All nitric acid (HNO₃) solutions were prepared from double-distilled 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 (following CDC IRB protocol 3994) and acidified to 1% v/v HNO₃ to be used as the base matrix. All radioactive source solutions were traceable to the National Institute for Standards and Technology (NIST) (Gaithersburg, MD, USA). We prepared urine pools for LOD determinations, as well as other urine solutions for accuracy testing, by spiking (volumetric determinations) base urine with dilutions of ²²⁶Ra isotope standard reference materials (SRM) from NIST 4967A. We used NIST traceable ¹⁹³Ir as an internal standard (High-Purity Standards, Charleston, SC). Serial dilutions of Pt, W, Pb, Ba, Sr, Bi, Os, and Ir single-element stock standards (Inorganic Ventures, Christiansburg, VA) were spiked into the base urine to verify high elimination factors for elements forming polyatomic interference by using the simple dilute and shoot sample procedure and analyzing them on ICP-QQQ-MS. We prepared different sets of external, aqueous-based stock calibrators from dilutions of ²²⁶Ra isotope SRM from the NIST and certified reference material (CRM) from Eckert & Ziegler Analytics, Inc. (Atlanta, GA) and Eckert & Ziegler Isotope Products (Valencia, CA). They were prepared by spiking 5% v/v HNO₃ with dilutions of ²²⁶Ra isotope SRM and CRM (7 calibration standards (S0–S6) with concentrations of 0.000, 0.006, 0.020, 0.060, 0.200, 0.600 and 2.00 ng/L). The sample diluent consisted of 2% v/v HNO₃ and 100 ng/L of ¹⁹³Ir, while the sample introduction system rinse solution consisted of 5% v/v HNO₃.

All samples, calibrators, and blanks were prepared at a 10x dilution into 15 mL polypropylene conical tubes. Matrix matched working calibrators were prepared by mixing an aliquot of each stock calibrator with base urine and sample diluent (0.5 mL of stock calibrator, 0.5 mL of base urine, and 4.0 mL of sample diluent). Urine samples (including quality control samples) were prepared in the same manner (0.5 mL of urine sample, 0.5 mL of 5% v/v HNO₃, and 4.0 mL of sample diluent). The aqueous blank (Reagent blank) was prepared similarly, replacing the urine volume with ≥ 18.2 MΩ·cm water (0.5 mL of 5% v/v HNO₃, 0.5 mL of water, and 4.0 mL of sample diluent), and was used as the blank for all patient samples, urine quality controls, and reference materials.

When detected results were greater than the calibration range verified by calibrators, we performed up to a 100x additional sample dilution with 5% v/v HNO₃ to dilute to bring the concentration within the calibration range. All dilutions have been validated.

Three levels of urine-based bench QC materials were prepared by spiking base urine with ²²⁶Ra at low, medium, and high concentrations (0.050, 0.450 and 1.50 ng/L), which are within the calibration range, by using NIST SRM 4967A in base urine. They were analyzed at the beginning and again at the end of each run. Modified Westgard rules as detailed in the Division of Laboratory Sciences Policies and Procedures Manual, NCEH, CDC were used to establish/determine whether runs were in control. Method precision, accuracy, and recovery were assessed using above prepared QC materials as well as urine samples spiked with NIST SRM 4967A in base urine.

For this rapid method, we used the Agilent 8800 Triple Quadrupole ICP-QQQ-MS (Agilent Technologies, Wilmington, DE) instrument to determine ²²⁶Ra concentrations. This device was the world’s first ICP Triple Quad meaning that it is a tandem MS that uses two hyperbolic profile quadrupoles separated by the octupole reaction system (ORS cell). Allowing operation in MS/MS, the distinctive tandem MS construction allows superlative control of interferences in reaction mode, providing extremely low background, greater accuracy and more consistent results [35–37]. A Micro Mist nebulizer, a quartz spray chamber double pass, quartz injector, nickel sampler cone, and nickel skimmer cone were used for all experiments (instrument and method parameters are listed in Table 1).

An ASX-520 autosampler (Elemental Scientific Inc., Omaha, NE) was used to access diluted urine samples for analysis. We used > 99.999% argon for the plasma and nebulizer gas (Specialty Gases Southeast, Atlanta, GA). All experimental on ICP-QQQ-MS instrument parameters are optimized to determine ²²⁶Ra concentrations by maximizing Tl ion intensity and minimizing the oxide formation rate using a 1 μg/L tuning solution (Agilent Technologies, Wilmington, DE). The method parameters were optimized with minimum relative standard deviations for ²²⁶Ra in a trade-off with minimal analysis time. The instrument sensitivity is ~0.26 cps per pg/L for ²²⁶Ra and the sample flow rate is ~0.8 mL/min. Table 1a and Table 1b summarize the optimized operating conditions and method parameters.

The determination of ²²⁶Ra by ICP-MS can be affected by spectral interferences caused by polyatomic species. The method we report here used a unique MS/MS function of Agilent 8800 to successfully eliminate these interferences. In MS/MS mode, Q1 of the Agilent 8800 operates as a mass filter, allowing only the target analyte mass to enter the cell and rejecting all other masses. Q2 is the second high-frequency hyperbolic quadrupole that filters the ions emerging from the cell exit, passing only the target analyte/product ions to the detector [35–37]. Interference removal experiments were performed using solutions containing the elements Pt, W, Pb, Ba and Sr at five concentration levels: 0.5 μg/L, 1.5 μg/L, 5.0 μg/L, 15 μg/L and 650 μg/L. These potential elements (Pt, W, Pb, Ba and Sr) along with argon and other elements from sample preparation (e.g., oxygen and hydrogen) can form polyatomic interferences in mass-charge 226. These spiked urine sample concentrations were well above the National Health and Nutrition Examination Survey (NHANES) 95^th percentile of urine Pt, W, Pb, Ba and Sr concentrations [38]. Although NHANES survey data is not available for Bi, Os and Ir, analysis of what were otherwise determined [39] to be high urine concentrations at 0.5 μg/L of Bi, 0.5 μg/L of Os and 1 μg/L of Ir did not implied in positive recoveries at mass-charge 226. On the other hand, when single-quad mode was chosen on Agilent 8800, though we obtained increased ²²⁶Ra sensitivity, high background on mass of 226 was observed for urine blank samples.

The LOD determination for ²²⁶Ra in urine specimens with this method is validated on an approach of considering both Type I and Type II error recommended by Clinical Laboratory Standards Institute (CLSI) [40], which is required by our Division of Laboratory Sciences in CDC. Based on ≥20 analytical runs, 0.5 mL of urine matrix-matched samples from four different low concentration pools that were spiked at concentrations close to the LOD - roughly the measured blank concentration plus 3 times the standard deviation (SD) of the measured blank concentration.

The LODs were calculated according to the following formula: ConcLOD=[meanb+1.645(Sb+int)]/[1–1.645(slope)] Where

mean_b = blank average

S_b = SD of blank average

int = intercept of the equation of SD versus concentration for LOD samples analyzed, and

slope = slope of the equation of SD versus concentration.

The LOD of this method was determined to be 0.007 ng/L (0.26 Bq/L) for ²²⁶Ra (Figure 1, Table 2, Spike 1 to Spike 4 were used for LOD determination). This LOD is well below 1/3 of the C/P CDG level (0.046 ng/L for ²²⁶Ra), and therefore acceptable for an emergency radiobioassay method to determine the concentration of ²²⁶Ra in urine collected at 5 days post-exposure.

We use linear calibration model for this method, which exhibits good linear regression signal response between concentrations of 0.006 ng/L and 2.00 ng/L (S1–S6) of ²²⁶Ra, with a linear fit coefficient >0.999 by using 1/x weighting for quantitation. If an assessed urine ²²⁶Ra concentration is above the concentration of the highest calibrator, the urine sample is diluted with 5% v/v HNO₃ to bring the concentration within the validated calibration range. Accuracy tests of diluted reference materials spiked in base urine show that ²²⁶Ra can be analyzed at up to a 100x extra dilution without significant effect (< ± 10% error) on the target values (Table 3).

We evaluated the analyte of ²²⁶Ra carryover by alternating the analysis of spiked urine samples (3 times the highest calibrator’s concentration) containing approximately 6.0 ng/L of analyte ²²⁶Ra and urine blanks over the period of ~ 2 hours (analysis time of 4.5 minutes per sample with 30 samples analyzed). The data showed no observed intensities or resulting concentration spikes of ²²⁶Ra in the urine blanks following high spiked urine samples.

To ensure the method’s run-to-run reproducibility, we evaluated in-house CDC QC samples spiked at desired concentrations as described above. Table 4 shows the calculated long-term precision observed among daily QCs analyzed at the beginning and at the end of each analytical run. The precision for the low, medium, and high QCs was within ±14% over 30 analytical runs, spanning a period of ~ 2 months, by using different sets of calibrators prepared from ²²⁶Ra stock solutions from different vendors. Compared to the spiking target values, the bias for these samples was in a range of −0.08% to 3.9%. Evaluation of the method’s accuracy was achieved by analyzing 18 urine samples prepared by using NIST SRM 4967A and using NIST traceable CRM from Eckert and Ziegler Analytics (EZA) spiked into base urine. These spiked samples were analyzed in 10 to 20 analytical runs spanning roughly one month by using different sets of calibrators prepared from ²²⁶Ra stock solutions from different vendors. ²²⁶Ra results for these samples analyzed on ICP-QQQ-MS are listed in Table 2. Compared to the spiking target values, results showed a bias of −4.7% to 6.2% for ²²⁶Ra. Additionally, 24 urine samples were prepared /spiked at 6 concentration levels using NIST traceable CRM from Eckert and Ziegler Isotope Products (EZIP) in order to evaluate the accuracy using spike recovery. Urine samples spiked with 0.060, 0.200, 0.600, 0.400, 1.00 and 1.60 ng/L had recoveries ranging from 93.3 to 102% for ²²⁶Ra in two analytical runs (Table 5) on two different days. The mean recovery was 99.7% with a SD of 3.3%.

To ascertain that the determined concentration of ²²⁶Ra in a urine sample was not altered by other factors during analysis, stability tests were conducted with QC materials to evaluate for conditions it would experience when the method is employed. These include sample collection and handling, short-term room temperature storage, long-term freezer storage at a specified temperature, and three freeze/thaw cycles. The results are shown in Table 6. The mean values from the replicates in stability testing results are within ± 11% of the values of initial measurement for each QC material.

In addition to high quality results, sample throughput is one of the most essential considerations in a radiological emergency response. For this method, the preparation of urine samples by simple dilution for a batch of 20 patient urine specimens, calibrators, blanks and QC samples, took ~30 minutes. The analysis run time for the above 20 samples was 2.5 hours (the average analysis time for each sample is ~4.5 minutes including rinsing). Samples may be prepared simultaneously with final ICP-QQQ-MS analysis, leading to a daily throughput of about 180 samples per day (24 hours) per instrument.