Determination of ²⁴¹Am in Urine Using Sector Field Inductively Coupled Plasma Mass Spectrometry (SF-ICP-MS)

Abstract

Quantification of ²⁴¹Am in urine at low levels is important for assessment of individuals’ or populations’ accidental, environmental, or terrorism-related internal contamination, but no convenient, precise method has been established to rapidly determine these low levels. Here we report a new analytical method to measure ²⁴¹Am as developed and validated at the Centers for Disease Control and Prevention (CDC) by means of the selective retention of Am from urine directly on DGA resin, followed by SF-ICP-MS detection. The method provides rapid results with a Limit of Detection (LOD) of 0.22 pg/L (0.028 Bq/L), which is lower than 1/3 of the C/P CDG for ²⁴¹Am at 5 days post-exposure. The results obtained by this method closely agree with CDC values as measured by Liquid Scintillation Counting, and with National Institute of Standards Technology (NIST) Certified Reference Materials (CRM) target values.

Introduction

Americium is a man-made, radioactive, metallic element produced when plutonium atoms undergo successive neutron capture events in nuclear reactors, in nuclear weapons, and during nuclear weapons’ detonations. Americium has several different isotopes, all of which are radioactive. The most important and prevalent americium isotope is ²⁴¹Am, with a half-life of 432.7 years. As it decays, ²⁴¹Am releases alpha particles at 5.44 MeV (13%) and 5.49 MeV (84.5%), becoming ²³⁷Np, which (in 35.9% of the decays) immediately emits gamma radiation at 59.5 keV. The ²⁴¹Am decay chain ends with ²⁰⁹Bi, a nonradioactive element. ²⁴¹Am in the environment originated from atmospheric testing of nuclear weapons during the 1950s and 1960s, as well as reprocessing plants and nuclear accidents. Facilities that are involved with nuclear weapons, well logging sources and manufacture smoke detectors are minor sources of ²⁴¹Am contamination [1].

The critical ²⁴¹Am exposure pathways are inhalation and ingestion. ²⁴¹Am poses significant health hazards, even in small concentrations, if it is taken into the body in a soluble form. Once in the body, ²⁴¹Am concentrates in the skeleton, liver, and muscle. It can stay in the body for decades and continue to expose the surrounding tissues to both alpha and gamma radiation. Long-term internal exposure to ²⁴¹Am may create an increased risk of developing cancer. Exposure to any significant amount of ²⁴¹Am is unlikely under normal circumstances [1, 2].

Several techniques exist for the determination of ²⁴¹Am concentration in environmental and human samples [3, 4]. Gamma spectrometry, using High Purity Germanium detectors, is the primary tool used to determine ²⁴¹Am at levels of 0.1−1 Bq/kg or higher; but to obtain accurate results, it requires that the user correct for the attenuation of gamma rays in the samples [5, 6]. Alpha spectrometry is the most commonly applied technique for determination of low-level ²⁴¹Am content. Its principal advantages are relatively low equipment costs, high sensitivity due to low background, and high selectivity for alpha particles against other types of radiation [3]. A limit of detection (LOD) of 10−20 mBq/kg has been reported for various sample matrices, depending on the counting time and count rate of the procedure blank [7–9]. However, tedious, time-consuming sample preparation procedures (e.g., precipitation, evaporation, elution, filtration, electroplating, etc.) and long measurement times limit throughput. Such preparation procedures are due to possible interference from other radionuclides with close alpha energies, and long counting times are necessary because of ²⁴¹Am’s relatively low specific activity.

SF-ICP-MS offers substantial advantages over conventional radiometric techniques and has recently been used for analysis of many long-lived radionuclides in various sample matrices. It is one of the fastest methods for ²⁴¹Am analysis [10–13]. An LOD of 1 pg/L for ²⁴¹Am has been reported on SF-ICP-MS [14]. This LOD is comparable to that of alpha spectrometry, assuming no interferences exist for SF-ICP-MS. However, the main analytical issue in SF-ICP-MS originates from isobaric and polyatomic interferences such as ²⁴¹Pu⁺, ²⁴⁰PuH⁺, ²⁰⁹Bi³²S⁺, ²⁰⁹BiO₂⁺, ²⁰⁶Pb³⁵Cl⁺, ²⁰⁴Pb³⁷Cl⁺, ²⁰⁵Tl³⁶Ar⁺, ²⁰⁷Pb³⁴S⁺, and ²⁰¹Hg⁴⁰Ar⁺ [11]. The major isobaric interference with ²⁴¹Am is ²⁴¹Pu. Since ²⁴¹Am is the decay product of ²⁴¹Pu (half-life is 14.33 years), in some samples of reactor origin the concentration of ²⁴¹Am is comparable to that of ²⁴¹Pu. Therefore, the method includes a thorough chemical separation of ²⁴¹Pu (which also removes most of the other interfering molecular ions) from the samples [11, 15].

Developing methods to determine exposure to ²⁴¹Am is within CDC’s public health mission. Quantitative analysis of ²⁴¹Am in urine is considered a useful, noninvasive way to assess levels of internal contamination. Our Emergency Response Analytical goal is to be able to detect threat-radionuclides in urine at levels well below (i.e., 1/3 of or lower) the levels for a general population or for special subgroups such as children or pregnant woman (C/P) at the National Council on Radiation Protection & Measurements (NCRP) Report No. 161 Clinical Decision Guide (CDG) based action level of 0.73 pg/L (0.093 Bq/L) for ²⁴¹Am (urine output expected at 5 days post intake) [16]. CDC’s IRATB recently developed a urine Gross Alpha/Gross Beta method using Liquid Scintillation Counting (LSC) [17]. However, the LOD of this method for ²⁴¹Am is 4.2 Bq/L, equivalent to 32.3 pg/L, which is much higher than the C/P CDG of 0.73 pg/L for ²⁴¹Am.

In this study, we report a novel and rapid analytical method for determination of ²⁴¹Am in urine samples. The Solid Phase Extraction (SPE) part of the method is based on preliminary studies carried out by Horwitz, et al. [18], Li, et al. [19] and Sadi, et al. [20] using a single DGA resin cartridge to separate Am from other actinides such as U and Pu. We further optimized the method to isolate ²⁴¹Am from a 10-mL volume of urine using simple extraction steps and used SF-ICP-MS for detection instead of LSC. The study’s purpose was to develop a rapid, simple method to address and respond to public health or other accidental, environmental, or terrorism-related exposures to ²⁴¹Am. This method is not designed to characterize the normal background level of ²⁴¹Am in the non-occupationally exposed population, but it does have a detection limit below the suggested CDG action levels. Thus it can serve as a means of rapidly identifying both adults and children who have been exposed to ²⁴¹Am and who might require medical intervention.

Experimental

Reagents and solutions

DGA Cartridges (normal, 1 mL) and a polycarbonate vacuum box (24 holes) were purchased from Eichrom Technologies (Darien, IL, USA). All nitric (HNO₃) and hydrochloric (HCl) acid solutions were prepared from double-distilled (DD) acids (GFS Chemicals Inc. Columbus, OH). Deionized water was used for all solutions (≥ 18 MΩ·cm, from an Aqua Solutions Ultrapure Water System, Aqua Solutions, Inc., Jasper, GA). “Base urine” was collected through anonymous human donations (CDC protocol 3994) and acidified to 1% v/v HNO₃. All radioactivity solution sources were traceable to the National Institute for Standards and Technology (NIST, Gaithersburg, MD, USA). Both low and high quality control (QC) solutions and other urine pools for LOD were prepared for determination by spiking base urine with dilutions of an ²⁴¹Am isotope standard (Eckert & Ziegler Analytics, Inc., Atlanta, GA). A series of aqueous ²⁴¹Am Certified Reference Materials (CRM) solutions were prepared by dilution of ²⁴¹Am radioactive source solutions from NIST. ²⁴³Am (Eckert & Ziegler Analytics Inc., Atlanta, GA) was used as an internal standard (tracer). Sodium nitrite (Sigma-Aldrich, St. Louis, MO) was used to adjust the oxidation states. Serial dilutions of uranium, lead, thallium, mercury, bismuth single-element stock standards (SPEX Industries, Inc., Edison, NJ) and a ²⁴²Pu radioactivity solution (U.S. Department of Energy, New Brunswick Laboratory, Argonne, IL) were spiked into the urine samples to verify that high separation factors for U, Pb, Tl, Hg, Bi and Pu were obtained using this SPE procedure.

Sample preparation

The urine sample volume for a single analysis is 10 mL. Allow urine specimens to reach ambient temperature, shake or vortex them to mix for 5 seconds before pipetting. Spike 400 µL of 1 ng/L ²⁴³Am solution as an internal standard (tracer) to every 10 mL of urine patient sample or QC sample. Add 4.76 mL of concentrated HNO₃ (68–70%, the final concentration in the sample is 5M) and then 0.13g of sodium nitrite to each sample as a valence adjuster to convert Pu to the tetravalent state. Shake or vortex to mix the samples for 5 seconds and let reaction occur at room temperature for at least 10 minutes. Load each sample on a DGA resin cartridge of 1 mL bed volume (cartridge preconditioned with 15 mL of 5 M HNO₃ using a vacuum box). Rinse the cartridge again with 15 mL of 5M HNO₃ followed by 15 mL × 3 of 0.5M HNO₃ using a vacuum box. Strip ²⁴¹Am from the column with 5 mL of 0.5M HCl. Transfer 1 mL of the purified samples into 4 mL polystyrene conical bottom sample cups for analysis (Figure 1). Prepare external, aqueous-based stock calibration standards by spiking 0.5M HCl with dilutions of ²⁴¹Am isotope standard, and then add 40 µL of internal standard solution (1 ng/L ²⁴³Am) to every 1 mL of standards to reach the same tracer concentration as the patient and QC samples. Prepare both calibration standards and sample blanks as 0.5 M HCl solutions, which match the elute solutions for the column of this method.

Figure 1.

Sequential sample preparation procedure for ²⁴¹Am determination

* Samples containing U concentrations greater than 10 µg/L, add steps 5 – 6.

Instrumentation

This method measures ²⁴¹Am concentrations using an extended dynamic range, high-resolution ICP-MS model Element XR (Thermo Fisher Scientific, Bremen, Germany), which is a double-focusing, magnetic sector, inductively-coupled-plasma mass spectrometer with a single discrete dynode detector (Mascom, Bremen, Germany). It uses the ICP-MS, equipped with nickel sampler and skimmer cones and a CD-2 guard electrode, in triple mode. The sample introduction system consists of a computer-controlled ASX-112 (Cetac, Omaha, NE) autosampler and an Aridus II™ (Cetac, Omaha, NE) desolvation unit. As discussed in our lab’s previous report [21], the Aridus II™ setup increases the sensitivity of the SF-ICP-MS by more than 10 times, enabling the measurement of ²⁴¹Am at the low level of < 1 pg/L. Samples self-aspirate from the autosampler into the desolvation unit through an Apex perfluoroalkoxy (PFA) 100 μL/minute nebulizer (ESI, Omaha, NE, or equivalent). The desolvation unit, equipped with an upgraded PFA spray chamber, operates at 110 °C. With the aid of argon sweep gas and nitrogen gas for sensitivity enhancement, the sample passes through a semi-permeable membrane coil in the unit that operates at 160°C. Optimize flow rates as needed, with argon sweep gas at ~ 3−7 L/min and nitrogen gas at ~ 3−7 mL/min. The desolvated sample exits the unit into a 1.8 mm I.D. sapphire injector and a standard quartz torch, and then into the mass spectrometer. All experimental parameters are optimized for ²⁴¹Am concentrations determination by SF-ICP-MS with respect to maximum ion intensity of ²³⁸U and minimum uranium oxide formation rate using a 5 ng/L natural uranium tuning solution. Table 1 contains a summary of our optimized operating conditions.

Table 1.

Instrumental conditions and data acquisition settings for SF-ICP-MS measurements

RF Power (KW)	1.2 – 1.3
Cooling Gas flow (L/min)	16
Auxiliary Gas flow (L/min)	0.9
Sample Gas flow (L/min)	0.7 – 0.8
Lenses (V)	Optimized as needed
Sample Take up time (min)	2.1
Wash (min)	3
Pump Speed During Wash (rpm)	1
LR Runs/Passes	3* 60
Detection Mode	Triple
Measurement Units	CPS
Scan Type	ESCAN
Scan Optimization	Speed
Number of Pre-Scans	5
Integration Type	Average
Res. Switch Delay (s)	2
Resolution	300
Mass Window (%)	15
Setting Time (s)	0.001
Sample Time (s)	0.001
Samples Per Peak	200
Search Window (%)	20
Integration Window (%)	15
Measured Isotopes	241Am, 243Am

Results and discussion

Removal of potential spectral interferences

Potential interferences for analysis of ²⁴¹Am include isobaric overlaps with anthropogenic ²⁴¹Pu and polyatomic overlaps with ²⁴⁰PuH⁺, ²⁰⁹Bi³²S⁺, ²⁰⁹BiO₂⁺, ²⁰⁶Pb³⁵Cl⁺, ²⁰⁴Pb³⁷Cl⁺, ²⁰⁵Tl³⁶Ar⁺, ²⁰⁷Pb³⁴S⁺, and ²⁰¹Hg⁴⁰Ar⁺. To test for complete removal of ²⁴¹Pu, a 50 pg/L solution of ²⁴²Pu isotope spike in base urine was prepared and tested. Experiments showed more than 99% of ²⁴²Pu is removed by the SPE portion of sample preparation. Using SPE sample preparation as described above, Pb, Tl, and Hg, spiked in base urine at concentrations of 3 µg/L, 0.5 µg/L, and 5 µg/L respectively, did not result in apparent (> 0.1 pg/L) ²⁴¹Am concentrations. These spiked urine samples’ concentrations were above the National Health and Nutrition Examination Survey (NHANES) 95^th percentile of urine Pb, Tl, and Hg concentrations [22]. Although no NHANES survey data was available for bismuth, analysis of what was otherwise determined [23, 24] to be a high urine concentration (5 µg/L) of Bi, produced no apparent ²⁴¹Am concentration.

Performance of a natural U-spike experiment determined that due to peak tailing, small interferences remained at m/z = 241 when the separated sample solutions contain high levels of U (> 0.5 µg/L). Analysis of urine samples with U =1.0 µg/L with this SPE method as part of the sample preparation procedure removed more than 99% of the U that might cause tailing into the m/z=241 region. Samples having U concentrations higher than 10.0 µg/L (the NHANES 95^th percentile of U concentration in urine of normal U. S. residents is 0.031 µg/L) [22] should be treated by the modified sample preparation procedure as shown in Figure 1, which will be described in more detail below.

Limit of detection

The LOD for ²⁴¹Am in urine specimens is based on 60 analytical runs of 4 different low-concentration samples close to the LOD (a first approximation of LOD is the measured blank concentration plus 3 times the Standard Deviation (SD) of the measured blank concentration) and was calculated according to the formula:

Conc_LOD= [meanb + 1.645(Sb + int)]/[1-1.645(slope)], where mean b = blank average, Sb = standard deviation of blank average, int = intercept of the equation of SD versus concentration for LOD samples analyzed at least 60 times, Slope = slope of the equation of SD versus concentration for LOD samples analyzed at least 60 times.

The LOD of this method is 0.22 pg/L (Figure 2). This LOD is < 1/3 of the C/P CDG (~0.734 pg/L), and is therefore acceptable for an emergency radiobioassay method for determining the concentration of ²⁴¹Am in urine collected at 5 days post-exposure.

Figure 2.

Plot for ²⁴¹Am LOD determination (60 runs per point).

Linearity

A linearity study determined the linear reportable range for this method. The method exhibits good linear signal response between concentrations of 0.3 pg/L and 1000 pg/L of ²⁴¹Am with a Coefficient of Determination of 1.000. The normal calibration range is from 0.3 pg/L to 30 pg/L, and the extended calibration range is from 30 pg/L to 1000 pg/L. If a urine ²⁴¹Am value is above the highest calibrator, the urine sample is diluted with 5% HNO₃ to bring the concentration within the validated calibration range.

Internal methods comparison study

A comparison of urine sample analysis results was performed between this method and our CLIA validated LSC method. The two samples LU-077203 and HU-077201 were prepared as QC material and, using LSC, analyzed for ²⁴¹Am at relatively high concentrations. They then were diluted 1:1000 to get within the desired ²⁴¹Am concentration range for the present method, purified and analyzed using SF-ICP-MS. The difference between the described methods is 2.1% to 3.0% (Table 2),

Table 2.

Observed ²⁴¹Am concentrations (pg/L) and among-run precision for reference materials and internal quality control materials

Sample	N	241Am
		Average	SD	Target Value	Bias (%)
LU-077203a	12	8,220	310	8,050d	2.1
HU-077201a	12	20,700	600	20,100d	3.0
Pool1b	59	0.335	0.058	0.3e	12
Low QCc	120	0.708	0.068	0.7e	1.2
Pool2b	60	1.44	0.093	1.4e	3.0
High QCc	120	10.1	0.56	10.0e	0.9
Extended High QCc	40	783	33.6	800e	−2.1
Dilution 1f	1	100	−	100	0.0
Dilution 2f	1	203	−	200	1.7
Dilution 3f	1	300	−	300	−0.1
Dilution 4f	1	399	−	400	−0.3
Dilution 5f	1	605	−	600	0.8
Dilution 6f	1	1007	−	1000	0.7

^a1:1000 dilution of urine QC materials used for the LSC Gross Alpha/Beta method at CDC.

^bUrine materials made at CDC by spiking certified reference material in pooled urine collected anonymously.

^cInternal quality control materials made at CDC by spiking certified reference material in pooled urine collected anonymously.

^dCharacterized results of co-worker by using the LSC Gross Alpha/Beta method at CDC[17].

^eTarget values of spiked urine pools using certified reference material.

^fAqueous dilutions of CRM from NIST.

Precision and accuracy

Analysis of serial aqueous dilutions of a Certified Reference Material (CRM) from NIST was also used to verify method accuracy. The observed ²⁴¹Am concentrations were in close agreement with the target values, with an analytical bias from −0.3% to 1.7% (Table 2). Table 2 also shows the typical precision observed at different concentrations of daily quality control materials analyzed at the beginning, in the middle, and at the end of each analytical run. Accuracy and precision of the reported results was assured based on adherence to the quality control/quality assurance program of the Division of Laboratory Sciences, NCEH, CDC [25].

Analysis of samples from the NIST Radiochemistry Intercomparison Program (NRIP)

NRIP is a performance evaluation program which provides high quality, traceable radionuclide materials to support low-level radioanalytical laboratories conducting environmental and radiobioassay radioactivity measurements. ²⁴¹Am is among the radionuclides used for testing. However, we found that the extraordinarily high concentrations of uranium present in these samples (intended for evaluation of environmental levels of uranium by alpha spectrometry) significantly affects the accuracy of trace level ²⁴¹Am determination by SF-ICP-MS. Further, these uranium concentrations would possibly produce significant, troublesome instrument contamination. To address this problem we developed and recommend a modified sample preparation procedure that is further optimized for samples with extremely high U content (usually higher than 10 µg/L).

In this procedure, after rinsing the cartridge with 15 mL of 5M HNO₃ followed by 15 mL × 3 of 0.5M HNO₃, replace both the cartridge reservoirs and tips to eliminate possible U deposits and rinse the cartridges with more 0.5M HNO₃ (15 mL × (3 - 6) of 0.5M HNO₃, see Figure 1). Table 3 and Table 4 show the results observed for ²⁴¹Am analysis of the NRIP samples. These samples were from two radiobioassay preparedness exercises during 2012 with different turnaround times (TATs): one 60 days, and one 8 hours. These synthetic urine samples typically have U concentrations ranging from 140 µg/L to 450 µg/L. After the more aggressive rinsing procedure, U concentrations in the elution solutions were under 0.20 µg/L, and did not result in apparent ²⁴¹Am signal contribution for these samples. All but one result had slight negative bias (average −2.1 +/− 2.4% at a 95% Confidence Level) compared with the NIST target values. Most of the observed results for the NRIP samples show a small negative bias compared to the NIST target values, indicating a slight negative systematic uncertainty. One result had a positive bias of 12.7%. We noted that analyses of this sample for other radionuclides yielded a similar positive bias, indicating an external sample preparation error, as opposed to method bias.

Table 3.

Comparison of CDC ²⁴¹Am results with NIST target values for NRIP12 60 days samples^*

Sample ID	Massic Activity (NIST Target Value)	Massic Activity (CDC Observed Results)	Relative Expanded Uncertainty (k=2)	Bias
	(Bq/g spike)	(Bq/g spike)	(%)	(%)
207	1.784	1.74	11.5	−2.52
212	1.784	1.74	11.2	−2.41
220	1.784	1.76	12.8	−1.57
224	1.784	1.74	12.5	−2.52
227	1.784	1.76	12.1	−1.63
214	1.784	1.74	12.3	−2.47
216	1.784	1.71	12.1	−4.09
228	1.784	1.75	12.2	−2.02
231	1.784	1.77	11.5	−0.95
232	1.784	1.77	11.2	−0.73
208	1.784	1.74	13.1	−2.75
211	1.784	1.74	13.0	−2.58
219	1.784	1.68	13.8	−5.61
223	1.784	1.76	11.7	−1.57
226	1.784	1.77	11.8	−1.01

^*All samples were diluted 1:2 before DGA (Eichrom’s extraction chromatographic materials in which the extractant system is N,N,N’,N’-tetra-n-octyldiglycolamide resin) separation

Table 4.

Comparison of CDC ²⁴¹Am results with NIST target values for NRIP12 8 hours samples^*

Sample ID	Massic Activity (NIST Target Values)	Massic Activity (CDC Observed Results)	Relative Expanded Uncertainty (k=2)	Bias
	(Bq/sample)	(Bq/sample)	(%)	(%)
215	0.146	0.14	11.0	−1.93
218	0.292	0.29	10.9	−0.62
222	0.149	0.15	11.1	−1.47
230	0.297	0.29	11.1	−1.99
234**	0.372	0.42	11.2	12.7

^*Samples 218 and 230 were diluted 1:2 and sample 234 was diluted 1:4 before DGA (Eichrom’s extraction chromatographic materials in which the extractant system is N,N,N’,N’-tetra-n-octyldiglycolamide resin) separation

^**Analyses for other radionuclides also produced unusually high results for this sample.

Sample turnaround time (TAT)

While maintaining high quality results, sample TAT is one of the important considerations in a radiological emergency. For this method, ~ 2.5 hours are required to pretreat the urine samples for a batch of 20 patient urine specimens plus QC samples. An additional 3.5 hours are required for final analysis of 20 patient samples by SF-ICP-MS, including calibrators, blanks, and QC samples. Samples may be pretreated concurrently with final SF-ICP-MS analysis, resulting in a daily throughput of approximately 120 samples per day (24 hours) per instrument.

Conclusions

We introduced a method for rapidly determining ultra-low levels of ²⁴¹Am in urine samples using a Solid Phase Extraction purification procedure and a high-sensitivity sample introduction system (Aridus II™), coupled with SF-ICP-MS. This method provides for analysis of ²⁴¹Am at very low levels, with a LOD of 0.22 pg/L (well below the C/P CDG level) and allows rapid throughput of samples. It attained good agreement, with a bias of 2.1%−3.0%, for urine samples in an internal comparison with a CDC LSC method. It also produced recoveries from 99.7% to 101.7% in analysis of aqueous dilutions of ²⁴¹Am SRM from NIST.

This method’s efficient urine sample separation scheme effectively eliminates most molecular ion interferences. However, if urine samples contain more than 10 µg/L of U, more aggressive rinsing procedures are required to eliminate U from the elution solutions. The results obtained by this method for NIST/NRIP reference materials with high-U levels are in close agreement with the NIST target values, with biases ranging from −0.62% to −5.61%.

A major advantage of this method over alpha spectrometry and other methods is that only a small, 10 mL volume of each urine sample is needed to perform the analysis, making successful analysis more likely, especially for young children and infants.

This procedure is appropriate for rapid identification and quantification of ²⁴¹Am in urine for emergency response involving accidental or terrorism-related elevated exposures, or for evaluating chronic environmental or other non-occupational exposures.