Spiked Plasma Samples Research Articles

HPLC assay of Lidocaine in plasma with solid phase extraction and UV detection

A sensitive and reliable method based on solid-phase extraction and reversed-phase liquid chromatography was developed and validated for the quantitation of Lidocaine (Lid) in dog plasma. Phenacemide was used as an internal standard (IS) in the extraction which employed C 18 solid-phase extraction cartridges. The washing and eluting solutions were 2 ml acetonitrile-pH 9.0 phosphate buffer (10:90 v/v) and 0.5 ml acetonitrile-pH 4.0 phosphate buffer (40:60 v/v), respectively. The eluent obtained from the cartridge was directly analyzed on a reversed-phase ODS column with UV detection at 210 nm. A clean chromatogram and high sensitivity were achieved at this wavelength. The mobile phase was acetonitrile and pH 5.9 phosphate buffer (20:80 v/v). The retention times were 6.4 and 7.2 min for Lid and IS, respectively, at a flow rate of 1.0 ml min −1. The mean absolute recovery was 96.6% ( n=9) with a CV of 3.8% for Lid and 81.7% with CV of 2.5% ( n=3) for IS. The limit of quantitation was 20 ng ml −1, with the intra- and inter-day precisions ( n=5) of 4.4 and 3.4%, respectively, and the intra- and inter-day accuracies ( n=5) of −4.3 and −5.0%, respectively. For the analyses of Lid in spiked plasma samples at 20, 100 and 200 ng ml −1, the overall mean intra- and inter-day precisions ( n=15) were 3.9 and 4.9%, respectively, and the overall mean intra- and inter-day accuracies ( n=15) were −3.7 and −4.6%, respectively. The correlation coefficients for calibration plots in the range 20–1000 ng ml −1 in plasma were typically higher than 0.998. The suitability of the method was demonstrated by the study in a beagle dog receiving a low intravenous dose of Lid.

Experimental design and multivariate calibration in the development, set-up and validation of a differential pulse polarographic and UV spectrophotometric method for the simultaneous plasmatic determination of the therapeutic metronidazole-pefloxacin combination.

Partial least squares regression (PLS1 and PLS2) and GOLPE variable selection procedures were used for the treatment of differential pulse polarographic and UV spectrophotometric data obtained from the analysis of the therapeutic combination of metronidazole and pefloxacin. The analytical method used for the determination was set up using experimental design strategies (Doehlert's design, full factorial design, fractional face center cube design, etc.) and by involving the simultaneous optimization of several responses (desirability function). Method validation was also performed, determining accuracy, precision, linearity and range, detection and quantification limits and robustness. The quantitative prediction abilities in determining metronidazole and pefloxacin plasma levels of the PLS1 and PLS2 models were tested on spiked plasma samples and good results were obtained (metronidazole, 97.5%, RSD = 4.8%, n = 3; pefloxacin, 100.6%, RSD = 3.6%, n = 3). The use of multivariate calibration was particularly useful for spectrophotometric quantification because of the highly overlapping spectra of the binary mixture.

Straightforward solid-phase extraction method for the determination of verapamil and its metabolite in plasma in a 96-well extraction plate

Straightforward solid-phase extraction (SPE) methods were developed for the determination of verapamil and its metabolite in a plasma matrix. The spiked plasma sample was pretreated with 2% phosphoric acid followed by two different SPE methods using a Waters Oasis™ HLB 96-well extraction plate. Recoveries greater than 90% were obtained using both a generic and a selective SPE methods. The generic method is a good starting protocol, and it is applicable to a wide range of compounds. This generic method consists of using 5% methanol as the wash solvent, and 100% methanol for the elution. The limitation of the non-specific method is that it does not remove all plasma constituents that interfere with the quantitation of the metabolite, norverapamil. A second, more specific method was developed using the same Oasis™ HLB sorbent which removes more plasma interferences and provides cleaner extracts for the HPLC–UV analysis. This selective method uses both the methanol concentration and the pH advantageously to preferentially isolate the analytes of interest from a complex sample matrix. Recoveries of greater than 90% with R.S.D.s less than 3.8% were obtained with this selective method.

Automated sample preparation for the determination of budesonide in plasma samples by liquid chromatography and tandem mass spectrometry

An automated bioanalytical method for the determination of the glucocorticosteroid drug budesonide in plasma samples at p M levels was investigated. The method was built using three separate automated analytical steps with manual transfer of samples between them. In the first step, a Tecan RSP150 (Genesis) pipetting robot was used to transfer 1 ml of centrifuged plasma samples and deuterated budesonide internal standard solutions into tubes and to homogenise the resulting admixture. In the second step, a solid-phase extraction was performed using an ASPEC Xli (Gilson) with 100 mg Isolute C 18 columns. In order to avoid conventional time-consuming evaporation and reconstitution steps, the solid-phase extraction was coupled on-line to a trace enrichment system for further purification and concentration of the sample extracts. The concentrated samples were eluted in 300 μl ethanol into injection vials, which were capped and transferred to the autosampler in the detection system. In the third step, the pre-treated samples were chromatographed in a gradient LC system and detected using a tandem MS system (Finnigan TSQ 7000), with an atmospheric pressure chemical ionisation interface. The described Analytical System consisting of one Tecan robot, two ASPEC systems and one LC–MS–MS system may analyse up to about 800 samples a week with less routine work for the analyst. The concentration range studied was 15 to 2500 p M in 1 ml spiked plasma samples and the limit of quantitation for the described method was determined as 15 p M, as defined by accuracy and precision better than 20%.

Comparison of p-fluoroketorolac and [ 18O 3]ketorolac for use as internal standards for the determination of ketorolac by gas chromatography/mass spectrometry (GC/MS)

A chemical and a stable-isotope analog, p-fluoroketorolac and [ 18O 3]ketorolac respectively, were directly compared for applicability as internal standards for the determination of ketorolac in plasma samples using gas chromatography/mass spectrometry (GC/MS) with selective-ion-monitoring detection, following derivatization to form the methyl esters. This comparison involved analyzing ketorolac calibration standards and spiked plasma samples that contained both internal standard candidates. The response for ketorolac and each internal standard was monitored simultaneously and electronically integrated peak heights were obtained. Thus, for each analysis performed, a response ratio was obtained for each internal standard relative to an identical ketorolac response. Linearity of response for ketorolac calibration standards and accuracy for spiked plasma sample analysis were compared using each internal standard. The use of [ 18O 3]ketorolac as the internal standard provided superior accuracy data for the analysis of ketorolac in plasma samples.

Simultaneous determination of an active metabolite and open-ring metabolites by high performance liquid chromatography and pharmacokinetic studies of a penem antibiotic, FCE22891, in dogs

A sensitive high performance liquid chromatographic method for the simultaneous determination of an active metabolite (FCE22101) and open-ring metabolites (P1, P2) of a penem antibiotic, FCE22891, in dog plasma was developed. Plasma samples were pretreated only by ultrafiltration for the determination of the metabolites. The filtrates were directly analyzed by a reversed-phase high-performance liquid chromatographic system using a two-sided bracketing injection technique. The quantitation limits of FCE22101, P1 and P2 were 0.03, 0.1 and 0.15 μg ml −1, respectively. Analysis of the spiked plasma samples demonstrated the good accuracy and precision of the method. The proposed method was applied to the pharmacokinetic studies of an active metabolite and open-ring metabolites after oral administration of a penem antibiotic, FCE22891, in dogs. In addition, the plasma levels of unchanged FCE22891 and the possible changes of formaldehyde and acyl- l-carnitine levels in plasma, which will be generated from the ester group of FCE22891, were also investigated.

Simple high-performance liquid chromatographic method with electrochemical detection for the simultaneous determination of artesunate and dihydroartemisinin in biological fluids

A simple, rapid, sensitive, selective and reproducible high-performance liquid chromatographic method with reductive electrochemical detection is described for the simultaneous quantification of artesunate (ARS) and dihydroartemisinin (DHA) in plasma. The procedure involved the extraction of ARS, DHA and the internal standard (artemisinin, ARN) with a mixture of dichloromethane and tert.-methyl butyl ether (8:2, v/v). Chromatographic separation consisted of the mobile phase (acetonitrile–water containing 0.1 M acetic acid, pH 4.8; 45:55, v/v) running through the column (Nova-Pak C 18, 150 cm×3.9 mm I.D., 5 μm particle size). The retention times of α-DHA, β-DHA, ARS and ARN were 2.9, 4.2, 4.5 and 6.0 min, respectively. The average recoveries of ARS, α-DHA and ARN in the concentration range of 10–800 ng/ml were 81.9, 88.2, 101.1 and 84.3%, respectively. The coefficients of variation (precision and repeatability) were below 10% for all three compounds at concentrations of 50, 200, 400 and 800 ng/ml, and below 20% at a concentration of 10 ng/ml. The limits of quantification for both ARS and α-DHA in spiked plasma samples were 5 and 3 ng/ml, respectively. The method was found to be suitable for application to pharmacokinetic studies of both ARS and DHA.

Determination of oestrogen concentrations in bovine plasma by a recombinant oestrogen receptor-reporter gene yeast bioassay.

A recombinant cell yeast bioassay (RCBA) was applied to the generic measurement of bovine plasma oestrogen concentration. Samples were prepared by diethyl ether extraction of plasma following addition of [3H]17 beta-oestradiol as internal standard; organic and aqueous phases were separated by freezing (recovery 97.1 +/- 0.7%) and dried extract reconstituted in culture medium (recovery 31.4 +/- 4.5%). Plasma oestrogen concentrations were measured by incubation of extracts with yeast containing a stable human oestrogen receptor (hER) and a reporter construct comprising an hER response element regulating beta-galactosidase expression. The linearity of response for the analysis of spiked plasma samples using the RCBA, following corrections, is described by y = 0.8994x - 0.111 (r2 = 0.9776, P < 0.0001). Inter-assay variation for endogenous oestrogen was 11.5% for > 1 pg ml-1. Plasma oestrogen concentrations for intact (n = 5) and castrated (n = 3) males were < 0.5 pg ml-1, and 3.7 +/- 2.6 pg ml-1 for luteal phase females (n = 10). Analysis by RCBA of sequential samples from heifers during the reproductive cycle failed to detect the pre-ovulatory increase in plasma 17 beta-oestradiol as determined by radioimmunoassay (RIA) (maximal concentrations 2.09 +/- 2.1 pg ml-1 and 32.6 +/- 14.6 pg ml-1, respectively). Interestingly, when samples were hydrolysed using Helix pomatia glucuronidase the RCBA gave concentrations (29.5 +/- 8.9 pg ml-1) not significantly different to those obtained by RIA. These preliminary findings suggest that a substantial proportion of plasma oestrogen during the pre-ovulatory period may be conjugated. These data indicate the potential of the RCBA to measure biologically active and physiological levels of plasma oestrogens in cattle. One potentially valuable application of this generic oestrogen assay could be in surveillance programmes to detect illegal use of anabolic oestrogens in live-stock where the identity of the analyte may be unknown.

Use of the ASTED system to determine l-N G-monomethylarginine (546C88) in human plasma by pre-column o-phthalaldehyde derivatisation and high-performance liquid chromatography

The use of the system automated sequential trace enrichment of dialysates (ASTED), to prepare plasma samples for the estimation of l-N G-monomethylarginine (546C88) by pre-column o-phthalaldehyde–2-mercaptoethanol derivatisation and HPLC is described. Calibration is achieved using purified albumin as a substitute matrix for plasma. Using this technique the procedure was observed to be specific for 546C88 and linear over the range 0.10 to 50.0 μmol/l. The within-run imprecision (C.V.) at four different spiked plasma 546C88 concentrations of 0.10, 1.0, 8.0 and 40.0 μmol/l was 6.48, 2.55, 2.79 and 3.37%, respectively, and the between-run imprecision (C.V.) estimated to be 8.50, 1.80, 2.10 and 3.30%, respectively, for the same spiked 546C88 concentrations. The overall accuracy (% bias) of the procedure using an albumin matrix for calibration was estimated to be −2.50, −5.25, −3.56, −3.53%, respectively, and the recovery of 546C88 from six different spiked plasma samples estimated to be 99.1±1.4%. © 1997 Elsevier Science B.V.

Simultaneous analysis of retinol, all- trans- and 13- cis-retinoic acid and 13- cis-4-oxoretinoic acid in plasma by liquid chromatography using on-column concentration after single-phase fluid extraction

A reversed-phase high-performance liquid chromatographic method for the simultaneous analysis of retinol, all- trans-retinoic acid, 13- cis-retinoic acid and 13- cis-4-oxoretinoic acid in human plasma and cell culture medium is described. Sample preparation involves precipitation of proteins and extraction of retinoids with 60% acetonitrile. After centrifugation, the acetonitrile content of the supernatant is reduced to 45%, allowing on-column concentration of analytes. Injection volumes up to 2.0 ml (equivalent to 0.525 ml of sample) can be used without compromising chromatographic resolution of all- trans-retinoic acid and 13- cis-retinoic acid. Retinoids were stable in this extract and showed no isomerization when stored in the dark in a cooled autosampler, allowing automated analysis of large series of samples. Recoveries from spiked plasma samples were between 95 and 103%. Although no internal standard was used, the inter-assay precision for all retinoids was better than 6% and 4% at concentrations of 30 n M and 100 n M, respectively. The method is a valuable tool for the study of cellular metabolism of all- trans-retinoic acid, as polar metabolites of this compound can be detected with high sensitivity in cell culture media.

Determination of Sufentanil in Human Plasma by Capillary Electrophoresis and Gas Chromatography–Mass Spectrometry

A fast capillary electrophoretic method and a gas chromatographic - mass spectrometric method were developed for the determination of sufentanil in human plasma. Spiked plasma samples and patient plasma samples were prepared by liquid-liquid extraction. In the capillary electrophoretic analysis, both inorganic and organic buffers were tested with and without surfactants, at various pHs. The final separation with fast analysis time was performed with organic 3–[N-morpholino]propanesulfonic acid solution at pH 7.00, with a bubble cell capillary and UV detection at 195 nm. The gas chromatographic separation was performed without sample derivatization, in a nonpolar column material with fentanyl as the internal standard. The mass spectrometric identification was done in electron impact mode. The limit of detection for sufentanil was lower in the gas chromatographic - mass spectrometric technique than in capillary electrophoresis with UV, therefore more sample was required in the capillary electrophoretic analysis. Comparison of the techniques was done with spiked plasma samples. Both techniques were found to be excellent for the determination of sufentanil. Advantages of the capillary electrophoretic analysis over the gas chromatographic method are better efficiency and the faster elution.

Mangafodipir trisodium injection, a new contrast medium for magnetic resonance imaging: detection and quantitation of the parent compound MnDPDP and metabolites in human plasma by high performance liquid chromatography

Manganese(II) dipyridoxyl diphosphate (MnDPDP) is the active component of mangafodipir trisodium injection (Teslascan ®), a new contrast medium for magnetic resonance imaging. A high performance liquid chromatography (HPLC) method was developed for the simultaneous determination of MnDPDP and its five major metabolites in human plasma, i.e. the dephosphorylation products MnDPMP (manganese(II) dipyridoxyl monophosphate) and MnPLED (manganese(II) dipyridoxyl ethylenediamine diacetate) and the corresponding substances obtained after transmetallation with zinc (ZnDPDP, ZnDPMP and ZnPLED). Heparinized blood samples from patients receiving mangafodipir trisodium injection were immediately mixed with solid trisodium phosphate dodecahydrate to obtain pH 10.0 ± 0.2 in order to inhibit further in vitro dephosphorylation and transmetallation. The plasma thus obtained was ultrafiltrated prior to HPLC analysis. The chromatographic separation was obtained using a mixed-bed resin with both anion exchange and reversed-phase functions (OmniPac ™ PAX-500) using isocratic elution and UV detection at 310 nm. With an injection volume of 50 μl, the limit of quantitation (LOQ) values were 0.8–2.3 μM for the Mn compounds and 0.1–0.8 μM for the Zn compounds. The between-run accuracy of spiked plasma samples was in the range 97.5–106.7%, with a precision in the range 3.1–9.0%. The best fit calibration curves were obtained using non-linear regression according to the equation Y = A + BX M in the concentration range from LOQ to 100 μM. Long-term storage of spiked plasma samples for three months at − 20°C demonstrated the required stability with recovery values within 85–115% of MnDPDP and its five metabolites.

Analysis of buprenorphine in rat plasma using a solid-phase extraction technique and high-performance liquid chromatography with electrochemical detection

A solid-phase extraction method and sensitive reversed phase high-performance liquid chromatography analysis with electrochemical detection of buprenorphine and its metabolite, norbuprenorphine, in rat plasma is described. Adequate separation of the compounds of interest was achieved on a Phenomenex C 18 reversed-phase column using a mobile phase comprising phosphate buffer: acetonitrile (75:25, pH 3.0) and 0.25 mM 1-octane-sulfonic acid, at a flow rate of 1 ml/min. Electrochemical detection was performed at a potential of 0.75 V and sensitivity of 2 nA. Buprenorphine and norbuprenorphine were extracted from plasma by solid-phase extraction technique using naltrindole as an internal standard (IS). Recoveries of buprenorphine and norbuprenorphine following the extraction method were high (70%–89%) over the concentration range used (25–100 ng/ml) and no endogenous substances in plasma interfered with any of the sample components. The retention times for norbuprenorphine, IS, and buprenorphine were 8, 12.5, and 30.5 min, respectively. The limits of detection of buprenorphine and norbuprenorphine in spiked plasma samples were 25 and 5 ng/ml, respectively. Using this method, buprenorphine was detected in rat plasma in animals acutely treated with the drug (5 mg/kg, s.c.).

Determination of vitamin E in human plasma by high-performance liquid chromatography.

The use of selective protein precipitation to enhance the recovery of vitamin E from plasma, by minimising binding with very-low-density lipoproteins, is reported. The procedure employed treatment of plasma with magnesium chloride and tungstate, followed by methanol protein precipitation. Separation of vitamin E was performed using reversed-phase high-performance liquid chromatography of the methanol extracts with subsequent UV detection of the compound. Using this technique the procedure was observed to be specific for vitamin E and linear over the range 1.0 to 40.0 micrograms/ml. The within-run imprecision (C.V.) at three different supplemented plasma vitamin E concentrations of 5.0, 10.0 and 20.0 micrograms/ml was 4.51, 3.33 and 2.58%, respectively, and the between-run imprecision (C.V.) estimated to be 5.19, 3.69 and 3.67%, respectively. With the same supplemented plasma vitamin E concentrations, the overall accuracy (bias) of the procedure, using an albumin matrix for calibration, was estimated to be 6.0, -5.0 and -3.5%, respectively, and the recovery of vitamin E from six different spiked plasma samples estimated to be 98.2 +/- 2.6%.

Evaluation of a solid-phase extraction procedure for the simultaneous determination of morphine, 6-monoacetylmorphine, codeine and dihydrocodeine in plasma and whole blood by GC/MS.

Different procedures of solid-phase extraction were re-examined and a new solid-phase extraction procedure was developed using gas chromatography-mass spectrometry for the simultaneous detection and quantitation of morphine, 6-monoacetylmorphine, codeine and dihydrocodeine in plasma and whole blood. The effects of different types of sorbent and buffer solutions on the recoveries and purity of the extracts were also studied. Some preparation techniques on whole blood samples were also investigated. The method developed using Chromabond C18 (100) with spiked plasma samples had good recoveries for all opiates of interest: morphine 93.1% +/- 7.4%, 6-monoacetylmorphine 68.0% +/- 6.7%, codeine 77.0% +/- 8.3% and dihydrocodeine 67.9% +/- 8.4%. The detection limit of all compounds was less than 5 micrograms/L. The blank plasma showed no interfering peaks in the GC/MS-analysis.

Liquid chromatographic analysis of penem antibiotic FCE22101 in biological fluids with a liquid-liquid extraction procedure.

A sensitive high-performance liquid chromatographic method has been developed for the determination of penem antibiotic FCE22101 in plasma and urine. FCE22101 was extracted from plasma and urine with ethyl acetate. After evaporating, the sample solutions were analyzed by a reversed-phase high-performance liquid chromatographic system using a two-sided bracketing injection technique. The determination limit of FCE22101 was 5 ng/ml in plasma. Analysis of the spiked plasma samples demonstrated the good accuracy and precision of the method. The proposed method improved from five- to ten-fold on the analytical sensitivity in comparison with the most commonly ultrafiltration method.

Sample Preparation Using a Miniaturized Supported Liquid Membrane Device Connected On-Line to Packed Capillary Liquid Chromatography

A miniaturized supported liquid membrane device has been developed for sample preparation and connected on-line to a packed capillary liquid chromatograph. The device consists of hydrophobic polypropylene hollow fiber, inserted and fastened in a cylindrical channel in a Kel-F piece. The pores of the fiber are filled with an organic solvent, in this study 6-undecanone, thus forming a liquid membrane. The sample is pumped on the outside of the hollow fiber (donor), and the analytes are selectively enriched and trapped in the fiber lumen (acceptor). With this approach, the volume of the acceptor solution can be kept as low as 1-2 μL. This stagnant acceptor solution is then transferred through capillaries attached to the fiber ends to the LC system. The system was tested with a secondary amine (bambuterol), as a model substance in aqueous standard solutions as well as in plasma. The best extraction efficiency in aqueous solution, with an acceptor volume of 1.9 μL, was 32.5% at a donor flow rate of 2.5 μL/min. At flow rates above 20 μL/min, the concentration enrichment per time unit was approximately constant, at 0.9 times/min, i.e., 9 times enrichment in about 10 min. The overall repeatability (RSD) for spiked plasma samples was ∼4% (n = 12). Linear calibration curves of peak area versus bambuterol concentration were obtained for both aqueous standard solutions and spiked plasma samples. The detection limit for bambuterol in plasma, after 10 min of extraction at a flow rate of 24 μL/min, was 80 nM.

Determination of benflumetol in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

A reversed-phase high-performance liquid chromatographic method for the determination of benflumetol in human plasma is described. Benflumetol in plasma samples was extracted with a glacial acetic acid-ethyl acetate (1:100, v/v) mixture at pH 4.0. Chromatography was performed on a Spherisorb C 18 column using a methanol-water-glacial acetic acid-diethyl amine (93:6:1:0.03, v/v) mixture as the mobile phase and UV-VIS detection at 335 nm. The identity and purity of the benflumetol peak were carefully examined, and the internal standard method was applied for its quantitation. The absolute recovery of benflumetol in spiked plasma samples was 92.91% over the concentration range 5–4000 ng/ml. The recovery of internal standard “8212” at a concentration of 300 ng/ml in spiked plasma was 84.85%. The detection limit of benflumetol was 11.8 ng/ml. Plasma concentration-time profiles in healthy volunteer adults were measured after a single-dose oral administration of 500 mg of benflumetol. The assay procedures were within the quality control limits.

Simultaneous determination of diltiazem and quinidine in human plasma by liquid chromatography

A novel method for simultaneous determination of diltiazem and quinidine in human plasma is described. Plasma is alkalinized and extracted with methyl tert.-butyl ether. The ether phase is separated and evaporated. The residue is reconstituted in 0.2 ml of mobile phase containing 56 m M octanesulfonic acid then washed twice with n-hexane. Aliquots are chromatographed on a silanol-deactivated reversed-phase column using a mobile phase containing aqueous H 2SO 4 (0.01 M, pH 2)-methanol-acetonitrile (45:45:10) and 10 m M octanesulfonic acid. Peaks are monitored with a UV detector set at 237 nm and a fluorescence detector using an excitation set at 247 nm and a 270 nm UV cut-off filter at the emission. Calibration and standard curves were linear from 1 to 130 ng on-column for diltiazem and from 2 to 600 ng on-column for quinidine. Limits of quantitation were 2 and 4 ng/ml for diltiazem and quinidine, respectively. Recoveries from spiked plasma were 94.0 to 102.5% (R.S.D. 6.0–11.4%) for diltiazem and 98.5% to 104.1 (R.S.D. 7.7–8.7%) for quinidine over the ranges studied. In vitro stability was studied in spiked plasma samples stored at −80°C for sixteen months. Both diltiazem and quinidine remained within 10% from nominal values. For ex vivo stability at −80°C, a plasma sample obtained from a volunteer 2 h after oral administration of diltiazem (60 mg) was analysed for two days after sampling and eighteen months later. The mean deviation from initial measured was 4.7%.

Analysis of inositol phosphates and derivatives using capillary zone electrophoresis-mass spectrometry

Capillary zone electrophoresis (CZE) has been combined with mass spectrometric detection for the separation and determination of inositol phosphates (IPs). Apart from IP1 through IP6 (inositol mono- through hexakisphosphate), an IP3 derivative has been analyzed and identified. The detection limits achieved are in the low micromolar range corresponding to an injected amount of ca. 900 fmol. In addition, an IP3 spiked plasma sample was analyzed after sample pretreatment using ultrafiltration.