Low-level (PPB) determination of cisplatin in cleaning validation (rinse water) samples. II. A high-performance liquid chromatographic method

A high-performance liquid chromatographic (HPLC) method is described for the determination of residual levels of cisplatin from extracts of surfaces with very low surface area; from extracts of surfaces of coupons made of Teflon® (polytetrafluoroethylene, PTFE), stainless steel, and glass; and in aqueous solution collected after rinsing equipment and parts. Initially, the method was developed to determine cisplatin at concentrations ranging from 20 to 200 ng/ml by direct injection. Retaining the same method conditions, the scope of the method was expanded by the addition of a sample preconcentration step, allowing analyses at levels ranging from 0.5 ng to 20 ng/ml. Preconcentration is necessary for the determination of cisplatin in rinse waters at a quantifiable concentration of about 2 PPB. Under these conditions, the detection limit is about 0.2 to 0.3 ng/ml. Residual cisplatin on different types of surfaces, including surfaces with very low surface area, can be determined by swabbing each test surface with a derivatizing solution. The cisplatin recovered in the swabbing solution can be analyzed by HPLC using direct injection or preconcentration, depending on the expected level of cisplatin in the sample. Initial methods were developed to quantitate at a cisplatin concentration of about 100 PPB or higher in solution extracted from surfaces. However, when surface areas are limited because of the size of the parts, solution concentration becomes very low as a result of the minimum volume required for extraction. To support the application of swabbing techniques to surface analysis, stainless steel, Teflon, and glass surfaces were spiked with cisplatin at 2.5 to 20 ng/cm². Satisfactory overall recoveries of 90% ± 10% were obtained from all surfaces. Cisplatin has no ultraviolet/visible (UV/Vis) spectral-active functional group that can be used to detect low levels of cisplatin. Hence, diethyldithiocarbamate (DDTC) was used as a derivatizing agent to increase sensitivity to UV absorption at 340 nm. Diethyldithiocarbamate forms complexes with the platinum in cisplatin to yield a platinum-DDTC (Pt-DDTC) complex with a high molar-extinction coefficient. The Pt(DDTC)² complex thus formed was chromatographically separated and then quantitated by comparison of its detector response to that of a similarly derivatized standard preparation. DDTC also has application as a cleaning agent for cisplatin (e.g., for production equipment cleaning, spill cleanup). Destruction of cisplatin can be effected by the reaction of cisplatin with this cleaning agent. Derivatization of cisplatin will convert active cisplatin to platinum-DDTC on surfaces or in solution. Final cleaning can be accomplished using a water-for-injection rinse. After such a cleaning process, the rinse water, when collected and analyzed, showed levels of free cisplatin less than the detection concentration of 0.2 PPB and a total platinum concentration less than 10 PPB as Pt-DDTC complex.     

Low-Level (PPB)Determination of Cisplatin in Cleaning Validation (Rinse Water) Samples. II. A High-Performance Liquid Chromatogrphic Method

A high-performance liquid chromatographic (HPLC) method is described for the determination of residual levels of cisplatin from extracts of surfaces with very low surface area; from extracts of surfaces of coupons made of Teflon® (polytetrafluoroethylene, PTFE), stainless steel, and glass; and in aqueous solution collected after rinsing equipment and parts. Initially, the method was developed to determine cisplatin at concentrations ranging from 20 to 200 ng/ml by direct injection. Retaining the same method conditions, the scope of the method was expanded by the addition of a sample preconcentration step, allowing analyses at levels ranging from 0.5 ng to 20 ng/ml. Preconcentration is necessary for the determination of cisplatin in rinse waters at a quantifiable concentration of about 2 PPB. Under these conditions, the detection limit is about 0.2 to 0.3 ng/ml. Residual cisplatin on different types of surfaces, including surfaces with very low surface area, can be determined by swabbing each test surface with a derivatizing solution. The cisplatin recovered in the swabbing solution can be analyzed by HPLC using direct injection or preconcentration, depending on the expected level of cisplatin in the sample. Initial methods were developed to quantitate at a cisplatin concentration of about 100 PPB or higher in solution extracted from surfaces. However, when surface areas are limited because of the size of the parts, solution concentration becomes very low as a result of the minimum volume required for extraction. To support the application of swabbing techniques to surface analysis, stainless steel, Teflon, and glass surfaces were spiked with cisplatin at 2.5 to 20 ng/cm². Satisfactory overall recoveries of 90% ± 10% were obtained from all surfaces. Cisplatin has no ultraviolet/visible (UV/Vis) spectral-active functional group that can be used to detect low levels of cisplatin. Hence, diethyldithiocarbamate (DDTC) was used as a derivatizing agent to increase sensitivity to UV absorption at 340 nm. Diethyldithiocarbamate forms complexes with the platinum in cisplatin to yield a platinum-DDTC (Pt-DDTC) complex with a high molar-extinction coefficient. The Pt(DDTC)² complex thus formed was chromatographically separated and then quantitated by comparison of its detector response to that of a similarly derivatized standard preparation. DDTC also has application as a cleaning agent for cisplatin (e.g., for production equipment cleaning, spill cleanup). Destruction of cisplatin can be effected by the reaction of cisplatin with this cleaning agent. Derivatization of cisplatin will convert active cisplatin to platinum-DDTC on surfaces or in solution. Final cleaning can be accomplished using a water-for-injection rinse. After such a cleaning process, the rinse water, when collected and analyzed, showed levels of free cisplatin less than the detection concentration of 0.2 PPB and a total platinum concentration less than 10 PPB as Pt-DDTC complex.     

Toward feedback-controlled anesthesia: voltammetric measurement of propofol (2,6-diisopropylphenol) in serum-like electrolyte solutions

Propofol is a widely used, potent intravenous anesthetic for ambulatory anesthesia and long-term sedation. The target steady state concentration of propofol in blood is 0.25-10 μg/mL (1-60 μM). Although propofol can be oxidized electrochemically, monitoring its concentration in biological matrixes is very challenging due to (i) low therapeutic concentration, (ii) high concentrations of easily oxidizable interfering compounds in the sample, and (iii) fouling of the working electrode. In this work we report the performance characteristics of an organic film coated glassy carbon (GC) electrode for continuous monitoring of propofol. The organic film (a plasticized PVC membrane) improved the detection limit and the selectivity of the voltammetric sensor due to the large difference in hydrophobicity between the analyte (propofol) and interfering compounds of the sample, e.g., ascorbic acid (AA) or p-acetamidophenol (APAP). Furthermore, the membrane coating prevented electrode fouling and served as a protective barrier against electrode passivation by proteins. Studies revealed that sensitivity and selectivity of the voltammetric method is greatly influenced by the composition of the PVC membrane. The detection limit of the membrane-coated sensor for propofol in PBS is reported as 0.03 ± 0.01 μM. In serum-like electrolyte solutions containing physiologically relevant levels of albumin (5%) and 3 mM AA and 1 mM APAP as interfering agents, the detection limit was 0.5 ± 0.4 μM. Both values are below the target concentrations used clinically during anesthesia or sedation.     

Speciation determination of chromium(III) and (VI) using preconcentration cloud point extraction with flame atomic absorption spectrometry (FAAS)

bis-[2-Hydroxy-1-naphthaldehyde] thiourea was synthesized and preconcentration cloud point extraction (CPE) for speciation determination of chromium(III) and (VI) in various environmental samples with flame atomic absorption spectrometry (FAAS) has been developed. Chromium(III) complexes with bis-[2-hydroxynaphthaldehyde] thiourea is subsequently entrapped in the surfactant micelles. After complexation of chromium(III) with reagent, the analyte was quantitatively extracted to the surfactant-rich phase in the non-ionic surfactant Triton X-100 after centrifugation. The effect of pH, concentration of chelating agent, surfactant, equilibration temperature and time on CPE was studied. The relative standard deviation was 2.13% and the limits of detection were around 0.18 μg L⁻¹.     

A novel slurry sampling analysis of lead in different water samples by electrothermal atomic absorption spectrometry after coprecipitated with cobalt/pyrrolidine dithiocarbamate complex

A preconcentration/separation technique based on the coprecipitation of lead with cobalt/pyrrolidine dithiocarbamate complex (Co(PDC)(2)) and subsequently its direct slurry sampling determination by electrothermal atomic absorption spectrometry (AAS) was described. For this purpose, at first, lead was coprecipitated with cobalt/pyrrolidine dithiocarbamate complex formed using ammonium pyrrolidine dithiocarbamate (APDC) as a chelating agent and cobalt as a carrier element. The supernatant was then separated and the slurry of the precipitate prepared in Triton X-100 was directly analyzed by electrothermal atomic absorption spectrometry with respect to lead concentration. The effects of experimental conditions on coprecipitation of lead with gathering precipitate as well as homogeneity and stability of the slurry were investigated. After the optimization of experimental parameters, a 100-fold enrichment of the analyte with quantitative recovery (>90%) and high precision (<10% R.S.D.) were obtained. By using the proposed technique, the lead concentrations in heavy matrices of Certified Sea-water and wastewater samples could be practically and rapidly determined in the range of 95% confidence level. The detection limit of the described method for lead using sample-matching blanks was 1.5ng/L (3sigma, N=10).     

On-line preconcentration and determination of mercury in biological and environmental samples by cold vapor-atomic absorption spectrometry

An on-line procedure for the determination of traces of total mercury in environmental and biological samples is described. The present methodology combines cold vapor generation associated to atomic absorption spectrometry (CV-AAS) with preconcentration of the analyte on a minicolumn packed with activated carbon. The retained analyte was quantitatively eluted from the minicolumn with nitric acid. After that, volatile specie of mercury was generated by merging the acidified sample and sodium tetrahydroborate(III) in a continuous flow system. The gaseous analyte was subsequently introduced via a stream of Ar carrier into the atomizer device. Optimizations of both, preconcentration and mercury volatile specie generation variables were carried out using two level full factorial design (2(3)) with 3 replicates of the central point. Considering a sample consumption of 25mL, an enrichment factor of 13-fold was obtained. The detection limit (3sigma) was 10ngL(-1) and the precision (relative standard deviation) was 3.1% (n=10) at the 5microgL(-1) level. The calibration curve using the preconcentration system for mercury was linear with a correlation coefficient of 0.9995 at levels near the detection limit up to at least 1000microgL(-1). Satisfactory results were obtained for the analysis of mercury in tap water and hair samples.     

A preconcentration system for determination of copper and nickel in water and food samples employing flame atomic absorption spectrometry

A separation/preconcentration procedure using solid phase extraction has been proposed for the flame atomic absorption spectrometric determination of copper and nickel at trace level in food samples. The solid phase is Dowex Optipore SD-2 resin contained on a minicolumn, where analyte ions are sorbed as 5-methyl-4-(2-thiazolylazo) resorcinol chelates. After elution using 1 mol L(-1) nitric acid solution, the analytes are determinate employing flame atomic absorption spectrometry. The optimization step was performed using a full two-level factorial design and the variables studied were: pH, reagent concentration (RC) and amount of resin on the column (AR). Under the experimental conditions established in the optimization step, the procedure allows the determination of copper and nickel with limit of detection of 1.03 and 1.90 microg L(-1), respectively and precision of 7 and 8%, for concentrations of copper and nickel of 200 microg L(-1). The effect of matrix ions was also evaluated. The accuracy was confirmed by analyzing of the followings certified reference materials: NIST SRM 1515 Apple leaves and GBW 07603 Aquatic and Terrestrial Biological Products. The developed method was successfully applied for the determination of copper and nickel in real samples including human hair, chicken meat, black tea and canned fish.     

Targeted analyte detection by standard addition improves detection limits in matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) has proven an effective tool for fast and accurate determination of many molecules. However, the detector sensitivity and chemical noise compromise the detection of many invaluable low-abundance molecules from biological and clinical samples. To challenge this limitation, we developed a targeted analyte detection (TAD) technique. In TAD, the target analyte is selectively elevated by spiking a known amount of that analyte into the sample, thereby raising its concentration above the noise level, where we take advantage of the improved sensitivity to detect the presence of the endogenous analyte in the sample. We assessed TAD on three peptides in simple and complex background solutions with various exogenous analyte concentrations in two MALDI matrices. TAD successfully improved the limit of detection (LOD) of target analytes when the target peptides were added to the sample in a concentration close to optimum concentration. The optimum exogenous concentration was estimated through a quantitative method to be approximately equal to the original LOD for each target. Also, we showed that TAD could achieve LOD improvements on an average of 3-fold in a simple and 2-fold in a complex sample. TAD provides a straightforward assay to improve the LOD of generic target analytes without the need for costly hardware modifications.     

Slurry analysis after lead collection on a sorbent and its determination by electrothermal atomic absorption spectrometry

In this study, in order to eliminate the drawbacks of elution step and to reach higher enrichment factors, a novel preconcentration/separation technique for the slurry analysis of sorbent loaded with lead prior to its determination by electrothermal atomic absorption spectrometry was described. For this purpose, at first, lead was collected on ethylene glycol dimethacrylate methacrylic acid copolymer (EGDMA-MA) treated with ammonium pyrolidine dithiocarbamate (APDC) by conventional batch technique. After separation of liquid phase, slurry of the sorbent was prepared and directly pipetted into graphite furnace of atomic absorption spectrophotometer. Optimum conditions for quantitative sorption and preparation of the slurry were investigated. A 100-fold enrichment factor could be easily reached.

The analyte element in certified sea-water and Bovine-liver samples was determined in the range of 95% confidence level. The proposed technique was fast and simple and the risks of contamination and analyte loss were low. Detection limit (3δ) for Pb was 1.67 μg l⁻¹.     

Speciation of Cr(III) and Cr(VI) after column solid phase extraction on Amberlite XAD-2010

A speciation procedure for Cr(III) and Cr(VI) based on column solid phase extraction on Amberlite XAD-2010 and flame atomic absorption spectrometry combination. Cr(VI) was quantitatively recovered on Amberlite XAD-2010 resin at pH range of 2.0-3.0 as its diethyldithiocarbamate complex, while the recoveries of Cr(III) was below 5%. The influences of the various parameters including amounts of the reagents, eluent type and its volume, sample volume, etc., on the quantitative recoveries were examined. The interference of matrix and coexistent elements for method were studied. The detection limit (corresponding to three times the standard deviation of the blank) and the enrichment factor for Cr(VI) were found to be 1.28 microg/L and 25, respectively. To verify the accuracy of the method, drinking water certified reference material (CRM-TMDW-500) was analyzed and the results obtained were in good agreement with the certified value. The proposed method has been successfully applied to the speciation of Cr(III) and Cr(VI) in water samples and preconcentration of total chromium in environmental samples.     

Solid phase extraction method for the determination of iron, lead and chromium by atomic absorption spectrometry using Amberite XAD-2000 column in various water samples

This work describes a procedure for the separation-preconcentration of Fe(III), Pb(II) and Cr(III) from some water samples using a column-filled Amberlite XAD-2000 resin. The analyte ions retained on the column were eluted with 0.5 mol L(-1) HNO(3). The analytes in the effluent were determined by atomic absorption spectrometry. Several parameters governing the efficiency of the method were evaluated including pH, resin amount, sample volume, flow rates, eluent type and divers ion effects. The recoveries under the optimum working conditions were found to be as 100+/-1% Fe, 96+/-1% Pb and 93+/-2% Cr. The relative standard deviations and errors were less than 2% and 5%, respectively. The detection limit based on three standard deviations of the blank was found to be 0.32, 0.51 and 0.81 microg L(-1), for Fe, Pb and Cr, respectively. The procedure was applied to the determination of Fe, Cr and Pb in hot spring water and drinking water samples.     

Optimization and application of homogeneous liquid-liquid extraction in preconcentration of copper (II) in a ternary solvent system

In this study a homogeneous liquid-liquid extraction based on the Ph-dependent phase-separation process was investigated using a ternary solvent system (water-acetic acid-chloroform) for the preconcentration of Cu(2+) ions. 8-Hydroxy quinoline was used as the chelating agent prior to its extraction. Flame atomic absorption spectrophotometry using acetylene-air flame was used for the quantitation of analyte after preconcentration. The effect of various experimental parameters in extraction step was investigated using two optimization methods, one variable at a time and central composite design. The experimental design was done at five levels of operating parameters. Nearly the same optimized results were obtained using both methods: sample size, 5 mL; volume of NaOH 10 M, 2 mL; chloroform volume, 300 microL; 8-hydroxy quinoline concentration more than 0.01 M and salt amount did not affect the extraction significantly. Under the optimum conditions the calibration graph was linear over the range 10-2000 microg L(-1). The relative standard deviation was 7.6% for six repeated determinations (C = 500 microg L(-1)). Furthermore, the limit of detection (S/N=3) and limit of quantification (S/N=10) of the method were obtained as 1.74 and 6 microg L(-1), respectively.     

Determination of trace lead in water samples by continuous flow microextraction combined with graphite furnace atomic absorption spectrometry

A new method of continuous flow microextraction (CFME) combined with graphite furnace atomic absorption spectrometry (GFAAS) was proposed for the determination of trace lead in water samples. A drop of 1-phenyl-3-methyl-4-benzoyl-5-pyrazolone (PMBP) dissolved in benzene is injected into a glass chamber by a microsyringe and held at the outlet tip of a PTFE connecting tube, the sample solution flows right through the tube and the glass chamber, the solvent drop interacts continuously with the sample solution, and the analyte was extracted into the drop and concentrated. After extracting for a period of time, the drop was retracted into the microsyringe and directly injected into graphite furnace for determination of Pb. Several factors affecting the extraction efficiency, such as solution pH, sample flow rate, drop volume and extraction time, were optimized. Under the optimized conditions, a concentration factor of 45 was achieved, and the detection limits for Pb were 12 pg mL(-1). The relative standard deviation for six replicate analyses of 10 ng mL(-1) Pb was 6.8%. The proposed method was applied to determine of trace Pb in water samples with satisfactory results.     

Ionic liquid ultrasound assisted dispersive liquid-liquid microextraction method for preconcentration of trace amounts of rhodium prior to flame atomic absorption spectrometry determination

In this article, we consider ionic liquid based ultrasound-assisted dispersive liquid-liquid microextraction of trace amounts of rhodium from aqueous samples and show that this is a fast and reliable sample pre-treatment for the determination of rhodium ions by flame atomic absorption spectrometry. The Rh(III) was transferred into its complex with 2-(5-bromo-2-pyridylazo)-5-diethylamino phenol as a chelating agent, and an ultrasonic bath with the ionic liquid, 1-octyl-3-methylimidazolium bis (trifluoromethylsulfonyl) imide at room temperature was used to extract the analyte. The centrifuged rhodium complex was then enriched in the form of ionic liquid droplets and prior to its analysis by flame atomic absorption spectrometry, 300 μL ethanol was added to the ionic liquid-rich phase. Finally, the influence of various parameters on the recovery of Rh(III) was optimized. Under optimum conditions, the calibration graph was linear in the range of 4.0-500.0 ng mL(-1), the detection limit was 0.37 ng mL(-1) (3S(b)/m, n = 7) and the relative standard deviation was ±1.63% (n = 7, C = 200 ng mL(-1)). The results show that ionic liquid based ultrasound assisted dispersive liquid-liquid microextraction, combined with flame atomic absorption spectrometry, is a rapid, simple, sensitive and efficient analytical method for the separation and determination of trace amounts of Rh(III) ions with minimum organic solvent consumption.     

Chemical vapor generation: atomic absorption by Ag, Au, Cu, and Zn following reduction of aquo ions with sodium tetrahydroborate(III)

Volatile species of Ag, Au, Cu, Zn, Cd, and As were generated at room temperature by the addition of sodium tetrahydroborate(III) to an acidified solution of the analytes. The vapor-phase species were rapidly transported to a heated quartz tube atomizer (QTA) for detection by atomic absorption spectrometry. A univariate approach was used to achieve optimized conditions and derive analytical figures of merit. The analytes are released from solution as molecular species (likely as metal hydrides) and are (partially) atomized in the QTA under nonoptimized conditions. Detection limits range from 1.8 (Zn) to 420 ng (Au). The efficiency of the generation process is estimated to be 92 +/- 4% for Au. Loss of analyte during transport to the QTA was minimized through use of the minimum length of narrow-bore Teflon transfer line possible.     

Enrichment of trace amounts of copper(II) ions in water samples using octadecyl silica disks modified by a Schiff base ionophore prior to flame atomic absorption spectrometric determination

Bis(5-bromo-2-hydroxybenzaldehyde)-1,2-propanediimine is synthesized by the reaction of 5-bromo-2-hydroxybenzaldehyde and 1,2-diaminopropane in ethanol. This ligand is used as a modifier of octadecyl silica disks for preconcentration of trace amounts of copper(II) ions, followed by nitric acid elution and flame atomic absorption spectrometric (FAAS) determination. The effect of parameters influencing the extraction efficiency, i.e. pH of the sample solutions, amount of the Schiff base, type and volume of stripping reagent, sample and eluent flow rates were evaluated. Under optimum experimental conditions, the capacity of the membrane disks modified by 4mg of the ligand was found to be 247.7 (+/-2.1)mug of copper. The detection limit and the concentration factor of the presented method are 2.4ng/l and greater than 400, respectively. The method was applied to the extraction, recovery and detection of copper in different synthetic and water samples.     

On-line solid phase selective separation and preconcentration of Cd(II) by solid-phase extraction using carbon active modified with methyl thymol blue

An on-line flow system was used to develop a selective and efficient on-line sorbent extraction preconcentration system for cadmium. The method is based on adsorption of cadmium ions onto the activated carbon modified with methyl thymol blue. Then the adsorbed ions were washed using 0.5M HNO(3) and the eluent was used to determine the Cd(II) ions using flame atomic absorption spectrometry. The results obtained show that the modified activated carbon has the greatest adsorption capacity of 80 microg of Cd(II) per 1.0 g of the solid phase. The optimal pH value for the quantitative preconcentration was 9.0 and full desorption is achieved by using 0.5M HNO(3) solution. It is established that the solid phase can be used repeatedly without a considerable adsorption capacity loss. The detection limit was less than 1 ngmL(-1) Cd(II), with an enrichment factor of 1000. The calibration graph was linear in the range of 1-2000 ngmL(-1) Cd(II). The developed method has been applied to the determination of trace cadmium (II) in water samples and in the following reference materials: sewage sludge (CRM144R), and sea water (CASS.4) with satisfactory results. The accuracy was assessed through recovery experiments.     

Speciation and determination of ultra trace amounts of chromium by solidified floating organic drop microextraction (SFODME) and graphite furnace atomic absorption spectrometry

Solidified floating organic drop microextraction (SFODME) method in combination with graphite furnace atomic absorption spectrometry (GFAAS) has been used for the determination of chromium species in water and urine samples. 1-undecanol containing 2-thenoyltrifluoroacetone (TTA) was used as a selective chelating agent for the extraction of Cr(III). The total Cr was determined after the reduction of Cr(VI) to Cr(III) with hydroxylamine. The concentration of Cr(VI) was determined from the difference between the concentration of total chromium and the Cr(III). Several variables such as the sample pH, concentration of TTA, salt concentration, extraction time and the sample volume were investigated in detail. Under the optimum conditions, the limit of detection of the proposed method was 0.006 μg l(-1) for Cr(III) and the relative standard deviation for six replicate determinations at 0.1 μg l(-1) Cr(III) was 5.1%. The proposed method was successfully applied for the determination of chromium species in tap water, well water, mineral water, and urine samples.     

Copper(II)-8-hydroxquinoline coprecipitation system for preconcentration and separation of cobalt(II) and manganese(II) in real samples

A separation-preconcentration procedure based on the coprecipitation of cobalt(II) and manganese(II) ions with copper(II)-8-hydroxquinoline system has been developed. The analytical parameters including pH, amount of copper(II) as carrier element, amount of 8-hydroxquinoline, sample volume, etc., was investigated for the quantitative recoveries of Co(II) and Mn(II). No interferic effects were observed from the concomitant ions which are present in real samples. The detection limits for analyte ions by three sigma criteria were 0.86microgL(-1) for cobalt and 0.98microgL(-1) for manganese. The validation of the presented preconcentration procedure was performed by the analysis of NIST SRM 2711 Montana soil and GBW 07605 Tea certified reference materials. The procedure presented was applied to the analyte contents of real samples including natural waters and some food samples with successfully analytical results.     

On-line coupling flow injection microcolumn separation and preconcentration to electrothermal atomic absorption spectrometry for determination of (ultra)trace selenite and selenate in water

A flow injection manifold with an air-segmented and air-transported operational sequence for on-line coupling of microcolumn separation and preconcentration to electro-thermal atomic absorption spectrometry (ETAAS) was developed for the determination of (ultra)trace selenite and selenate in water. The determination of selenite was achieved by selective reaction with pyrrolidine dithiocarbamate (PDC), sorption of the resultant Se-PDC compound onto a conical microcolumn (10.2 microL) packed with RP C18 sorbent, elution with ethanol, and detection by ETAAS. The concentration of selenate was obtained as the difference between the concentrations of selenite after and before prereduction of selenate to selenite. With the developed manifold and operation sequence,the dispersion during elution and eluate transport and the eluent volume required for complete elution of the sorbed analyte were minimized. As a result, the sorbed analyte was quantitatively eluted from the column with only 26 microL of ethanol, and all the eluate was automatically introduced into the graphite tube by an air flow without the need of preheating the graphite tube or precise timing. Pretreatment of the graphite tube with iridium as a long-term "permanent" modifier effectively prevented analyte loss arising from the high volatility of the Se-PDC compound and greatly improved the precision, sensitivity, and detection limit. One thermal pretreatment of the graphite tube with injection of 150 microgram of iridium made possible at least 200 repetitive atomization cycles. With a preconcentration time of 180 s and a sample flow rate of 1.4 mL min(-1), an enhancement factor of 112 was achieved in comparison with direct injection of 30 microL of aqueous solution. The detection limit (3s) was 4.5 ng L(-1)Se. The RSD (n = 7) was 3.8% at 20 ng L(-1)Se. The concentrations of selenite and selenate determined in synthetic aqueous mixtures were in good agreement with the expected values. The recoveries for selenite from spiked seawater samples ranged from 98 to 102%. The concentrations of selenite in several seawater reference materials obtained with simple aqueous standard solutions for calibration agreed well with the certified and information values, respectively. In addition, the developed method was successfully applied to the certification of selenite and selenate in water.