Sequential cloud point extraction of trace elements from biological samples and determination by inductively coupled plasma mass spectrometry

A two-step sequential cloud point extraction (CPE) of trace elements from small sample volumes of human serum, animal blood, and food diet is proposed to gain analytical information in the analysis by inductively coupled plasma mass spectrometry. The first CPE was attained by adding O,O-diethyldithiophosphate, the non ionic surfactant Triton® X-114 followed by heating at 40 oC, centrifugation and cooling at 0 oC. The resulting surfactant-rich phase was separated to determine Cd, Pb and Cu by isotope dilution. Isotope ratio measurements presented RSD < 0.7%. The residual surfactant-poor phase solution had the pH adjusted in the range 4 to 5 before the chelating reagent, 4-(2-pyridylazo) resorcinol plus surfactant Triton® X-114 were added followed by the sequence to attain the CPE. Co and Ni were quantified in the second extracted surfactant-rich phases by standard additions method with RSD < 2%. Recoveries from 85 to 96% were obtained for all elements. Analyzing reference materials with certified and recommended values assessed accuracy.


Introduction
Surfactant mediated phase separation constitutes the basis for the approach known as cloud point extraction 1 which is a liquid-liquid extraction alternative to that of organic solvents.The main parameter to attain a surfactant monomer agglomeration in a micelle-rich phase is the surfactant concentration at the cloud point temperature.The temperature-concentration phase diagram is specific for each surfactant. 2 The effect of additives such as salts and other surfactants also has to be considered. 1,3he first application of cloud point extraction for analytical purposes described the micelle aggregation of hydrophobic anionic metal complexes. 2The ability of surfactant micelles to bind organic compounds separated by CPE was applied for pre-concentration of organic compounds prior to the separation of species by capillary electrophoresis 3 and chromatography. 4The surfactants octylphenoxy polyethoxyethanol (Triton ® X-114) and polyoxyethylene (7.5)nonylphenyl ether (PONPE ® 7.5) with cloud point temperatures (CPT) below 40 °C and 20 °C, respectively, have been used preferentially for cloud point extraction of soluble metal ions complexes. 5Both surfactants are characterized by forming a surfactant-rich phase denser than the aqueous solution which interacts with metallic species, mainly in the chelated form.This facilitates the phase separation by gravity or centrifugation at room temperature.Another advantage of surfactants attaining the critical micelle concentration (CMC) at low temperature is the minimum redissolution of micelles into the bulk aqueous solution occurring during phase separation. 1 Theoretical aspects and applications of phase separations using aqueous solutions of surfactants have been described. 5,6ialkyl phosphorodithiotic acids and the sodium salt diethyldithiophosphate were ligands proposed for solvent extraction of several metals. 7,8][11][12][13] The formation of micelles at low temperature using PONPE ® 7.5 allowed the high-efficiency extraction of Pb(II) ions from saliva samples without any complexant. 14he cloud point extraction of water-soluble metal complexes with 4-(2-pyridylazo) resorcinol (PAR) has been described before. 15,16A surfactant media is necessary to dissolve chelating reagents such as 1-(2-thiazolylazo)-2naphtol (TAN), 17 dithizone 18 and 1-(2-pyridylazo)2-naphtol (PAN). 1 Cloud point extraction has been extensively described as a separation and pre-concentration procedure for metals prior to their determination by atomic spectrometric techniques. 19Specific applications of CPE with Triton ® X-114 to determine traces of Bi-dithizonate in urine and human hair by electrothermal atomic absorption spectrometry (ET AAS) have been reported. 18The CPE with DDTP and Triton ® X-114 was applied to determine Cd in tobacco and water samples with flame atomic absorption spectrometry (F AAS) detection 10 and Cd, Pb and Pd in blood by ET AAS. 11The same reagents were employed for the simultaneous determination of noble metals in urine by electrothermal vaporization inductively coupled plasma mass spectrometry (ETV-ICP-MS) 12 and Ag, As, Au, Cd, Cu, Pb and Se in water samples using ultrasonic nebulization ICP-MS. 13The micellar phases are very viscous, and commonly they require dilution.Methanol or ethanol plus nitric acid have been described to facilitate the introduction of micellar phases into F AAS, 10 ET AAS, 11 ETV-ICP-MS 12 and ICP-MS. 13Tetrahydrofuran has been used for introduction into ETAAS, 18 or just water has been applied for dilution. 15,20The combination of electrothermal devices using appropriately programmed vaporization temperatures avoided matrix interferences in the atomic spectrometry detection of micellar phases. 11,12,18The flow injection of small sample volumes (100 µL) by using an ultrasonic nebulizer with a dessolvation unit avoided the main drawbacks of introducing micelles dissolved in methanol into an ICP-MS.Seven trace elements were determined by ICP-MS in the transient signals. 13Precision characterized by RSD ≤ 6% was reported.To overcome matrix effects the extracted analytes by CPE has been quantified by isotope dilution, 20 and standard additions. 14he application of isotope dilution (ID) is convenient to determine trace elements in problematic samples, especially when recoveries are variable or matrix dependent.Nevertheless, quantification by ID requires precise isotope ratio measurements to avoid error magnification on results. 21Typical RSD's from 0.1 to 0.5% for isotope ratio measurements by quadrupole ICP-MS and from 0.05 to 0.2% using sector-field mass spectrometers ICP-SFMS have been reported. 22Corrections for mass bias and detector dead time are required to attain accurate results when applying ID.
In this research, a two-step cloud point extractions are planned that produces two surfactant-rich phases in sequence.The micelle extracts are then subjected to oxidative digestion before analysis to facilitate sample introduction and to improve precision on isotope ratio measurements.Quantification by applying either isotope dilution or standard additions is proposed.

Instrumentation
An inductively coupled plasma mass spectrometer, Element 1 (Finnigan MAT, Bremen, Germany), was used in low-resolution mode with a low-flow (0.6 mL min -1 ) concentric nebulizer (Meinhard, Santa Ana, CA, USA) and a Scott-type spray chamber cooled to 10 °C.The ICP-MS operating conditions are summarized in Table 1.The medium and high resolution modes were not practiced for presenting worsen sensitivity and precision. 23Samples were digested in a microwave oven (Ethos 1600, Milestone, Italy) installed in a class-100 clean room.A heating block (Tecnal, Piracicaba, Brazil) and a centrifuge (Cientec model 55, Piracicaba, Brazil) were used to accelerate the phase separation.Conical Pyrex centrifuge tubes (15 mL) Vol. 19, No. 3, 2008   (Corning, New York, USA) were used.Spikes were applied from solutions at 20 °C by using a 20.0 ± 0.04 to 200.0 ± 0.03 µL micropipette (model NPX-200, Nichipet EX, Tokio, Japan).

Samples and sample preparation
Certified reference materials including IRMM Human Serum IMEP-17 (Institute for Reference Materials and Measurements) and HPS Mixed Food Diet (High Purity Standards) were analyzed.Blood samples (0.1 mL) collected from rats contaminated with Cd were analyzed.
Samples solutions in triplicate received the spiked amounts presented in Table 2 calculated for each sample by the reverse isotope dilution equation.Standards containing 0.5 and 1.0 mg L -1 of Co and Ni were used for spiking the samples to perform the standard additions method.The samples and blanks received 3 mL of nitric acid and 2 mL of H 2 O 2 in sequence before digestion in microwave oven in closed PTFE vessels.Three heating steps of 2, 4, and 10 min at temperatures of 60 °C, 85 °C and 120 °C, respectively were used.

Preliminary experiments of cloud point extraction using DDTP and PAR
The application of cloud point extraction to a 200 µg L -1 of QCS-21 in 0.1 mol L -1 HCl solution was performed.The digested micelles were analyzed by ICP-MS in

Sequential cloud point extraction procedure
The proposed sequential CPE scheme is presented on the right side diagram shown in Figure 1.The first extraction was performed from spiked, digested samples at pH < 1 by adding 0.5 mL of the 10% m/v DDTP and 0.1 mL of the 10% surfactant Triton ® X-114 to attain 0.2% m/v in the final solution.
Tubes containing the solutions were immersed in a thermostatic bath heated at 40 °C for 15 min.Immediately after heating, the warm solutions exhibiting turbidity were centrifuged at 4800 rpm for 10 min, and finally the flasks were immersed in an ice bath for 5 min.Two phases were clearly observed: a transparent solution and a white or pale yellow depleted residue.Inverting the tubes separated the supernatant solution corresponding to the surfactantpoor phase.The pH of the surfactant-poor solutions was adjusted to a pH range of 4 to 5 with acetate buffer.The PAR mixed with Triton ® X-114 solution was added as the chelating reagent to perform another cloud point extraction.A second micelle-rich phase was formed just by heating to 40 °C for 15 min, followed by centrifugation and cooling in an ice bath.The depleted organic phase exhibited an orange color.After separation, the micelle-rich surfaces and the tube walls were washed twice with 0.5 mL of water to remove external residues.The separated micelles were easily dissolved by adding 500 µL of HNO 3 plus 200 µL of H 2 O 2 and heated at 120 °C for 15 min in a digestion block to obtain a clear final solution.The micelle containing the DDTP complexes was analyzed by isotope dilution without a volumetric adjustment.After digestion of the second micelle, the volume was made up to 3 mL.The elements in the sample solutions were quantified by standard additions with the addition of 0.0; 50.0 and 100.0 µg L -1 Co and Ni.The surfactant-poor phase separated from the second CPE was also analyzed to calculate recoveries.

Isotope dilution
In preliminary tests the possibility of quantifying Cd, Pb and Cu by isotope dilution in the micelle produced by DDTP and Triton ® X-114 was evaluated.
The instrumental conditions were adjusted to measure isotope ratios 112 Cd/ 114 Cd and 65 Cu/ 63 Cu in standard solutions accurately.Due to the Pb isotopic variability a solution of the Pb isotopic standard NIST SRM 982 was analyzed to determine the instrumental mass bias factor.Detector dead time of 20 ms was experimentally determined for the m/z of interest.

Preliminary results
Preliminary results of the cloud point extraction of Co and Ni complexes using PAR are presented in Figure 2. The efficiency of PAR to form chelates with Co could be affected by high concentrations of concomitants, mainly for samples with high concentrations of transition metals in the +2 oxidation state. 24In effect, PAR in acetate medium at pH 4-6 is also efficient for Zn, Cd, Ni, Cr, V, Ti, Cu and other metals.
The serial scheme was defined for extracting first several transition elements with DDTP.After separating the micelle, the CPE performed on the surfactant-poor solution in acetate media adding PAR previously mixed with Triton ® X-114.Higher stability of PAR in the organized media was observed.An excess of (1 mmol L -1 ) of PAR was used to overcome effects of concomitants guarantying for Co and Ni recoveries higher than 95%.
Results of preliminary tests of cloud point extraction performed on standard solutions and blanks demonstrated that more than 90% of As, Cd, Fe and Pb and 60% of Cu were extracted with DDTP and close to 100% of Co, Ni, V, Ti, Cr and Zn with PAR.No effect from the main matrix constituents in biological samples, such as Na, K, Ca and Mg was noticed.However, the cloud point extraction of blanks and spiked blanks yielded contamination levels of 0.1 ng mL -1 for Cd, Ni and Co of 3.0 ng mL -1 for Cu and Pb.Those levels were related to the reagents impurities.To decrease Cu and Pb contaminations the purification of DDTP was provided.Impurities from 206 Pb-certificated material (CIL PBLM-3662) of Cd < 0.05% and Cu < 0.01% becomes important for isotope dilution quantification.
Preliminary tests of sequential cloud point extraction performed in 3 mL of biological samples allowed estimating the analytes concentration in the residual solution of both micelles.

Quantitative results
The spiked samples were submitted to the sequential CPE scheme represented in Figure 1.The mole fraction ratio 25 was used for calculating the spikes.The simplest case was for 65 Cu with natural ratio 65 Cu/ 63 Cu (0.44563) and highly enriched spike material with isotope ratio R s > 300, allows projecting a final isotope ratio closed to 1.In the case of 112 Cd, the natural isotopic ratio is 112 Cd/ 114 Cd (0.84009) and the R s ~100; then the choice for ratios close to 10 was preferred to obtain highly precise results.For Cd and Pb the isotope ratios were variable, increasing when the sample mass was over estimated and a higher mass was spiked.In all situations precise isotope ratio measurements were attained (RSD < 0.7%) indicating the effectiveness of micelles digestion procedure.The precision of isotopic ratio measurements on CPE micelles just diluted with nitric acid plus methanol reported elsewhere 13 was characterized by RSD from 3-8 %, thus not appropriate for applying isotope dilution quantification.This behavior was attributed to poor aerosol formation and plasma instabilities resulting from the high organic content.The C content of the digested micelle solutions was less than 0.3%.Uncertainties of isotope ratio measurements on Table 2 were calculated by the counting statistic equation proposed elsewhere 25 that considers the square root of the sum of the inverse of counts s -1 on both isotopes.Detection limits of 0.10 µg L -1 for 65 Cu/ 63 Cu, 0.06 µg L -1 for 112 Cd/ 114 Cd and 0.05 µg L -1 for 206 Pb/ 208 Pb  were calculated. 26,27The high isotopic enrichment of the spikes led to lowering detection limits of isotope dilution once the spiked blanks presented isotopic ratios closed to the spike solution. 26The interferences of Ar Na + on 63 Cu were avoided by washing twice the rich-surfactant surface and the tube walls.
The quantification of Co and Ni was performed in micelle-rich produced by CPE using PAR plus Triton ® X-114 in the original samples and spiked samples.The LOD for Co and Ni were 0.04 and 0.06 µg L -1 , respectively.Table 3 lists the average concentrations and uncertainties obtained analyzing three independent sub-samples of different materials.The sample of mixed food diet presented very low concentrations for Co (1.1 µg L -1 ) and Cd (12 µg L -1 ) with precision deteriorated probably by blank corrections.In the case of Cd uncertainties could also due to mass fraction effects and spiking accuracy. 28he sequential CPE procedure generates two surfactantrich extracts containing specific trace elements and a residual solution containing most of the matrix elements.
The analysis of the extracted solutions was performed with no matrix effects since alkaline and alkaline earth elements are not complexed by DDTP or PAR. 29The CPE procedure applied to the CRM´s, presented recoveries for Cd, Pb, Co and Ni in the range from 85 to 96% of the total content.

Conclusions
The sequential CPE procedure is convenient for trace elements determination in low-volume samples, which elements are separated in small volumes of extracted solutions containing specific analytes.The cloud point extraction performed in biological fluids with Triton ® X-114 allowed producing two rich surfactant phases: the first extracting DDTP complexes with Cd, Cu, Pb and the second containing PAR chelates of Co and Ni.The advantage of the two-step CPE separation procedure over sample mineralization and direct analysis results from the possibility of doubling the volume without diluting the analytes and also performing the analysis with less matrix and interferences effects.The high analytes recoveries reveal the right choice of selective ligands and extraction efficiencies.The subsequent digestion of micelles produced by CPE resulted in highly precise isotope ratio measurements making feasible the quantification by isotope dilution ICP-MS.

Figure 1 .
Figure 1.Schematic representation of the sequential cloud point extraction and the quantification procedures.Isotope dilution performed to quantify analytes on the first CPE by measuring isotope ratios (IR) and the standard additions method (SAM) for Co and Ni quantification in the second CPE.

Figure 2 .
Figure 2. Recoveries attained for different elements in a 200 µg L -1 solution chelated with PAR and extracted by Cloud Point Extraction with Triton ® X-114 (0.2% m/v).

Table 1 .
ICP-MS operating conditions

Table 2 .
16,17d amounts and experimental isotope ratios and precision (n = 3) The feasibility of promoting CPE for Co by chelating with several pyridine and thiazole azo compounds was reported before.16,17PARforms anionic HR -water-soluble complexes with Co and Ni at pH from 4.2 to 7. 17 However, the capability for simultaneous cloud point extractions using PAR was not established before.

Table 3 .
Results found in samples, in triplicate, and their reported/certified values