhodamine B in Beverages Using a Polystyrene-Coated Magnetite Nanocomposite for Magnetic Solid Phase Extraction

ABSTRACTAn efficient magnetic sorbent based on a polystyrene-coated magnetite nanocomposite was prepared for the preconcentration of rhodamine B from beverages. The synthesized nanocomposite was characterized by scanning electron microscopy, transmission electron microscopy, and Fourier transform-infrared spectroscopy. Determination of rhodamine B was performed by high-performance liquid chromatography. The optimized adsorption conditions were 70 mg of sorbent, 250 mL of sample, pH 4, 35°C, and 20 min. The optimized elution of analyte was performed using 2.5 mL of methanol at pH 8, 20°C, and 5 min. Under these conditions, the preconcentration factor for rhodamine B was 100, and the calibration curve was linear from 1 to 250 µg/L. The limit of detection was 0.0021 µg/mL, the limit of quantification was 0.0035 µg/mL, and the relative standard deviation was 0.87%. The developed method was used for the determination of rhodamine B in beverages.

Transient Ion-Pair Separations for Electrospray Mass Spectrometry.

We report a novel ion-pair chromatography (IPC) approach for liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS), where the eluent does not contain any ion-pairing reagent (IPR). The IPR is injected on the column, much like the sample, and moves down the column. Significant amounts of a high retention factor IPR is injected, resulting in a transient but reproducible regional coating that progresses along the column. The sample is injected after a brief interval. The sample components interact with the IPR coated region during their passage; the chosen eluent gradient elutes the analytes of interest into the mass spectrometer before the IPR. Following analyte elution, the gradient is steeply raised, the IPR is washed out, and the effluent is sent to waste via a diverter valve until it is fully removed. As the nature of the analyte retention continuously changes along the column and with time, we call this transient ion-pair separation (TIPS). As the IPR never enters the MS, TIPS addresses two major drawbacks of IPC for ESI-MS: it avoids both ion suppression and ion source contamination. The potential of the generic approach for other modes of separation is discussed. An illustrative separation of two small inorganic ions, iodate and nitrate, is demonstrated on a reverse phase column by a transient prior injection of hexadecyltrimethylammonium chloride as IPR.

Ionization Suppression and Recovery in Direct Biofluid Analysis Using Paper Spray Mass Spectrometry.

Paper spray mass spectrometry is a method for the direct analysis of biofluid samples in which extraction of analytes from dried biofluid spots and electrospray ionization occur from the paper on which the dried sample is stored. We examined matrix effects in the analysis of small molecule drugs from urine, plasma, and whole blood. The general method was to spike stable isotope labeled analogs of each analyte into the spray solvent, while the analyte itself was in the dried biofluid. Intensity of the labeled analog is proportional to ionization efficiency, whereas the ratio of the analyte intensity to the labeled analog in the spray solvent is proportional to recovery. Ion suppression and recovery were found to be compound- and matrix-dependent. Highest levels of ion suppression were obtained for poor ionizers (e.g., analytes lacking basic aliphatic amine groups) in urine and approached -90%. Ion suppression was much lower or even absent for good ionizers (analytes with aliphatic amines) in dried blood spots. Recovery was generally highest in urine and lowest in blood. We also examined the effect of two experimental parameters on ion suppression and recovery: the spray solvent and the sample position (how far away from the paper tip the dried sample was spotted). Finally, the change in ion suppression and analyte elution as a function of time was examined by carrying out a paper spray analysis of dried plasma spots for 5 min by continually replenishing the spray solvent. Graphical Abstract ᅟ.

Continuous online determination of 226Ra in liquid effluents using automated column chromatography-ICP-MS

A measurement system capable of continuous on-line matrix removal, pre-concentration and analysis of 226Ra using pre-packed columns coupled to a flow injection system and an ICP-MS was developed. Full instrumental control of both the ICP-MS and the flow injection system provided automatic integration of the transient signals. The flow injection system was programmed to control column conditioning, sample loading, column rinsing, analyte elution and column cleaning operations employing appropriate solutions. The application of this system to the 226Ra analysis of an industrial liquid effluent was demonstrated. Using this particular instrument together with pre-concentration and matrix removal procedures, a limit of detection of 5.4 fg L−1 (2 mBq L−1) and a method detection limit of 16.2 fg L−1 (6 mBq L−1) were achieved for the measurement of 226Ra using a 25 mL sample volume. Total time for sample handling and analysis is approximately 10 minutes. The concentration of 226Ra in a discharged effluent sample was 0.73 pg L−1 (27 mBq L−1), which is in good agreement with the value of 0.81 pg L−1 (30 mBq L−1) measured using conventional alpha counting techniques.

Extraction and pre-concentration of platinum and palladium from microwave-digested road dust via ion exchanging mesoporous silica microparticles prior to their quantification by quadrupole ICP-MS.

We report on the use of mesoporous silica microparticles (μPs) functionalized with quarternary amino groups for the isolation of platinum and palladium tetrachloro complexes from aqueous road dust digests. The μPs have a size ranging from 450 to 850 nm and are suspended directly in the aqueous digests, upon which the anionic Pt and Pd complexes are retained on the cationic surface. Subsequently, the μPs are separated by centrifugation. Elements that cause spectral interferences in ICP-MS determination of Pt and Pd can be quantitatively removed by adding fresh 0.240 mol L−1 HCl to the μPs and by repeating the centrifugation step. The analyte-loaded μPs are then dissolved in 0.1 mL of 2 mol L−1 HF, diluted to 2 mL, and the solutions thus obtained are analyzed by quadrupole ICP-MS. This method avoids analyte elution from the sorbent. This “dispersed particle extraction” approach yielded a run-to-run relative standard deviation ≤ 5 % for Pt and ≤ 4 % for Pd (at 0.1 ng mL−1, n = 4 road dust digests). Method detection limits (expressed as concentrations in the dust samples) are 2 and 1 ng g−1 for Pt and Pd, respectively. The method was validated by analysis of a reference material (BCR CRM 723) and applied to the analysis of road dust samples collected in downtown Vienna. Pt and Pd concentrations in samples collected in summer and in winter were compared, with concentrations ranging from 205 to 1445 ng g−1 for Pt and from 201 to 1230 ng g−1 for Pd.Graphical Mesoporous silica microparticles (μPs) functionalized with quarternary amino groups were used for isolating platinum and palladium from aqueous road dust digests. The μPs were suspended directly in the aqueous digests, and the analyte-loaded μPs were analyzed using “dispersed particle extraction”.

Application of ion chromatography for the determination of biogenic amines in food samples

Ion chromatography procedure for the simultaneous determination of histamine, tyramine, phenylethylamine, cadaverine and putrescine in different food samples is described. The simple procedure based on isocratic elution, 5 mM HNO3 without organic modifier as mobile phase and conductometric detection without suppression was proposed and discussed in details. The linearity of the tested procedure was between 0.5–5 and 5–100 mg/L, average recoveries for the standard solutions of amines varied between 98–100%, whereas in standard addition method between 96–99%, that indicated acceptable accuracy of the discussed method. The satisfactory reproducibility was proved by values of coefficient of variations (CV) in the range 0.2–5.0% for all tested food samples. The proposed method reveals a number of advantages: fast elution of analytes without mobile phase conductivity suppression, lack of derivatization step, simple sample preparation and environmentally friendly character.

Determination of Trace Amounts of Cd(II), Cu(II), and Ni(II) in Food Samples Using a Novel Functionalized Magnetic Nanosorbent

This work describes a novel sorbent based on functionalization of magnetic nanoparticles by 2-aminobenzothiazole and its application in the extraction and preconcentration of trace amount of Cd(II), Cu(II), and Ni(II) ions. This nanosorbent was characterized by Fourier transfer infrared spectroscopy, thermal analysis, X-ray powder diffraction, elemental analysis, and scanning electron microscopy. The effects of various factors such as pH value, sorption time, sorbent dosage, type, volume, and concentration of the eluent as well as the elution time were investigated. Following the sorption and the elution of target analytes, the Cd(II), Cu(II), and Ni(II) ions were determined by flame atomic absorption spectrometry. Under the optimal conditions, the limits of detection (LODs) were 0.03, 0.009, and 0.1 μg L−1 for Cd(II), Cu(II), and Ni(II), respectively. Linearity was within the range of 0.1–75 ng mL−1 for Cd(II), 0.03–50 ng mL−1 for Cu(II), and 0.5–100 ng mL−1 for Ni(II) in the initial solution with r 2 values greater than 0.9978. The relative standard deviations of the method were less than 8.4 %. The preconcentration factor of the method was 277. The sorption capacity of this new sorbent was 65, 78, and 49 mg g−1 for Cd(II), Cu(II), and Ni(II), respectively. The proposed method was validated using two certified reference materials (LGC 6010 hard drinking water and NIST SRM 1515 apple leaves) in order to exhibit its applicability. Ultimately, this method was applied to the rapid extraction of the trace quantities of Cd(II), Cu(II), and Ni(II) ions in different food samples, and satisfactory results were obtained.

Quantitation of the anaesthetic xylazine in ovine plasma by LC–MS/MS

Removal of the wool-bearing skin around a young lamb's rump (mulesing) provides long term health benefits for the animal, and the use of a sedative and analgesic agent such as xylazine may assist with pain relief to reduce discomfort and stress. Sensitive analytical methods are essential for monitoring pharmaceuticals and their metabolites in animals destined for human consumption. The following work reports a method that is 200 times more sensitive for xylazine detection than previously published methods, with lower limits of quantitation for xylazine and its primary metabolite in animals of 0.5pg and 2pg on-column, respectively. The use of a square wave solvent gradient immediately prior to analyte elution resulted in larger MS/MS peaks and a reduction in baseline noise, allowing reliable detection of lower analyte concentrations. The method uses as little as 1mL of plasma which allows replication within a sample if required, and requires simple sample preparation, minimising the introduction of matrix components into the MS/MS.

Determination of phytosterols in oenological matrices by liquid chromatography-atmospheric pressure chemical ionization and ion-trap mass spectrometry

The aim of the present study is the identification of plant sterols and the development of an analytical method that allows for the quantification of such family of compounds in oenological matrices. The application of liquid chromatography-atmospheric pressure chemical ionization ion-trap mass spectrometry (LC-APCI-ITMS) to sterol characterization is a useful tool and was selected to perform this research. Sterol separation was achieved using a C8 column with a mobile phase consisting of water and acetonitrile under gradient conditions and column temperature of 35°C, which leads to analyte elution in less than 25min. Retention times, precursor ions and MRM transitions of analytes allowed for the identification and sensitive quantitative determination of phytosterols in oenological matrices at trace levels. The method showed a dynamic linear range over the concentration ranges from 0.02 to 320mgkg−1 for the different parts of grapes and from 8 to 100ngmL−1 in case of wine. The most abundant phytosterol in all samples was β-sitosterol. The seeds are the richest source of phytosterols having a great amount of β-sitosterol, 314mgkg−1 fresh berry mass, followed by stigmasterol, fucosterol and campesterol at much lower concentrations (ranging from 3 to 10mgkg−1).

Application of Sea Sand Disruption Method for HPLC Determination of Quercetin in Plants

This study deals with the sea sand disruption method (SSDM) in sample preparation for chromatographic analysis of quercetin in different plants. Several SSDM variables were tested in relation to quercetin recovery: sample to sand mass ratio, type of dispersing liquid, volume of the solvent applied for analyte elution, and the time of grinding. The optimized procedure has been applied for the extraction of quercetin from the edible and inedible parts of different varieties of onion, apples, and tea. The SSDM results are comparable with those obtained by pressurized liquid extraction, regarded as one of the most effective sample preparation techniques. The presented results show that the hydrolysis of quercetin glycoside form in SSDM conditions does not occur or is negligible. Hence, the method is especially useful when the estimation of the real amount of aglycones in plants is required.

Retention prediction and separation optimization under multilinear gradient elution in liquid chromatography with Microsoft Excel macros

A package of Excel VBA macros have been developed for modeling multilinear gradient retention data obtained in single or double gradient elution mode by changing organic modifier(s) content and/or eluent pH. For this purpose, ten chromatographic models were used and four methods were adopted for their application. The methods were based on (a) the analytical expression of the retention time, provided that this expression is available, (b) the retention times estimated using the Nikitas-Pappa approach, (c) the stepwise approximation, and (d) a simple numerical approximation involving the trapezoid rule for integration of the fundamental equation for gradient elution. For all these methods, Excel VBA macros have been written and implemented using two different platforms; the fitting and the optimization platform. The fitting platform calculates not only the adjustable parameters of the chromatographic models, but also the significance of these parameters and furthermore predicts the analyte elution times. The optimization platform determines the gradient conditions that lead to the optimum separation of a mixture of analytes by using the Solver evolutionary mode, provided that proper constraints are set in order to obtain the optimum gradient profile in the minimum gradient time. The performance of the two platforms was tested using experimental and artificial data. It was found that using the proposed spreadsheets, fitting, prediction, and optimization can be performed easily and effectively under all conditions. Overall, the best performance is exhibited by the analytical and Nikitas-Pappa's methods, although the former cannot be used under all circumstances.