Separation of Fluoroquinolones by MEKC Modified with Hydrophobic Ionic Liquid as a Modifier

The hydrophobic ionic liquid [BMIM]PF₆ (1-butyl-3-methylimidazolium hexafluorophosphate) can interact with sodium dodecyl sulfate (SDS) micelles in aqueous solution and modify their physicochemical properties to produce a unique separation efficiency in micellar electrokinetic chromatography (MEKC). An MEKC method was developed using [BMIM]PF₆ as a modifier for separating eight fluoroquinolone compounds (ciprofloxacin, enrofloxacin, gatifloxacin, ofloxacin, norfloxacin, enoxacin, pazufloxacin, and tosufloxacin). The effects of several parameters on the separation selectivity, e.g., pH, concentration of background electrolyte, concentration ratio and amount of [BMIM]PF₆ and SDS, were investigated. Under the optimal conditions of 10 mmol L⁻¹ sodium borate, pH 7.1, 1.7% (w/w) SDS, 1.5% (w/w) [BMIM]PF₆ with 18 kV as running voltage, the eight investigated quinolone compounds were baseline separated within 15 min. The selectivity of the developed method differed from that of the simple SDS micelles system containing no ionic liquid. The results suggest that hydrophobic ionic liquids should be promising modifiers in capillary electrophoresis, especially in MEKC analysis.

     

Simultaneous Determination of Eight Typical Biogenic Amines by CZE with Capacitively Coupled Contactless Conductivity Detection

A novel method has been developed for simultaneous determination of eight typical biogenic amines (BAs) based on CZE with capacitively coupled contactless conductivity detection (CZE–C⁴D). On-column C⁴D was used for direct quantification of these nonUV-absorbing amine compounds without derivatization. The effects of various experimental factors on separation and detection were investigated. Under the optimum conditions, spermine, spermidine, histamine, putrescine, cadaver, β-phenylethylamine, tyramine, and tryptamine can be well separated within 24 min at the separation voltage of 16 kV in 150 mmol L^?1 18-crown-6/500 mmol L^?1 acetic acid running buffer, and the excitation voltage and frequency of C⁴D were 60 V and 550 kHz, respectively. A good linear relationship could be obtained between the peak area and the concentration of each BA at three orders of magnitude; the limits of detection were in the range of 44.3–149 ng mL^?1. This proposed method has been successfully applied for the analysis of BAs in water and hard liquor samples.     

Use of Surfactant as Mobile Phase Additive in LC for Simultaneous Determination of Metal-Pyrrolidine Dithiocarbamate Chelates

Reversed phase liquid chromatography using UV detection was developed for the simultaneous analysis of Hg(II), Pb(II), Cd(II), Ni(II), Fe(III) and V(V) ions after their complexation with pyrrolidine-dithiocarbamate (PDC). Optimum chromatographic conditions were a μ-Bondapak C₁₈ column and an isocratic mobile phase consisting of 40 mmol L^?1 SDS, 34 mmol L^?1 TBABr and 68% acetonitrile in 10 mmol L^?1 phosphate buffer pH 3.5. The separation of six PDC complexes was achieved within 8 min. Analytical performances and method validation were investigated. The detection limits ranged from 0.16 μg L^?1(Fe(III)) to 5.40 μg L^?1(Pb(II)). Recoveries obtained for all the studied samples including tap water, whole blood and vegetables were 72–98%. The results obtained from the proposed method were not significantly different compared to those obtained from atomic absorption spectrometry (P = 0.05).     

Determination of the Camptothecin Derivatives CPT-11 and SN-38 in Urine and Saliva by Micellar Electrokinectic Chromatography

The anti-cancer synthetic drug irinotecan (CPT-11) and its active metabolite SN-38 have been determined by micellar electrokinetic capillary chromatography (MEKC). The detection of the analytes was made at 368 nm and their separation took less than 7 min using a borate buffer (pH 8.8 at 25 mmol L^?1) solution containing sodium dodecyl sulfate (45 mmol L^?1) and acetonitrile (13.5% v/v). On-line analyte concentration (normal stacking mode) and the use of a highly sensitive cell (Z shaped cell) improved detection limits (at the 10^?8 mol L^?1 level). Recovery in fortified human saliva was 108 ± 5%, in agreement with the result achieved with the reference HPLC method. For the analysis of urine from rats submitted to a single dose of CPT-11 and SN-38, camptothecin was used as internal standard enabling recoveries close to 100% when compared to the results achieved using HPLC.     

Determination of Triazines by Ultrasonic-Assisted Ionic Liquid Microextraction Coupled with High Performance Liquid Chromatography

The determination of triazine herbicides by ultrasonic‐assisted ionic liquid microextraction coupled with high‐performance liquid chromatography was described. 1‐Hexyl‐3‐methylimidazolium hexafluorophosphate ([C₆MIm][PF₆]) was used as the extraction solvent and some extraction parameters, including volume of [C₆MIm][PF₆], extraction temperature and time, salt concentration and pH values of sample solution, were examined and optimized. The isolation of the target compounds from the matrix was found to be efficient when triazines in 10 mL of sample solution was extracted with 100 µL of [C₆MIm][PF₆] for 40 min at 50°C. The detection limits for the triazine range from 0.36 to 1.41 µg·L^?1. The satisfactory recoveries (82.3% –120.3%) with relative standard deviations ≦10.1% were obtained for the four triazine herbicides from six kinds of practical water samples.     

Analysis of Carbamazepine in Tablet and Human Serum by Sweeping‐Micellar Electrokinetic Chromatography Method

Abstract

A sensitive and simple micellar electrokinetic chromatography (MEKC) method was developed for the determination of the antiepileptic drug carbamazepine (CBZ) using a sweeping on‐line concentration method with photodiode array detection. The effect of pH, concentration of the running buffer solution, organic modifier, applied voltage and injection time on the concentration efficiency and separation was investigated. An untreated fused‐silica capillary was used (50 cm; effective length, 40 cm, 75 µm i.d.) for the analysis. The background solution (BGS) was 50 mmol · L^?1 NaH₂PO₄ (pH 3.0) containing 100 mmol · L^?1 SDS and 20% acetonitrile (5.82 ms · cm^?1) with an applied voltage of ?20 kV at 25°C. Sample introduction was performed at 0.5 psi for 90 s with diode array detection at 214 nm. For the method, the calibration curve was linear over a range of 0.5–40 µg · mL^?1 for CBZ with a correlation coefficient of 0.998. The detection limit (S/N=3∶1) of CBZ was 0.10 µg · mL^?1. About 100‐fold improvement in concentration sensitivity was achieved in terms of peak height by the sweeping method compared to conventional injection method. The sweeping‐MEKC method has been successfully applied to the analysis of CBZ in tablet and human serum.     

Rapid Analysis of Nitrate and Nitrite by Ion-Interaction Chromatography on a Monolithic Column

Ion-interaction chromatography with direct conductivity detection has been used for analysis of nitrate and nitrite. Chromatographic separation was performed on a monolithic silica-based C₁₈ column dynamically modified with tetrabutylammonium (TBA⁺). Using the optimized mobile phase, containing 2.0 mmol L^?1 TBA⁺ and 0.8 mmol L^?1 citrate (pH 6.0), delivered at a flow rate of 6.0 mL min^?1, separation of five anions (chloride, nitrite, bromide, nitrate, and sulfate) was achieved in only 40 s at a column temperature of 30 °C. The detection limits for nitrate and nitrite were 0.74 and 0.92 mg L^?1, respectively. The relative standard deviation (RSD, n = 5) of the retention times of nitrate and nitrite was 0.1% and RSD of chromatographic peak areas were 0.4 and 0.2%, respectively. The method was successfully used for analysis of the anions in groundwater. Recovery of nitrate and nitrite was 99.1 and 105%, respectively.     

Ionic liquids versus ionic liquid-based surfactants in dispersive liquid–liquid microextraction for determining copper in water by flame atomic absorption spectrometry

This work compares the performance of dispersive liquid–liquid method (DLLME) as a prior step for determining copper by flame atomic absorption spectrometry (FAAS), when using the ionic liquid (IL) 1-butyl-3-methylimidazolium hexafluorophosphate (C₄MIm-PF₆) or the IL-based surfactant 1-hexadecyl-3-butylimidazolium bromide (C₁₆C₄Im-Br) as extractant solvents. For the water-insoluble C₄MIm-PF₆, the most conventional DLLME mode using acetonitrile as dispersive solvent was employed. For the water-soluble C₁₆C₄Im-Br, the in situ DLLME mode with lithium bis[(trifluoromethane)sulfonyl]imide (Li-NTf₂) as metathesis reagent was employed. In both approaches, some effective parameters such as volumes of extractant and dispersive solvents, concentration of complexing agent, pH of sample solution, salting-out effect and final diluting solvent to ensure compatibility with FAAS, were properly optimised. The optimum conditions for the IL-DLLME method using C₄MIm-PF₆ were: 100 μL of neat C₄MIm-PF₆, 1 mL of acetonitrile, 10 mL of water, no control of pH for environmental waters, NaCl content of 23 g L^?1, diethyl dithiocarbamate (DDTC) as complexing agent at 10 mg L^?1 and final dilution of the micro-droplet with acetonitrile up to 70 µL. The optimum conditions for the in situ IL-DLLME method using C₁₆C₄Im-Br were: 0.8 mL of acetonitrile, 10 mL of water containing C₁₆C₄Im-Br at 25.2 mmol L^?1, final dilution step of the micro-droplet with 200 µL of acetonitrile and remaining conditions as those of C₄MIm-PF₆. The analytical performance of both methods was similar, being slightly better for the IL-DLLME method using C₄MIm-PF₆, with limits of detection (LOD) of 3.3 µg L^?1 (versus 5.1 µg L^?1 when using C₁₆C₄Im-Br), precision values as intraday relative standard deviation (RSD in %) lower than 8.8% (being of 10% for the C₁₆C₄Im-Br method) and an enrichment factor of 54 (being 27 when using C₁₆C₄Im-Br). The DLLME-FAAS method with C₄MIm-PF₆ was used in the analysis of environmental waters with successful performance, with relative recoveries of 110% and 105%, and interday precision with RSD values of 21% and 7.4% for spiked levels of 60 and 160 µg L^?1, respectively. The results obtained when analysing an urban wastewater sample coming from an inter-laboratory exercise was comparable to those obtained for other 93 laboratories. The method was also valid for the determination of Cu²⁺ in presence of foreign ions commonly found in natural waters.     

A Simple Method for Determination of Homologue Imidazolium Cations in Ionic Liquids Using IC with Direct Conductivity Detection

An investigation was carried out into the fast determination of five homologue imidazolium cations in ionic liquids by ion chromatography using a cation-exchange column and direct conductivity detection. Ethylenediamine, complex organic acid (citric acid, oxalic acid and tartaric acid) and organic modifiers (acetonitrile) were used as mobile phase. The influences of the eluent types, eluent concentration, eluent pH and column temperature on separation of the cations were discussed. Simultaneous separation and determination of the five homologue imidazolium cations in ionic liquids were achieved under an optimum condition. The optimized mobile phase was consisted of 0.25 mmol L^?1 ethylenediamine + 0.5 mmol L^?1 citric acid + 3% acetonitrile (v/v) (pH 4.1), set at a flow rate of 1.0 mL min^?1. The column temperature was 40 °C and detection limits were obtained in the range of 1.1–45.6 mg L^?1. The relative standard deviations of the chromatographic peak areas for the cations were <3.0% (n = 5). This method was successfully applied to separate imidazolium cations in ionic liquids produced by organic synthesis. The recoveries of spiked components were 92.5–101.9%.     

Influence of sodium addition on taurine adduct formation generated in acetic acid/acetate salt buffer applied in LC–MS/MS analysis

To investigate the influence of sodium ion addition on analyte adduct formation generated in acetic acid/acetate salt buffer appropriate infusion experiments of a 1 and 10 μg/mL taurine solution prepared both in methanol with a 1 mmol L^?1/10 mmol L^?1 CH₃COONH₄/CH₃COOH buffer and with a 1 mmol L^?1/10 mmol L^?1 CH₃COONa/CH₃COOH buffer were performed. The results achieved revealed that sodium ion concentration has a relevant influence on taurine adduct formation in the negative electrospray ionisation mode since depending on conditions applied different analyte adducts are favored. Sodium ions which originate from the glassware/chemicals make an effective detection of taurine dimer adducts possible, but taurine adducts with sodium acetate are not formed effectively. On the other side sodium ion addition enables an effective taurine adduct formation with sodium acetate, but taurine dimer based adducts can be observed only at higher analyte concentrations.     

LC Analysis of Aliphatic Primary Amines and Diamines After Derivatization with 2,6-Dimethyl-4-quinolinecarboxylic Acid N-Hydroxysuccinimide Ester

A method for sensitive simultaneous analysis of aliphatic primary amines and diamines has been developed and validated. The compounds were analyzed by reversed-phase high-performance liquid chromatography after pre-column derivatization with 2,6-dimethyl-4-quinolinecarboxylic acid N-hydroxysuccinimide ester as fluorescent probe. The derivatization reaction was performed at 50 °C for 40 min in 0.1 mol L^?1 borax buffer solution (pH 7.5). The resulting fluorophores were separated to baseline on a C₁₈ column and fluorimetrically detected at λ _ex/λ _em = 326/409 nm. Detection limits were in the range 0.50–0.02 nmol L^?1. The method was successfully used for analysis of aliphatic amines in water, human urine, and serum.     

CE, with Hydroxypropyl-β-Cyclodextrin as Chiral Selector, for Separation and Determination of the Enantiomers of Amlodipine in the Serum of Hypertension Patients

A capillary electrophoretic method for separation of the enantiomers of amlodipine in the serum of hypertension patients has been established and validated. The two enantiomers were separated in a fused-silica capillary with phosphate running buffer (75 mmol L^?1, pH 2.5) containing 15 mmol L^?1 hydroxypropyl-β-cyclodextrin (HP-β-CD). The effects on the separation of buffer pH and concentration, separation potential, and concentration of HP-β-CD were investigated. The range of quantitation for both enantiomers was 2.0–16.0 μg mL^?1. Intra-day and inter-day relative standard deviation (RSD; n = 5) was <10%. The limits of detection (LOD) and quantification (LOQ) of the amlodipine enantiomers, at 214 nm, were approximately 0.5 and 0.7 μg mL^?1, respectively (S/N = 3 and 10, respectively; 5-s injection). Recovery was always >85%. Results from enantiomer separation and quantification showed that concentrations of the enantiomers of amlodipine in serum from an elderly patient were higher than in serum from a young patient administered the same dose. The method was useful for determining the concentration of the enantiomers of amlodipine in hypertension patient serum and for monitoring the transition behavior of the enantiomers in humans. The method proved suitable for application to the separation of the enantiomers of amlodipine and analysis of clinical samples.     

Use of Capillary Zone Electrophoresis and Micellar Electrokinetic Chromatography for Separations of Anthraquinone Derivatives

The behavior of seven hydroxy anthraquinone derivatives was studied by capillary zone electrophoresis and micellar electrokinetic chromatography. The effects of buffer pH (6.5–10.8) and sodium dodecyl sulfate concentration (10–20 mmol L^?1) on the effective mobilities of the analytes and their separation were tested. A comparison of the two optimized separation systems showed that micellar electrokinetic chromatography was superior as it permits separation of all the seven analytes within 15 min, using 15 mmol L^?1 sodium dodecyl sulfate in 10 mmol L^?1 tetraborate buffer, pH 8.5, at a voltage of 20 kV. The calibration curves were linear in the concentration range from 5.0 · 10^?7 to 5.0 · 10^?4 mol L^?1 for most of the analytes, at a detection wavelength of 254 nm. LOD and LOQ values of the analytes were in the ranges of 2.10 · 10^?7–1.28 · 10^?6 mol L^?1and 6.99 · 10^?7–4.25 · 10^?6 mol L^?1, respectively. The proposed separation conditions were applied to determination of 1,2-dihydroxy anthraquinone (alizarin) and 1,2,4-trihydroxy anthraquinone (purpurin) in Rubia tinctorum aglycone and of the recently described 1-acetyl-2,4,5,7-tetrahydroxy-9,10-anthraquinone and 1-acetyl-2,4,5,7,8-pentahydroxy-9,10-anthraquinone in the mycelium of fungi Geosmithia lavendula.     

Liquid–Liquid Equilibria for Extraction of Citrus Essential Oil Using Ionic Liquids

The object of this study was to measure the liquid–liquid equilibria (LLE) data of binary mixtures containing ionic liquids and citrus essential oil. We investigated linalool as the citrus essential oil, and 1-alkyl-3-methyl-imidazolium bis(trifluoromethanesulfonyl)imide ([C _n MIM]⁺[TFSI]^?) as the ionic liquid. Firstly, the experimental apparatus and procedure for the LLE measurement of mixtures containing ionic liquids were verified by measuring the LLE of the binary mixture 1-hexyl-3-methyl-imidazolium bis(trifluoromethanesulfonyl)imide ([C₆MIM]⁺[TFSI]^?) + 1-hexanol as a reference test system recommended by Marsh et al. (Pure Appl Chem 81:781–789, 2009). Next, the LLE data for IL + linalool were obtained, and the LLE data of two binary mixtures 1-butyl-3-methyl-imidazolium bis(trifluoromethanesulfonyl)imide ([C₄MIM]⁺[TFSI]^?) or [C₆MIM]⁺[TFSI]^? + linalool were determined. The experimental LLE data were satisfactorily represented by the non-random two-liquid model.     

Temperature-assisted ionic liquid-based dispersive liquid–liquid microextraction with following back-extraction for HPLC/UV–Vis determination of 3-indole acetic acid in pea plants

In this work, the ionic liquid (IL)[C₆mim][PF₆] was used as IL-based extractant for dispersive liquid–liquid microextraction, followed by back-extraction and HPLC/UV–Vis determination of 3-indole acetic acid (IAA) in pea plant. The effects of some crucial factors such as chemical structure and volume of IL, pH adjustment, dissolution temperature, extraction time, centrifugation time, and ionic strength of aqueous sample were studied. The linear range of the HPLC method for IAA quantification was 17.5 × 10^?2–36.8 mg L^?1. LOD, LOQ, method recovery, and preconcentration factor values were 0.170 mg L^?1, 0.175 mg L^?1, 98.3, and 40 %, respectively. The RSD for the suggested method was calculated as 0.93 % at 35.04 mg L^?1 of IAA and each IL phase was able to be reused for at least four DLLME/back-extraction cycles. To evaluate the applicability of the suggested method, IAA was determined in pea plant samples.     

LC Determination of Malondialdehyde Concentrations in the Human Umbilical Vein Endothelial Cell Culture Medium: Application to the Antioxidant Effect of Vitexin-2″-O-rhamnoside

Vitexin-2″-O-rhamnoside (VOR) presents the leaves of Crataegus pinnatifida Bge. var. major which plays a role in preventing human pathologies related to oxidative stress, but as the principal component in the leaves, little attention has been devoted to its study of antioxidation of VOR. A simple, rapid and sensitive high-performance liquid chromatography method was developed to determine malondialdehyde (MDA) in ECV304 cell culture medium induced by tert-butyl-hydroperoxide (TBHP). The preparation of analyzed samples involved a one-step derivatization with thiobarbituric acid (TBA). The separation was achieved using the Synergi Hydro-RP, a polar endcapped C₁₈ column (250 × 4.6 mm, 4 μm pore size) with a linear gradient elution of acetonitrile and 10 mmol L^?1 of ammonium acetate aqueous solution (pH 6.8). The calibration curve was linear over the ranges 0.0125–1.25 μmol L^?1 MDA (r = 0.9962). The lower limit of quantification of MDA was 0.0125 μmol L^?1. Relative standard deviations of intra-day and inter-day precision were less than 5.2 and 4.4%. The method with high recovery (95.4 ± 1.3%) was successfully applied to the investigation of antioxidation of VOR by determining MDA in ECV304 cell culture medium.     

UPLC a Powerful Tool for the Separation of Imidazolium Ionic Liquid Cations

Ultra-performance liquid chromatography (UPLC) in reversed-phase (RP), ion pair (IP) and hydrophilic interaction chromatography (HILIC) has been investigated for the separation of imidazolium-based ionic liquid (IL) cations. Among the three stationary phases (i.e., C18, C8 and phenyl) studied under RP conditions the phenyl phase provided much stronger retention for the IL cations. Four acids (hydrochloric, methanesulfonic, perchloric and trifluoroacetic) as mobile phase additives were compared in light of their effects on the retention of IL cations. It was shown that the retention of all IL cations decreased upon acidification of the mobile phase, possibly due to suppression of residual silanol ionization. Very fast (~3 min) and efficient RP-UPLC separation of six cations was achieved by gradient elution with acetonitrile?Cwater mobile phase containing 2.5 mmol L^?1 perchloric acid. In IP-UPLC all solutes were well resolved in about 4 min by gradient elution with acetonitrile?Cwater mobile phase containing 1 mmol L^?1 sodium 1-octanesulfonate as ion pairing reagent. Finally, under HILIC conditions by using isocratic elution with acetonitrile?Cwater (85:15, v/v) mobile phase containing 5 mmol L^?1 ammonium formate (pH 3.2) the separation time was reduced to less than 2 min while maintaining excellent peak shapes and sufficient resolution. Compared to current LC systems UPLC allowed considerably faster separations with better peak shapes.     

Sensitive Analysis of Wilforidine in Human Plasma by LC-APCI-MS/MS

Wilforidine is a potentially efficient medicine to cure autoimmune diseases. In this paper, a sensitive and selective liquid chromatographic method coupled with atmospheric -pressure chemical ionization mass spectrometry (LC–APCI–MS/MS) has been developed for quantification of wilforidine in human plasma. Samples were deproteinized with acetonitrile and cleaned by solid-phase extraction. The chromatographic separation was performed on an analytical RRHD C₁₈ column (50 × 2.1 mm) using ammonium acetate solution (10.0 mmol L^?1)/acetonitrile (30/70, v/v) as the mobile phase at a flow rate of 0.7 mL min^?1. Detection was carried out by the positive multiple reaction monitoring mode with transitions of m/z 780 → 684 for wilforidine, and 646 → 586 for aconitine (internal standard), respectively. The calibration curve was linear (r = 0.9991) in the concentration range of 0.5–100.0 μg L^?1 with a lower limit of quantification of 0.5 μg L^?1 in plasma. Intra- and inter-day relative standard deviations were less than 6.8 and 13.1 %, respectively, and the recoveries were between 88.0 and 96.0 %. This accurate and highly specific assay provides a useful method for evaluating the pharmacokinetics of wilforidine in human plasma.     

Ionic Liquid-Induced Structural and Activity Changes in Hen Egg White Lysozyme

Lysozyme crystals in the presence of 1-butyl-3-methylimidazolium tetrafluoroborate ([C₄mim]BF₄), 1-butyl-3-methylimidazolium chloride ([C₄mim]Cl), 1-butyl-3-methylimidazolium bromide([C₄mim]Br), and 1,3-dimethylimidazolium iodine([dmim]I) were prepared, and the influence of ionic liquids (ILs) on the structure and activity change of lysozyme was investigated. Fourier transform infrared spectroscopy revealed the major secondary structures of α-helix and β-sheet for lysozyme. It was interesting to note that increases of the band near 2,935 and 1,656 cm^?1 from Raman spectroscopy are attributed to the unfolding of lysozyme molecules. A shift in amide III from 1,230 to 1,270 cm^?1 in adding [dmim]I occurs, indicating a transformation from β-sheet to random coil. With regard to adding [C₄mim]BF₄, [C₄mim]Cl, and [C₄mim]Br, α-helix and β-sheet are the predominant structures for lysozyme. The activity study showed that the ILs used brought a positive effect. Especially, [dmim]I leads to a drastic increase in relative activity, and its value reaches 50 %.     

Determination of Fatty Acids (C1 – C10) from Bryophytes and Pteridophytes

A simple and mild method for the determination of fatty acids (C₁ – C₁₀) based on a condensation reaction using 7-aminonaphthalene-1,3-disulfonic acid (ANDSA) as labeling reagent with capillary zone electrophoresis has been developed. The detection was performed with a diode array detector at 254 nm. A 58.5 cm × 50 μm i.d. (50 cm effective length) untreated fused-silica capillary was used. To optimize the separation conditions, the background electrolyte concentration, column temperature, voltage and other factors were evaluated. The optimal separation conditions were as follows: 30 mmol L⁻¹ borate buffer (pH 9.5), 15 mmol L⁻¹ β-CD, temperature at 20 °C, pressure 50 mbar and injection time 8 s. Under the established conditions, 10 fatty acid derivatives could be well-separated within 17 min. The linearity was in the range of 0.07–5.0 μmol L⁻¹. Detection limits (at a signal-to-noise ratio of 3) were in the range of 0.027–0.042 μmol L⁻¹. The fatty acids from the extracted Funaria Hedw. and Selaginella samples were determined with satisfactory results.