Determination of ethylenediaminetetraacetic acid, ethylenediaminedisuccinic acid and iminodisuccinic acid in cosmetic products by capillary electrophoresis and high performance liquid chromatography

A capillary electrophoresis (CE) and a high performance liquid chromatography (HPLC) method are described for the simultaneous determination of ethylenediaminetetraacetic acid (EDTA), S,S′-ethylenediaminedisuccinic acid (EDDS) and R,S-iminodisuccinic acid (IDS) complexing agents as their Fe(III) complexes in cosmetics like shower cream and foam bath. The non-biodegradable EDTA is used in combination with biodegradable analogues like EDDS and IDS in many commercial products. The HPLC method involves separation by reversed-phase ion pair chromatography on a C₁₈ column using methanol-formate buffer (20 mM tetrabutylammonium hydrogen sulfate, 15 mM sodium formate adjusted to pH 4.0 with formic acid) (10:90, v/v) as mobile solvent at a flow rate of 0.8 mL min⁻¹ at 24 °C using UV detection at 240 nm. The CE separation was performed in a fused silica capillary of 50 μm i.d. with the total length of 50 cm with a 10 mM MES and MOPSO (pH 5.5) at an applied voltage of −25 kV. The samples were introduced by applying a 50 mbar pressure for 2 s. Absorbances at 215 and 225 nm were monitored for the detection of the complexes. The methodology performance of the two methods was evaluated in terms of linearity, limit of detection (LOD), limit of quantitation (LOQ) and reproducibility. The LOD values obtained from HPLC are low when compared with CE. The applicability of both the methods was demonstrated for the analysis of cosmetic products such as shower cream and foam bath. The results obtained by both CE and HPLC were found to be comparable and in good agreement.     

Fast determination of flavonoids in Hippophae rhamnoides and its medicinal preparation by capillary zone electrophoresis using dimethyl-beta-cyclodextrin as modifier

A fast capillary zone electrophoresis (CZE) method, using dimethyl-β-cyclodextrin (DM-β-CD) as modifier, has been developed for the determination of three flavonoids (quercetin (QU), kaempferol (KA) and isorhamnetin (IS)) in the Chinese herbal extract from Hippophae rhamnoides and its medicinal preparation (Sindacon tablet). Optimum separation was achieved with 20 mM borate buffer at pH 10.0 containing 5 mg ml⁻¹ of DM-β-CD. The applied voltage was 15 kV and the capillary temperature was kept constant at 25 °C. Regression equations revealed linear relationships (correlation coefficients: 0.9973, 0.9992 and 0.9996) between the peak area of each compound (QU, KA and IS) and its concentration. The relative standard deviations of migration times and peak areas were <1.53 and 4.14%, respectively. The effects of several CE parameters on the resolution were studied systematically. The contents of three flavonoids in H. rhamnoides were successfully determined with 4.5 min, with satisfactory repeatability and recovery. It was also tested that the possibilities of using this method for the determination of flavonoids in Chinese medicinal preparation.     

Trace determination of short-chain aliphatic amines in biological samples by micellar electrokinetic capillary chromatography with laser-induced fluorescence detection

In this study, a new capillary electrophoresis (CE) method is described originally for the sensitive and selective determination of short-chain aliphatic amines in biological samples. These amines were converted into their N-hydroxysuccinimidyl fluorescein-O-acetate (SIFA) derivatives and measured by micellar electrokinetic capillary chromatography with laser-induced fluorescence detection. The derivatization conditions and separation parameters for the aliphatic amines were optimized in detail. The SIFA-labeled amines were fully separated within 8.5 min using 25 mM pH 9.6 boric acid electrolyte containing 60 mM sodium dodecyl sulfate (SDS). The parameters of validation such as linearity of response, precision and detection limits were determined. The detection limits were obtained in the range from 0.02 to 0.1 nM, which was the lowest value reported by CE methods. The developed method was successfully employed to monitor aliphatic amines in serum and cells samples. After comparison of other CE methods using different fluorescent probes, the present method represents a powerful tool for the trace determination of aliphatic amines in complex biological samples.     

Fast simultaneous determination of 14 nucleosides and nucleobases in cultured Cordyceps using ultra-performance liquid chromatography

Determination of nucleosides and their metabolic compounds is important for physiological and pharmacological studies. Herein, a rapid ultra-performance liquid chromatography (UPLC) method was developed for the simultaneous determination of 14 nucleosides and nucleobases, namely adenine, adenosine, cytosine, cytidine, uracil, uridine, guanine, guanosine, hypoxanthin, inosine, thymine, thymidine, 2′-deoxyuridine and cordycepin. The separation was performed on Waters Acquity UPLC system with Acquity UPLC BEH C₁₈ column and gradient elution of 0.5 mM acetic acid and acetonitrile in 5 min. The correlation coefficients of 14 analytes were high (R² > 0.9995) within the test ranges. The LOD and LOQ were lower to 11.9 and 47.0 ng/ml with 1 μl of injection volume, respectively. The overall R.S.D. for intra- and inter-day of 14 analytes were less than 1.8%. The developed method was applied for the analysis of nucleosides and nucleobases in cultured Cordyceps, which also could be used for the fast determination of the analytes in pharmaceutical products and biological fluids.     

Determination of quinolizidine alkaloids in Sophora medicinal plants by capillary electrophoresis

A new capillary electrophoresis (CE) method for the determination of quinolizidine alkaloids in Sophora medicinal plants was developed. A total of seven alkaloid components (cytisine, sophocarpine, matrine, lehmannine, sophoranol, oxymatrine and oxysophocarpine) were separated within 15 min. The running buffer was a 50 mM phosphate buffer containing 1%HP-β-CD and 3.3% isopropanol. The linear calibration ranges were 5.50-88.0 μg ml⁻¹ for cytisine and lehmannine, 5.00-88.0 μg ml⁻¹ for sophocarpine and sophoranol, 5.60-89.6 μg ml⁻¹ for matrine and oxysophocarpine, and 24.0-384 μg ml⁻¹ for oxymatrine. The recoveries of the seven alkaloids were 96.0-102.9% with relative standard deviations from 1.50 to 3.00% (n = 5). The method was successfully applied to different Sophora medicinal plants including Sophora flavescens, Sophora tonkinensis and Sophora alopecuroides.     

Flow-injection catalytic spectrophotometic determination of molybdenum(VI) in plants using bromate oxidative coupling of p-hydrazinobensenesulfonic acid with N-(1-naphthyl)ethylenediamine

A novel flow-injection spectrophotometry has been developed for the determination of molybdenum(VI) at nanograms per milliliter levels. The method is based on the catalytic effect of molybdenum(VI) on the bromate oxidative coupling of p-hydrazinobenzenesulfonic acid with N-(1-naphthyl)ethylenediamine to form an azo dye (λ_max = 530 nm). Chromotropic acid (4,5-dihydroxy-2,7-naphthalenedisulfonic acid) acted as an effective activator for the molybdenum(VI)-catalyzed reaction and increased the sensitivity of the method. The reaction was monitored by measuring the change in absorbance of the dye produced. The proposed method allowed the determination of molybdenum(VI) in the range 1.0-20 ng mL⁻¹ with sample throughput of 15 h⁻¹. The limit of detection was 0.5 ng mL⁻¹ and a relative standard deviation for 10 ng mL⁻¹ molybdenum(VI) (n = 10) was 2.5%. The interfering ions were eliminated by using the combination of a masking agent and on-line minicolumn packed with cation exchanger. The present method was successfully applied to the determination of molybdenum(VI) in plant foodstuffs.     

Separation of tyrosine enantiomer derivatives by capillary electrophoresis with light-emitting diode-induced fluorescence detection

Capillary electrophoresis (CE) coupled with fiber-optic light-emitting diode-induced fluorescence detection has been developed for the separation of tyrosine (Tyr) enantiomers. R(−)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole was used as a chiral fluorescence tagged reagent for derivatization of Tyr. The effect of pH, running buffer concentration and applied voltage on enantioselectivity has been investigated. The optimum CE conditions are 15 mmol/L borate running buffer (pH 10.5) and 14-kV applied voltage. Good reproducibility was obtained with coefficient of variation (n = 7) of migration time and peak area less than 0.2 and 2.0%, respectively. The limits of detection of d- and l-Tyr derivatives were 2.9 and 2.2 μmol/L (S/N = 3), respectively. The proposed method has been successfully applied to the determination of Tyr in a commercial amino acid oral solution.     

Fast,sensitive and highly discriminant gas chromatography–mass spectrometry method for profiling analysis of fatty acids in serum

A method for the determination of fatty acids in serum based on GC–MS (micro-SIS detection mode) has been developed and the separation and cis/trans isomers have been identified. A prior two-step extraction/derivatization procedure accelerated by ultrasound allows individual determination of esterified (EFAs) and non-esterified fatty acids (NEFAs), and shortening of the derivatization steps to 5 min for EFAs and 15 min for NEFAs. The total analysis time for 39 fatty acids was 61 min. The minimum LOD and LOQ values were 0.002 and 0.006 μg/ml, respectively. The proposed method was validated for EFAs and NEFAs using two different methods and the results show no statistical differences between the proposed method and those used as reference. The proposed derivatization–extraction methodology is suitable for fatty-acid analysis of human serum, and can be applied to nutritional and epidemiological studies.     

Development and validation of a capillary zone electrophoresis method for the determination of propranolol and N-desisopropylpropranolol in human urine

A simple, rapid and sensitive procedure using solid phase extraction and capillary zone electrophoresis for the determination of propranolol (a beta-blocker) and one of its metabolites, N-desisopropylpropranolol, has been developed and validated. The optimum separation of both analytes was obtained in a 37 cm × 75 μm fused silica capillary using 20 mmol/L phosphate buffer (pH 2.2) as electrolyte, at 25 kV and 30 °C, and hydrodynamic injection for 5 s. Prior to the electrophoretic separation, the samples were cleaned up and concentrated using a C₁₈ cartridge and then, eluted with methanol, allowing a concentration factor of 30.Good results were obtained in terms of precision, accuracy and linearity. The limits of detection were 28 and 30 μg/L for N-desisopropylpropranolol and propranolol, respectively. Additionally, a robustness test of the method was carried out using the Plackett-Burman fractional factorial model with a matrix of 15 experiments.The presented method has been applied to the determination of both compounds in human urine.     

Plastified poly(ethylene terephthalate) (PET)-toner microfluidic chip by direct-printing integrated with electrochemical detection for pharmaceutical analysis

Rapid separation and determination of acetaminophen and its hydrolysate with end-channel electrochemical (EC) detection integrated on a plastified poly(ethylene terephthalate) (PET)-toner microchip capillary electrophoresis (CE) system was investigated. In this separation and detection system, a Pt ultramicroelectrode integrated on a three-dimensional adjustor was used as working electrode. Factors influencing the separation and detection were investigated and optimized. Results show that acetaminophen and p-aminophenol can be well separated within 84 s with R.S.D. < 1% for migration time and R.S.D. < 3.6% for detection current for both analytes. Detection limits for both analytes are determined to be 5.0 μM (S/N = 3). This method has been successfully applied to the detection of trace p-aminophenol in paracetamol tablets. The results demonstrate that the PET-toner microchips can obtain better performance than PDMS microfluidic devices but at much lower cost.     

In-vial liquid–liquid microextraction-capillary electrophoresis method for the determination of phenolic acids in vegetable oils

An in-vial liquid–liquid microextraction method was developed for the selective extraction of the phenolic acids (caffeic, gallic, cinnamic, ferulic, chlorogenic, syringic, vanillic, benzoic, p-hydroxybenzoic, 2,4-dihydroxybenzoic, o-coumaric, m-coumaric and p-coumaric) in vegetable oil samples. The optimised extraction conditions for 20 g sample were: volume of diluent (n-hexane), 2 mL; extractant, methanol: 5 mM sodium hydroxide (60:40; v/v); volume of extractant, 300 μL (twice); vortex, 1 min; centrifugation, 5 min. Recoveries for the studied phenolic acids were 80.1–119.5%. The simultaneous determination of the phenolic acid extracts was investigated by capillary electrophoresis (CE). Separations were carried out on a bare fused-silica capillary (50 μm i.d. × 40 cm length) involving 25 mM sodium tetraborate (pH 9.15) and 5% methanol as CE background electrolyte in the normal polarity mode, voltage of 30 kV, temperature of 25 °C, injection time of 4 s (50 mbar) and electropherograms were recorded at 200 nm. The phenolic acids were successfully separated in less than 10 min. The validated in-vial LLME-CE method was applied to the determination of phenolic acids in vegetable oil samples (extra virgin olive oil, virgin olive oil, pure olive oil, walnut oil and grapeseed oil). The developed method shows significant advantages over the current methods as lengthy evaporation step is not required.     

Fast determination of paeonol in plasma by headspace solid-phase microextraction followed by gas chromatography-mass spectrometry

Paeonol is the active component in the traditional Chinese medicines (TCMs), such as Cynanchum paniculatum, which has been used to treat many diseases, such as eczema. In this work, a simple, rapid and sensitive method was developed for the determination of paeonol in rabbit plasma, which was based on headspace solid-phase microextraction (HS-SPME) followed by gas chromatography-mass spectrometry (GC-MS). The extraction parameters of fiber coating, sample temperature, extraction time, stirring rate and ion strength were systemically optimized; the method linearity, detection limit and precision were also investigated. It was shown that the proposed method provided a good linearity (0.02-20 μg mL⁻¹, R² > 0.990), low detection limit (2.0 ng mL⁻¹) and good precision (R.S.D. value less than 8%). Finally, GC/MS following HS-SPME was applied to fast determination of paeonol in rabbit plasma at different time point after oral demonstration of Cynanchum paniculatum essential oil. The experimental results suggest that the proposed method provided an alternative and novel approach to the pharmacokinetics study of paeonol in the TCMs.     

Hollow fiber liquid-phase microextraction for the determination of trace amounts of rosiglitazone (anti-diabetic drug) in biological fluids using capillary electrophoresis and high performance liquid chromatographic methods

A three-phase hollow fiber liquid-phase microextraction (HF-LPME) coupled either with capillary electrophoresis (CE) or high performance liquid chromatography (HPLC) with UV detection methods was successfully developed for the determination of trace levels of the anti-diabetic drug, rosiglitazone (ROSI) in biological fluids. The analyte was extracted into dihexyl ether that was immobilized in the wall pores of a porous hollow fiber from 10 mL of aqueous sample, pH 9.5 (donor phase), and was back extracted into the acceptor phase that contained 0.1 M HCl located in the lumen of the hollow fiber. Parameters affecting the extraction process such as type of extraction solvent, HCl concentration, donor phase pH, extraction time, stirring speed, and salt addition were studied and optimized. Under the optimized conditions (extraction solvent, dihexyl ether; donor phase pH, 9.5; acceptor phase, 0.1 M HCl; stirring speed, 600 rpm; extraction time, 30 min; without addition of salt), enrichment factor of 280 was obtained. Good linearity and correlation coefficients of the analyte was obtained over the concentration ranges of 1.0–500 and 5.0–500 ng mL⁻¹ for the HPLC (r² = 0.9988) and CE (r² = 0.9967) methods, respectively. The limits of detection (LOD) and limits of quantitation (LOQ) for the HPLC and CE methods were (0.18, 2.83) and (0.56, 5.00) ng mL⁻¹, respectively. The percent relative standard deviation (n = 6) for the extraction and determination of three concentration levels (10, 250, 500 ng mL⁻¹) of ROSI using the HPLC and CE methods were less than 10.9% and 13.2%, respectively. The developed methods are simple, rapid, sensitive and are suitable for the determination of trace amounts of ROSI in biological fluids.     

Rapid capillary electrophoretic determination of glutaraldehyde in photographic developers using a cationic polymer coating

A new capillary electrophoretic (CE) method was developed for the rapid and simple determination of glutaraldehyde in photographic developing solutions. The proposed system is based on the selective pre-capillary derivatization of glutaraldehyde with sulfite and CE determination of anionic glutaraldehyde-bisulfite adduct formed. Since the developing solutions contain large amounts of sulfite ions, the only procedure required for derivatization is a dilution of the sample with carrier electrolyte. For separations of sulfite, acetate, glutaraldehyde-bisulfite and hydroquinone sulfonate anions, capillaries coated with poly(diallyldimethylammonium chloride) prior to analysis were used. Separation of four anions was accomplished in less than 3 min in 5 mmol l⁻¹p-hydroxybenzoate-imidazole (p-HB) electrolyte at pH 6.0 using indirect UV detection at 254 nm. The optimal conditions for the derivatization reaction were established by varying concentration of sulfite, pH and reaction time. The recovery tests established for photographic developer samples were within the range 94.0-98.4%.     

Direct spectrofluorimetric determination of glutathione in biological samples using 5-maleimidyl-2-(m-methylphenyl) benzoxazole

A new thiol fluorescence probe, 5-maleimidyl-2-(m-methylphenyl)benzoxazole (MMPB) has been developed for the direct determination of reduced glutathione (GSH) in real samples. Compared to the reported N-substituted maleimide type of thiol reagents, the main advantage of MMPB is its rather high selectivity for GSH to cysteine (Cys), which often coexists with GSH in biological samples. Under mild conditions similar to the physiological environment, MMPB reacted with GSH to give a highly fluorescent derivative with the excitation and emission wavelengths of 299.2 and 355.8 nm, respectively. In the presence of 0.40-fold (molar ratio) of Cys, a linear relationship was found in the range of 0-1.62×10⁻⁷ mol l⁻¹ with the detection limit (3σ) of 3.23×10⁻¹⁰ mol l⁻¹ for GSH determination. Many other amino acids (100-fold) did not interfere with the determination. Since the molar ratio of Cys to GSH in mammalian tissues and blood does not exceed the value of 0.40:1, the proposed method has been used in the direct determination of GSH in these kinds of biological samples, such as human blood, pig’s liver and heart with the recoveries of 94.3-104.5%     

Speciation of dissolved Fe(II) and Fe(III) in environmental water samples by micro-column packed with N-benzoyl-N-phenylhydroxylamine loaded on microcrystalline naphthalene and determination by electrothermal vaporization inductively coupled plasma-optical emission spectrometry

A novel solid phase extraction technique for the speciation of trace dissolved Fe(II) and Fe(III) in environmental water samples was developed by coupling micro-column packed with N-benzoyl-N-phenylhydroxylamine (BPHA) loaded on microcrystalline naphthalene to electrothermal vaporization inductively coupled plasma-optical emission spectrometry (ETV-ICP-OES). Various influencing factors on the separation and preconcentration of Fe(II) and Fe(III), such as the acidity of the aqueous solution, sample flow rate and volume, have been investigated systematically, and the optimized operation conditions were established. At pH 3.0 Fe(III) could be selectively retained by micro-column (20 mm × 1.4 mm, i.d.) packed with BPHA immobilized on microcrystalline naphthalene, and Fe(II) passed through the micro-column. Both Fe(II) and Fe(III) could be adsorbed by the micro-column at pH 6.5. Thus, the total Fe could be determined without the need for preoxidation of Fe(II) to Fe(III). The retained Fe(III) or the Fe(II) and Fe(III) was subsequently eluted by 0.1 ml of 1 mol l⁻¹ HCl. The adsorption capacity of the solid phase adsorption material was found to be 45.0 mg g⁻¹ for Fe(III) at pH 3.0 and 65.3 mg g⁻¹ for Fe(II) at pH 6.5, respectively. The detection limit (3σ) of 0.053 μg l⁻¹ was obtained with a practical enrichment factor of 156 at a sample volume of 17 ml. The relative standard deviations of 4.2% and 4.6% (C_Fe(III) = C_Fe(II) = 10 μg l⁻¹, n = 7) for Fe(III) and total iron were found, respectively. The method was successfully applied to the determination of trace Fe(II) and Fe(III) in environmental water samples (East Lake water, local tap water and mineral water). In order to validate the method, the developed method was applied to the determination of total iron in certified materials of NIES NO.10-b rice flour and GBW07605 tea leaves, and the results obtained were in good agreement with the certified values.     

Universal fluorescent tri-probe ligation equipped with capillary electrophoresis for targeting SMN1 and SMN2 genes in diagnosis of spinal muscular atrophy

This is the first ligase chain reaction used for diagnosis of spinal muscular atrophy (SMA). Universal fluorescent tri-probe ligation (UFTPL), a novel strategy used for distinguishing the multi-nucleotide alternations at single base, is developed to quantitatively analyze the SMN1/SMN2 genes in diagnosis of SMA. Ligase chain reaction was performed by adding three probes including universal fluorescent probe, connecting probe and recognizing probe to differentiate single nucleotide polymorphisms in UFTPL. Our approach was based on the two UFTPL products of survival motor neuron 1 (SMN1) and SMN2 genes (the difference of 9 mer) and analyzed by capillary electrophoresis (CE). We successfully determined various gene dosages of SMN1 and SMN2 genes in homologous or heterologous subjects. By using the UFTPL-CE method, the SMN1 and SMN2 genes were fully resolved with the resolution of 2.16 ± 0.37 (n = 3). The r values of SMN1 and SMN2 regression curves over a range of 1–4 copies were above 0.9944. Of the 48 DNA samples, the data of gene dosages were corresponding to that analyzed by conformation sensitive CE and denatured high-performance liquid chromatography (DHPLC). This technique was found to be a good methodology for quantification or determination of the relative genes having multi-nucleotide variants at single base.     

Enzyme-linked immunosorbent assay for the determination of 20(S)-protopanaxatriol

A highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) method has been developed for the determination of 20(S)-protopanaxatriol (PPT), one of the major aglycones of dammarane-type ginseng saponins. Polyclonal antibodies raised against ginsenoside F₁ (GF₁)-bovine serum albumin showed high reactivities to PPT and GF₁, whereas they exhibited minor or even no cross-reactivities to other ginsenosides and protopanaxadiol (0.19%). The working range of this method extends from 50 pg ml⁻¹ to 20 ng ml⁻¹ of PPT. The assay reported here has been validated against an HPLC technique using PPT-containing samples and was shown to correlate closely (γ=0.993). This ELISA could be a useful tool for the determination of PPT contained in biological fluids and plant materials.     

Determination of mannitol and three sugars in Ligustrum lucidum Ait. by capillary electrophoresis with electrochemical detection

A method based on capillary electrophoresis with electrochemical detection has been developed for the separation and determination of mannitol, sucrose, glucose, and fructose in Ligustrum lucidum Ait. for the first time. Effects of several important factors such as the concentration of NaOH, separation voltage, injection time, and detection potential were investigated to acquire the optimum conditions. The detection electrode was a 300 μm diameter copper disc electrode at a working potential of +0.65 V (versus saturated calomel electrode (SCE)). The four analytes can be well separated within 13 min in a 40 cm length fused-silica capillary at a separation voltage of 12 kV in a 75 mM NaOH aqueous solution. The relation between peak current and analyte concentration was linear over about three orders of magnitude with detection limits (S/N = 3) ranging from 1 to 2 μM for all analytes. The proposed method has been successfully applied to monitor the mannitol and sugar contents in the plant samples at different growth stages with satisfactory assay results.     

Determination of glyphosate and aminomethylphosphonic acid in natural water using the capillary electrophoresis combined with enrichment step

A previously elaborated capillary electrophoresis (CE) method used for the determination of glyphosate and aminomethylphosphonic acid (AMPA) was slightly modified in order to improve the sensitivity. However, detection limits attained (5 μg mL⁻¹ for glyphosate and 4 μg mL⁻¹ for AMPA) were still not satisfactory for analytical purposes, thus the addition of a preconcentration step before the CE analysis was proposed. AMBERLITE^®IRA-900, a strong anion-exchange resin, was used to preconcentrate both analytes in environmental aqueous samples. The experimental conditions optimised in a previous work were readapted, by decreasing the eluent concentration due to CE limitations. Satisfactory results were attained when spiked ultrapure water was applied, with recoveries from 84 to 87% for glyphosate (R.S.D. < 6%) and from 85 to 98% for AMPA (R.S.D. < 5%). Enrichment factors up to 65 were achieved with this system, allowing the determination of 85 ng mL⁻¹ of glyphosate and 60 ng mL⁻¹ of AMPA. The extraction efficiency varied when four different natural water samples of varying conductivity were applied. Especially the strong dependence on ion concentration in samples on AMPA recovery was found. For glyphosate, good recoveries (86-99%) were obtained for samples of low and medium conductivity (0-800 μS). The effect of sample salt content on extraction efficiency was studied and a linear relationship could be established for AMPA (r² = 0.996). An important improvement on recoveries was observed when lower volumes of sample were treated.A HPLC method with UV-vis detection and pre-column derivatisation with p-toluensulphonyl chloride was compared to the CE method. No significant differences in results were found when t- and F-statistical tests were applied.