Supplementary Material

Section 1: Magnetic Sector (SF) ICP-MS Operating Conditions and Quality Assurance Program Summary

Supplementary Table 1 presents a list of the elements quantified, along with the primary analytical isotope (m/z) and mass resolution applied for each element. Global ICP-MS operating conditions are outlined below Supplementary Table 1. Supplementary Table 2 summarizes the suite of QA/QC checks employed and the corresponding acceptance criteria.

Supplementary Table 1. SF-ICP-MS Elements, Isotopes and Resolutions.

Element / Isotope (m/z) / Resolution#
Al / 27 / MR
Ba / 138 / MR
Ca / 44 / MR
Cd / 111 / LR
Ce / 140 / LR
Co / 59 / MR
Cr / 52 / MR
Cs / 133 / LR
Cu / 63 / MR
Fe / 56 / MR
K / 39 / HR
Li / 7 / LR
Mg / 25 / MR
Mn / 55 / MR
Mo / 95 / MR
Na / 23 / MR
Ni / 60 / MR
P / 31 / MR
Pb / 208 / LR
Rb / 85 / MR
S / 32 / MR
Sb / 121 / MR
Sr / 88 / MR
Ti / 49 / MR
Tl / 203 / LR
U / 238 / LR
V / 51 / MR
Zn / 66 / MR

# LR= Low Resolution MR=Medium Resolution HR=High Resolution

RF power = 1150 W; Cool gas flow = 16 L/min; Auxiliary gas flow = 0.81 L/min; Nebulizer gas flow = 0.96 L/min; Sample uptake rate = 0.4 mL/min; Nickel sampler and skimmer cones.

Supplementary Table 2. Summary of Quality Assurance and Control Program

QA/QC Metric / Acceptance Criterion
ICP-MS Continuing Calibration Verification / 80-120% recovery
Reagent Blank Element Spikes / 80-120% recovery
Full Method Blank Element Spikes / 80-120% recovery
Sample Spikes / 80-120% recovery
CRM Recovery (reference blood) / 70-130% recovery
Element Spiked Wet Blood / 70-130% (Cr >60%) recovery
Element Spiked Spotted (dried blood) / 70-130% (Cr >60%) recovery
Sample Duplicate Precision / <20% RPD

Section 2: Outline of Protocols Evaluated for Extraction of Elements from NBS

The goal of the extraction method evaluation/optimization was to develop a protocol that maximized recovery of as many target elements as possible from spotted blood yet minimized the filter blank contribution. We also performed a rigorous microwave-assisted total digestion of the filter paper in a three-acid matrix to establish benchmark maximum levels of each target element. Blood reference materials were incorporated into the optimization trials in an effort to identify potential problematic elements and analytical steps and to ultimately ensure a more robust protocol. These materials included: certified reference blood materials (UTAK and NIST), run both neat and spotted/dried on filter paper and a multi element-spike (at several levels) into certified reference blood, run both neat and spotted/dried on filter paper.

The three general extraction protocols evaluated in detail are summarized below.

  1. A dilute acid + surfactant extraction solution was tested. Trials included:
  2. Nitric acid at 5% (v/v) (0.8 M) + 0.1% (v/v) Triton X-100 solution
  3. Nitric acid at 10% (v/v) (1.6M) + 0.1% (v/v) Triton X-100 solution
  4. Nitric acid at 20% (v/v) (3.2M) + 0.1% (v/v) Triton X-100 solution
  5. Nitric acid at 10% (v/v) (1.6M) + Hydrochloric acid at 4% (v/v) (0.5M) + 0.1% (v/v) Triton X-100 solution

Samples were sonicated for periods of both 15 and 30 minutes with 5.0 mL of extraction solution and then filtered. Each trial and sonication period was performed in triplicate. The 5% (v/v) nitric acid matrix was further tested in follow-up experiments with the addition of either: 0.5% (v/v) IPA, 0.005% APDC, or 0.5% (v/v) IPA + 0.005% APDC.

2. A base extraction with TMAH and alcohol + metal chelator + surfactant. Trials included 1% (v/v) TMAH + 2% (v/v) ETOH + 0.2% APDC + 0.01% (v/v) Triton X-100 solution and 1% (v/v) TMAH + 4% (v/v) ETOH + 0.2% APDC + 0.01% (v/v) Triton X-100 solution. Samples were sonicated for 15 minutes with 4.5 mL of extraction solution, diluted to 5.0 mL with nitric acid to pH 2, vortexed, and then filtered. This method is modeled after a protocol published by McShane et al. 2008 (citation below) for analysis of Cd, Hg, and Pb in whole blood.

3. A base digestion in 10% (v/v) TMAH, followed by extraction with alcohol and metal chelator. Samples were digested with 0.5 ml of a 10% (v/v) TMAH solution (with sonication) for 10 minutes, followed by extraction for 5 minutes (with sonication) with the following solutions:

  1. 1.0 mL of a solution of 20% (v/v) ETOH and 2% APDC.
  2. 0.5 mL of a solution of 20% (v/v) ETOH and 1% APDC
  3. 0.5 mL of a solution of 20% (v/v) ETOH and 0.01% APDC

Extracts were then diluted to 5.0 mL with either (a) acidification - nitric acid to pH 2, vortexed and filtered, or (b) without acidification – MQ, vortexed and filtered.

Section 3: Outline of Methodused for correcting NBS element measurements for the element contribution of filter paper.

The correction procedure estimates the filter paper weight in each NBS sample and uses element measurements from either three different lots of blank filter paper(W-031 (n=5), W-041 (n=5), and W-051 (n=15) or adjacent filter paper to correct each sample element measurement for filter paper element as outlined in the steps below.

  1. The ng element / mg of sample was calculated by dividing the mass of element determined by SF-ICP-MS (ng) by the gravimetrically determined mass of filter (mg) extracted (for all NBS samples and blank filter paper samples).
  1. The average and standard deviation in the ng element / mg of paper for the three lots of blank filter paper was calculated. The standard deviation was used to estimate 2 for correction method A.
  1. The ng element in paper was calculated by multiplying the ng element / mg of paper by the mg of the sample used for extraction to arrive at a sample-specific filter correction. Since the measured mass of the section included contributions from both the paper and blood, the contribution of blood to the mass was subtracted as described in step IV.
  1. From separate dedicated experiments, we determined that the mass of dried blood that results from the application of 80 microliters of whole blood (the volume that was determined to saturate the inscribed circle on the filter paper card) to filter paper is 20.16 milligrams. The mass in mg of filter paper alone under each blood spot section analyzed was calculated according to the formula: [(total mass of section analyzed (i.e. filter+blood)) – (blood mass per 80 microliters (20.16 mg)) x (mass of spot section analyzed / total mass of spot)].
  1. To estimate the ng element in the filter paper underlying each sample spot, the mass of filter paper used (value from IV) was multiplied by the ng/mg element in filter paper (from step I for Correction Method B or step II for Correction Method A). This formula was also applied to the ICP-MS uncertainty for filter paper element measurements in Correction method B and was used as the estimated uncertainty, 2, for correction method B.
  1. The total element in each sample (element mass (ng) measured by ICP-MS) was next corrected for the filter blank (ng element in filter from step V) by subtracting the two masses to yield the element mass (ng) per spot fraction analyzed (blank corrected).
  1. Finally, the value in VI was equated to the full blood spot by dividing by the ratio: (mass of spot section analyzed / total mass of spot) to yield the element mass (ng) per blood spot (blank corrected).

References

McShane, WJ, Pappas RS, Wilson-McElprang V, Paschal D. 2008. A rugged and transferable method for determining blood cadmium, mercury, and lead with inductively coupled plasma mass spectrometry. Spectrochimica Acta B – Atomic Spectroscopy 63:638-644.