New column separation method for emergency urine samples

The Savannah River Site Environmental Bioassay Lab participated in the 2007 NRIP Emergency Response program administered by the National Institute for Standards and Technology (NIST) in May, 2007. A new rapid column separation method was applied directly to the NRIP 2007 emergency urine samples, with only minimal sample preparation to reduce preparation time. Calcium phosphate precipitation, used to preconcentrate actinides and ⁹⁰Sr in NRIP 2006 urine and water samples, was not used for the NRIP 2007 urine samples. Instead, the raw urine was acidified and passed directly through the stacked resin columns (TEVA+TRU+SR-Resins) to separate the actinides and ⁹⁰Sr from the NRIP urine samples more quickly. This improvement reduced sample preparation time for the NRIP 2007 emergency urine analyses significantly. This approach works well for small volume urine samples expected during an emergency response event. Based on initial feedback from NIST, the SRS Environmental Bioassay Lab had the most rapid analysis times for actinides and ⁹⁰Sr analyses for NRIP 2007 emergency urine samples.     

An improved actinide separation method for environmental samples

This paper presents a rapid method of separation of five actinide elements (Th, U, Np, Pu, and Am) for aqueous media samples. This separation method utilizes the unique chemistries of the actinides at low concentrations^1,2 and the properties of the EIChroM TRU-Resin^TM extraction resin. In order to cleanly recover the five actinides from aqueous samples or solubilized soil samples, the sample is passed through the column twice. The sample is first loaded in an HCl solution with hydrogen peroxide. This allows the Am and most matrix ions to pass through the column. Then the Th is eluted using dilute HCl followed by the Np and Pu which are eluted together with oxalic acid in dilute HCl solution. Finally, the U is eluted with ammonium oxalate solution. A calcium-oxalate coprecipitation is performed on the original load solution containing the Am ions and the dissolved precipitate is then reloaded onto the TRU-Resin^TM column in HNO₃ with ascorbic acid. The procedure requires approximately 1.5 working days for experienced technicians, greatly reduces waste, and generally results in actinide recoveries of 80–100%.     

Rapid separation method for emergency water and urine samples

The Savannah River Site Environmental Bioassay Lab participated in the 2008 NRIP Emergency Response program administered by the National Institute for Standards and Technology (NIST) in May, 2008. A new rapid column separation method was used for analysis of actinides and ⁹⁰Sr in the NRIP 2008 emergency water and urine samples. Significant method improvements were applied to reduce analytical times. As a result, much faster analysis times were achieved, less than 3 hours for determination of ⁹⁰Sr and 3–4 hours for actinides. This represents a 25%–33% improvement in analysis times from NRIP 2007 and a ∼100% improvement compared to NRIP 2006 report times. Column flow rates were increased by a factor of two, with no significant adverse impact on the method performance. Larger sample aliquots, shorter count times, faster cerium fluoride microprecipitation and streamlined calcium phosphate precipitation were also employed. Based on initial feedback from NIST, the SRS Environmental Bioassay Lab had the most rapid analysis times for actinides and ⁹⁰Sr analyses for NRIP 2008 emergency urine samples. High levels of potential matrix interferences may be present in emergency samples and rugged methods are essential. Extremely high levels of ²¹⁰Po were found to have an adverse effect on the uranium results for the NRIP-08 urine samples, while uranium results for NRIP-08 water samples were not affected. This problem, which was not observed for NRIP-06 or NRIP-07 urine samples, was resolved by using an enhanced ²¹⁰Po removal step, which will be described.     

Ammonia abatement in an enzymatic flow system for the determination of creatinine in blood sera and urine

The abatement of ammonia in standard solutions, and in human blood and urine samples is achieved by adding suitable amounts of NADPH and α-ketoglutarate to the sample and passing it through a 2-m nylon tube with glutamate dehydrogenase immobilized on the inner wall. The procedure provides removal of 98% of the ammonia (1–5 × 10^?4 M) in the original sample in 50 s. The abatement of ammonia permits the use of an ammonia probe coupled with an immobilized degradative enzyme for the determination of creatinine. Creatinine was determined in clinical blood and urine samples by first removing the ammonia from the sample and then cleaving the creatinine to N-methylhydantoin and ammonia with immobilized creatininase. Only 200 μl of sample is needed and the entire process is conducted in a single flow stream.     

Rapid analysis of emergency urine and water samples

There is a need for fast, reliable methods for the determination of actinides and ^89/90Sr analysis on environmental and bioassay samples in response to an emergency radiological incident. The Savannah River Site (SRS) Environmental Laboratory participated in the National Institute of Standards and Technology Radiochemistry Intercomparison Program (NRIP-06) and analyzed water and urine samples within 8 hours of receipt. The SRS Environmental Laboratory was the only lab that participated in the program that analyzed these samples for both actinides and ^89/90Sr within an eight hour turnaround time. A rapid actinide and ^89/90Sr separation method was used for both urine and water samples. This method uses stacked TEVA Resin®, TRU Resin® and Sr-Resin® cartridges from Eichrom Technologies (Darien, IL, USA) that allows the rapid separation of plutonium (Pu), neptunium (Np), uranium (U), and americium (Am), curium (Cm) and thorium (Th) using a single multi-stage column combined with alpha-spectrometry. Vacuum box cartridge technology with rapid flow rates was used to minimize sample preparation time. This paper discusses the technology and conditions employed for both water and urine samples and presents the SRS performance data on the NRIP-06 samples.     

Matrix interference free determination of perchlorate in urine by ion association-ion chromatography-mass spectrometry

Quantitative measurement of perchlorate in biological fluids is of importance to assess its toxicity and to study its effects on the thyroid gland. Whenever possible, urine samples are preferred in toxicologic/epidemiologic studies because sample collection is non-invasive. We present here a pretreatment method for the determination of perchlorate in urine samples that lead to a clean matrix. Urine samples, spiked with isotopically labeled perchlorate, are exposed to UV to destroy/decompose organic molecules and then sequentially treated with an H⁺-form cation exchange resin to remove protolyzable compounds, with ammonia to raise the pH to 10-11 and finally passed through a mini-column of basic alumina to remove the color and other organic matter. After filtration through a 0.45 μm syringe filter, the sample thus prepared can be directly injected into an ion chromatograph (IC). We use ion association-electrospray ionization-mass spectrometry (ESI-MS) to detect and quantify perchlorate. The proposed sample preparation method leads to excellent limits of detection (LOD's) for perchlorate since there is essentially no dilution of sample and the matrix effects are eliminated. Results of urine samples from both men and women volunteers are reported for perchlorate, as well as for iodide and thiocyanate, which are generally present at much higher concentrations and for which a “dilute and shoot” approach is adequate. The limit of detection (S/N = 3) for iodide, thiocyanate and perchlorate by the present method was 0.40, 0.10 and 0.080 μg l⁻¹, respectively.     

Determination of betaines as ultraviolet-absorbing esters

A method is described for the estimation of fully N-methylated amino acids (betaines) in plant extracts. After partial purification of aqueous extracts with ion-exchange resins, a crown ether and potassium phosphate buffer are used to facilitate the esterification of betaines with α, p-dibromoacetophenone, p-nitrobenzyl bromide or α-bromo-p-tolunitrile. The esters are isolated with dilute sulphuric acid and chloroform; 10–200 nmol can be determined spectrophotometrically, and smaller amounts by high-performance liquid chromatography.     

Assay of actinides in human urine by rapid method

A method using DGA resin (N,N,N′,N′-tetra-n-octyldiglycolamide on an inert support) was developed for the rapid analysis of actinides in urine samples. Samples acidified with HCl to 4 M were loaded directly (without digestion) onto a DGA column. Actinides were stripped simultaneously, α-sources were prepared by co-precipitation with NdF₃. Americium, plutonium and uranium were separated with acceptable high recoveries (40–80%). The americium, plutonium and uranium content of 100–200 ml urine samples was determined within 24 h with detection limits as low as 0.01 Bq l^?1. Based on model experiments using ¹⁴C-spiked urea, it was proven that high urea content can affect americium separation deleteriously due to irreversible fixing of americium on DGA resin.     

Separation and determination of <Superscript>241</Superscript>Am in urine samples from radiation workers using PC88-A and alpha spectrometry

Bioassay technique is used for the estimation of actinides present in the body based on their excretion rate through body fluids. For occupational radiation workers urine assay is the preferred method for monitoring of chronic internal exposure. Determination of low concentrations of actinides such as plutonium, americium and uranium at low level of mBq in urine by alpha spectrometry requires pre-concentration of large volumes of urine. This article deals with standardization of analytical method for the determination of ²⁴¹Am isotope in urine samples using Extraction Chromatography (EC) and ²⁴³Am tracer for radiochemical recovery. The method involves oxidation of urine followed by co-precipitation of americium along with calcium phosphate. This precipitate after treatment is further subjected to calcium oxalate co-precipitation. Separation of Am was carried out by EC column prepared by PC88-A (2-ethyl hexyl phosphonic acid 2-ethyl hexyl monoester) adsorbed on microporous resin XAD-7 (PC88A-XAD7). Am-fraction was electro-deposited and activity estimated using tracer recovery by alpha spectrometer. Ten routine urine samples of radiation workers were analyzed and consistent radiochemical recovery was obtained in the range 44–60% with a mean and standard deviation of 51 and 4.7% respectively.     

Rapid determination of actinides in urine by inductively coupled plasma mass spectrometry and alpha spectrometry: A hybrid approach

A new rapid separation method that allows separation and preconcentration of actinides in urine samples was developed for the measurement of longer lived actinides by inductively coupled plasma mass spectrometry (ICP-MS) and short-lived actinides by alpha spectrometry; a hybrid approach. This method uses stacked extraction chromatography cartridges and vacuum box technology to facilitate rapid separations. Preconcentration, if required, is performed using a streamlined calcium phosphate precipitation. Similar technology has been applied to separate actinides prior to measurement by alpha spectrometry, but this new method has been developed with elution reagents now compatible with ICP-MS as well. Purified solutions are split between ICP-MS and alpha spectrometry so that long- and short-lived actinide isotopes can be measured successfully. The method allows for simultaneous extraction of 24 samples (including QC samples) in less than 3 h. Simultaneous sample preparation can offer significant time savings over sequential sample preparation. For example, sequential sample preparation of 24 samples taking just 15 min each requires 6 h to complete. The simplicity and speed of this new method makes it attractive for radiological emergency response. If preconcentration is applied, the method is applicable to larger sample aliquots for occupational exposures as well. The chemical recoveries are typically greater than 90%, in contrast to other reported methods using flow injection separation techniques for urine samples where plutonium yields were 70-80%. This method allows measurement of both long-lived and short-lived actinide isotopes. ²³⁹Pu, ²⁴²Pu, ²³⁷Np, ²⁴³Am, ²³⁴U, ²³⁵U and ²³⁸U were measured by ICP-MS, while ²³⁶Pu, ²³⁸Pu, ²³⁹Pu, ²⁴¹Am, ²⁴³Am and ²⁴⁴Cm were measured by alpha spectrometry. The method can also be adapted so that the separation of uranium isotopes for assay is not required, if uranium assay by direct dilution of the urine sample is preferred instead. Multiple vacuum box locations may be set-up to supply several ICP-MS units with purified sample fractions such that a high sample throughput may be achieved, while still allowing for rapid measurement of short-lived actinides by alpha spectrometry.     

Evaluation of mass spectrometry and radiation detection for the analysis of radionuclides

Summary The feasibility of measuring picogram levels of actinides in a urine matrix using ion chromatography coupled on-line to an inductively coupled plasma quadrupole mass spectrometer (IC-Q-ICPMS) was investigated. A chelation column for separation of matrix ions and preconcentration of the actinides was combined with a cation-exchange column for separation of the actinides. Sample preparation required simple addition of ammonium acetate to adjust the pH of the urine matrix. Spike solutions containing ²³² Th, ²³⁷Np, ²³⁸U, ²³⁹Pu, and ²⁴¹Am were added to undiluted urine, diluted urine (1 : 9) and water. Results showed that this approach enhanced the signal sensitivities of all the tested actinides over two orders of magnitude in the water matrix, while certain elements (especially Am) can still be effectively concentrated in undiluted urine.     

SPE for Endo- and Exo-Iohexol Analysis with HPLC in Canine Serum and Rat Urine

A new solid-phase extraction, using LiChrolut^® EN cartridges, was developed and validated as sample preparation for analysis of the iodinated contrast medium iohexol in canine serum and rat urine. Samples were further analyzed by high-performance liquid chromatography (HPLC) and UV detection at 246 nm. Iohexol ((±)-N,N′-Bis (2,3-dihydroxypropyl)-5-[N-(2,3-dihydroxypropyl)-acetamido]-2,4,6-triiodisophthalamid, trade name Omnipaque^®) consists of the two isomers endo- and exo-iohexol. Excellent separation of these isomers could be achieved with the C30 column (Prontosil^®200, 250 × 4.6 mm, pore size 5 μm). A multistep gradient elution was followed, using methanol and a phosphate buffer at pH 7.4 (15 mM dipotassium hydrogen phosphate trihydrate and 1.5 mM tetrabutylammonium chloride) and a flow rate of 1 mL min^?1. Validation of the method showed good sensitivity, reproducibility, and precision. The analysis was linear from 20 to 200 μg mL^?1. Recovery rates for serum samples ranged from 73.6 to 82.6%. For urine samples, recovery rates of 77.0–83.1% were observed. Throughout sample preparation and HPLC-UV analysis, the contrast medium sodium diatrizoate dihydrate served as a control substance.     

Evaluation of chelation concentration and cation separation of actinides at ultra-trace levels in urine matrix

Summary The feasibility of measuring picogram levels of actinides in a urine matrix using ion chromatography coupled on-line to an inductively coupled plasma quadrupole mass spectrometer (IC-Q-ICPMS) was investigated. A chelation column for separation of matrix ions and preconcentration of the actinides was combined with a cation-exchange column for separation of the actinides. Sample preparation required simple addition of ammonium acetate to adjust the pH of the urine matrix. Spike solutions containing ²³² Th, ²³⁷Np, ²³⁸U, ²³⁹Pu, and ²⁴¹Am were added to undiluted urine, diluted urine (1 : 9) and water. Results showed that this approach enhanced the signal sensitivities of all the tested actinides over two orders of magnitude in the water matrix, while certain elements (especially Am) can still be effectively concentrated in undiluted urine.     

Analysis of Anisodine and Identification of Twenty of Its Metabolites in Rat Urine by Liquid Chromatography–Tandem Mass Spectrometry

A simple and rapid high-performance liquid chromatographic-electrospray ionization (ESI) tandem mass spectrometric method has been developed for elucidation of the structures of the metabolites of anisodine in rat urine after administration of a single dose (20 mg). Different extraction techniques (free fraction, acid hydrolysis, and enzyme hydrolysis) were compared for investigation of the metabolism of anisodine. After extraction the pretreated samples were injected into a reversed-phase C₁₈ column with 60:40 (v/v) methanol–0.01% triethylamine solution (2 mM, adjusted to pH 3.5 with formic acid) as mobile phase. Detection was by on-line MS-MS. Identification of the metabolites and elucidation of their structure were performed by comparing changes in molecular masses (ΔM), retention-times, and spectral patterns of product ions with those of the parent drug. At least twenty metabolites (norscopine, scopine, α-hydroxytropic acid, aponoranisodine, apoanisodine, noranisodine, anisodine N-oxide, hydroxyanisodine, hydroxyanisodine N-oxide, methoxyanisodine, hydroxymethoxyanisodine, trihydroxyanisodine, dihydroxymethoxyanisodine, hydroxydimethoxyanisodine, glucuronide conjugates, and sulfate conjugates of noranisodine, hydroxyanisodine and the parent drug) and the parent drug were found in the urine after ingestion of 20 mg anisodine by healthy rats. Anisodine N-oxide, hydroxyanisodine, and the parent drug were detected in rat urine for up 120 h after ingestion of the drug.     

The use of calix[6]arene molecules for actinides analysis in urine and drinking water: an alternative to current procedures

In this work the applicability of calix[6]arene columns for actinides analysis in urine samples and drinking water was investigated. A radiochemical procedure has been developed for U, Pu, Am analysis in urine. A simple and effective method has also been proposed on a specific column named AQUALIX, for the separation and preconcentration of U from drinking water. These procedures are suitable for routine analysis and require a considerably reduced number of steps of sample treatment as compared to current procedures.     

On-line sample stacking of peptides in capillary electrophoresis for the study of prebiotic reactions between α,α-dialkylated amino acids and amino acid N-carboxyanhydrides

The reaction between α,α-dialkylated amino acids and amino acid N-carboxyanhydrides is slow leading to low concentrations of products (peptides). The detection by capillary electrophoresis of the analytes contained in such samples is therefore a challenging issue. In this work, on-line sample pre-concentration methods based on field-amplified sample stacking have been implemented and compared. Because of the high ionic strength present in the sample matrix, samples were diluted with an organic solvent prior to analysis to decrease the sample conductivity. Different modes of sample injection (field amplified sample injection (FASI), hydrodynamic normal sample stacking (NSS) or large volume sample stacking (LVSS)) were compared. Pre-concentration factors of 20 for FASI, about 30–40 for NSS and 60 for LVSS were obtained for the analysis of (l,l) dipeptide of valine in a large excess of isovaline and 0.2 M of ionic strength. For LVSS application and resolution optimisation, a new non-covalent coating based on the partial modification of the capillary surface was used to tune the electroosmotic flow magnitude and to pump the sample matrix out of the capillary. This on-line sample pre-concentration step allowed confirming that oligopeptides including α,α-dialkylated amino acids are formed during the reaction between α,α-dialkylated amino acids and N-carboxyanhydride amino acids.     

Analysis of cocaine and benzoylecgonine in urine by using multisyringe flow injection analysis‐gas chromatography‐mass spectrometry system

In this paper, a method was described to determine cocaine (COC) and benzoylecgonine (BZE) in human urine samples by GC‐MS detection. The extraction of analytes from urine samples was achieved in an Oasis hydrophilic–lipophilic balance column (20 mm×3.9 mm id, dp=25 μm; Waters, USA), incorporated in a multisyringe flow injection system, used for the sample treatment. Finally, to improve the volatility of the BZE, an in‐line derivatization reaction with N,O‐bis (trimethylsilyl) trifluoroacetamide with 1% trimethylchlorosilane was made microwave‐assisted in order to reduce the reaction time. The results showed that the proposed method is a good alternative for the analysis of COC and BZE in urine samples because it offers advantages compared with those described in the literature, which include simplicity in the sample treatment, the sensitivity and selectivity necessary to determine the analytes of interest at low levels in the urine and high sample throughput.     

Liquid chromatography-electrospray ionization tandem mass spectrometry for on-line characterization, monitoring and isotopic profiling of the main selenium-metabolite in human urine after consumption of Se-rich and Se-enriched food

The metabolism of selenium (Se) in the human body has yet not completely been unravelled and hence, an efficient method for characterization and on-line monitoring of the main Se-compound in human urine after consumption of Se-rich food was developed. Total Se-concentration in human urine after consumption of several Se-rich products was measured with inductively coupled plasma mass spectrometry (ICP-MS). The highest Se concentration in urine was observed after 4-10 h. The urine samples were brought onto a reversed phase column and the Se was detected by ICP-MS. Parameters for liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS) measurements were optimized by using commercially available sugars, because it is known that some of the urinary metabolites contain a sugar moiety. In order to characterize the predominant Se-metabolite, it was necessary to extensively clean-up the sample and preconcentrate the species. The main metabolite was measured on its precursor ion on three different m/z according to three isotopes of Se. Relative peak surfaces matched the relative abundances of the isotopes. The product ions could be measured in a human urine sample in accordance to the product ions of the commercially available sugars. Moreover, the evidence of a selenosugar was demonstrated by the use of the Se-isotopes when measuring the product ions. LC-ESI-MS-MS was proven to be very efficient for the characterization of the main urinary Se-metabolite and can be used for on-line monitoring of the compound in urine samples. The method can be extended for clinical screening after consumption of Se-(en)rich(ed) food by use of the Se-isotopic profile and/or of the typical product ions of (methyl)-N-acetyl-hexosamines.     

Pre-concentration and separation of thorium, uranium, plutonium and americium in human soft tissues by extraction chromatography

An extraction chromatographic method is described for the pre-concentration and separation of thorium, uranium, plutonium and americium in human soft tissues. Tissues such as lung and liver are oven dried at 120°C, ashed at 450°C and the ashed sample is alternately wet (HNO₃/H₂O₂) and dry ashed, and then dissolved in 8M HCl. Because of the complex matrix and large sample samples (up to 1500 g), the actinides were preconcentrated from the tissue solution using the TRU^TM resin (EIChroM) prior to elemental separation by extraction chromatography and determination of americium, plutonium, uranium and thorium by alpha spectrometry. The actinides were eluted from the preconcentration column and each actinide was individually eluted on TEVA^TM and TRU^TM resin columns in a tandem configuration. Actinide activities were then determined by alpha spectrometry after electrodeposition from a sulfate medium. The method was validated by analyzing human tissue samples previously analyzed for americium, plutonium, uranium and thorium in the United States Transuranium and Uranium Registries (USTUR). Two National Institute of Standards and Technology (NIST) Standard Reference Materials, SRM 4351-Human Lung and SRM 4352-Human Liver were also analyzed. United States Transuranium and Uranium Registries, Washington State University, Pullman, WA, 99163, USA.     

Capillary electrophoretic study of thiolated α-cyclodextrin-capped gold nanoparticles with tetraalkylammonium ions

Capillary zone electrophoresis (CZE) has been employed to characterize nanometer-sized thiolated α-cyclodextrin-capped gold nanoparticles (α-CD-S-AuNPs). The addition of tetrabutylammonium (Bu₄N⁺) ions to the run buffer greatly narrows the migration peak of α-CD-S-AuNP. The optimal run buffer was determined to be 10 mM Bu₄N⁺ in 30 mM phosphate buffer at pH 12 and an applied voltage of 15 kV. The effect of various tetraalkylammonium ions on the peak width and electrophoretic mobility (μ_e) of α-CD-S-AuNP was studied in detail. Bu₄N⁺ ions assist in inter-linking the α-CD-S-AuNPs and narrowing the migration peak in CZE. This observation can be explained by the fact that each Bu₄N⁺ ion can simultaneously interact with several hydrophobic cavities of the surface-attached α-CDs on AuNPs. The TEM images show that α-CD-S-AuNPs with Bu₄N⁺ are linked together but in the absence of Bu₄N⁺, they are more dispersed. The migration mechanism in CZE is based on the formation of inclusion complexes between Bu₄N⁺ and α-CD-S-AuNPs which induces changes in the charge-to-size ratio of α-CD-S-AuNPs and μ_e. An inverse linear relationship (r² > 0.998) exists between the μ_e and size of α-CD-S-AuNPs in the core range 1.4–4.1 nm. The CZE analyses are rapid with migration time less than 4 min. A few nanoliters of each of the α-CD-S-AuNP samples were injected hydrodynamically at 0.5 psi for 5 s. Our work confirms that CZE is an efficient tool for characterizing the sizes of α-CD-S-AuNPs using Bu₄N⁺ ions.