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A Method for the Determination of Iodide in Dried Blood Spots from Newborns by High Performance Liquid Chromatography Tandem Mass Spectrometry Un-Jung Kim, and Kurunthachalam Kannan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04827 • Publication Date (Web): 07 Feb 2018 Downloaded from http://pubs.acs.org on February 8, 2018

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Analytical Chemistry

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A Method for the Determination of Iodide in Dried

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Blood Spots from Newborns by High Performance

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Liquid Chromatography Tandem Mass Spectrometry

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Un-Jung Kima and Kurunthachalam Kannana,b*

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a

Wadsworth Center, New York State Department of Health, and Department of Environmental

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Health Sciences, School of Public Health, State University of New York at Albany, Empire State

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Plaza, P.O. Box 509, Albany, New York 12201-0509, United States

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b

Biochemistry Department, Faculty of Science and Experimental Biochemistry Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia

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*Corresponding author: [email protected]

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For submission to: Analytical Chemistry

1 Environment ACS Paragon Plus

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT

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Dried blood spots (DBS), collected for newborn screening programs in the United States, have

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been used to screen for congenital metabolic diseases in newborns for over 50 years. DBS

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provide an easy and inexpensive way to collect and store peripheral blood specimens, and

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present an excellent resource for studies on the assessment of chemical exposures in newborns.

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In this study, a selective and sensitive method was developed for the analysis of iodide in DBS

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by high performance liquid chromatography electrospray tandem mass spectrometry. Accuracy,

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inter- and intra-day precision, matrix effects, and detection limits of the method were

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determined. Further validation of the method was accomplished by concurrent analysis of whole

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blood and fortified blood spotted on a Whatman 903 filter card. A significant positive correlation

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was found between measured concentrations of iodide in venous whole blood and the same

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blood spotted as DBS. The method limit of detection was 0.15 ng/mL iodide. The method was

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further validated by the analysis of a whole blood sample certified for iodide levels (proficiency

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testing sample) by spotting on a filter card. Twenty DBS samples collected from newborns in

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New York State were analyzed to demonstrate the applicability of the method. The measured

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concentrations of iodide in whole blood of newborns from New York State ranged between

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100 µg/L, and no more than 20% of the population

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should have values

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127 [127I-]) and 18O-labeled-perchlorate (107 [Cl18O4-] > 89 [Cl18O3-]; because the molecular ion

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m/z 127 does not fragment further, the precursor ion selected for Q1 was also selected as the



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product ion for Q3). Nitrogen was used both as curtain and collision gas. The optimized source

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parameters were as follows: curtain gas = 20 psi, collision gas = 12 psi, nebulizing gas = 52.5

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psi, heater gas = 55 psi, ionization voltage = - 1500 V, and source heater temperature = 500 ˚C.

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Detailed MRM parameters are shown in the supporting information (Table S1).

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Quality Assurance/Quality Control (QA/QC). Ten-point matrix-matched calibration

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standards (0.5–200 ng/mL for I- and 50 ng/mL for Cl18O4-) were prepared, and the instrumental

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responses were compared with a solvent-based calibration standard to correct for matrix effects.

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As a check for background levels of contamination, equipment blank (EB), laboratory reagent

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blank (LRB), procedural blank (PB) and two types of matrix blanks (MB) were analyzed. The

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accuracy and precision of the method were examined by the analysis of iodide-fortified DBS

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(i.e., in-house DBS) samples. The EB consisted of injection of reagent water by LC/MS/MS (i.e.

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without any extraction or purification step). The LRB was the reagent water that was injected

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between samples to verify that there was no carry-over of iodide between injections. The PB was

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reagent water that was processed similarly to samples by passing through the entire analytical

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procedure. The MBs were unspotted areas from Whatman 903 filter paper and whole blood

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samples (used in fortification), which were analyzed similarly to those of samples by passing

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them through the entire analytical procedure; the MB-1 was (unspiked) whole blood used in the

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preparation of in-house DBS and the MB-2 was the unspotted area of DBS filter card. This

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analysis was performed to determine the background levels of iodide in DBS filter card. Matrix

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spike (MS) samples were prepared at three different concentrations of iodide: low (MS-L; 5

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ng/mL), medium (MS-M; 40 ng/mL) and high (MS-H; 100 ng/mL). These concentrations

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covered the range of expected levels found in human blood.10 Two whole blood samples certified


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by the Trace Element PT Program of Wadsworth Center for total iodine were spiked onto DBS

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cards and analyzed as quality control samples (QC-A and QC-B).

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RESULTS AND DISCUSSION

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Chromatographic Separation. Four different HPLC columns, namely the Dionex AS-21

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anion exchange, the Acclaim Trinity P1 (Thermo Scientific), the Agilent Zorbax Sb-Aq, and the

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Luna HILIC column (Phenomenex) (see supporting information) were tested for their ability to

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resolve iodide peak from other matrix interferences with various combinations of mobile phases

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and gradient programs.30-33 Although the intensity of the iodide signal from solvent and blood

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extracts was higher with the HILIC column than the anion exchange column, the sensitivity and

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selectivity of iodide in both whole blood and DBS extract were higher with the anion exchange

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column. Figure 1 shows chromatograms of iodide (100 ng/mL) injected onto the anion exchange

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column with the optimized mobile phase gradient and MS/MS parameters in reagent water,

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blood serum, whole blood, and whole blood spotted in-house DBS specimen. In reagent water

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(Fig. 1-A), iodide eluted faster (8.5 min for iodide, 11.5 min for Cl18O4-) than that from other

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sample matrixes (11 – 13 min for iodide and 15.5 – 17.5 min for Cl18O4-). The retention time of

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iodide from the in-house DBS specimen was reproducible within a 10% standard deviation.

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Fig. 1. Typical HPLC-ESI-MS/MS MRM chromatograms of iodide (blue, 100 ng/mL, 2 µL

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injection volume) and

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blood serum (B), human whole blood (C), DBS filter card (D), and whole blood spotted in-house

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DBS specimen (E).

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O-labeled perchlorate (red, 50 ng/mL) in reagent water (A), human

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Limits of Detection and Quantification. The limit of detection (LOD) was defined as the

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statistically calculated minimum concentration of iodide at the 99% confidence interval. The

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LOD was calculated by analyzing a matrix spike sample (DBS) fortified at 1 ng iodide/mL (MS-

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L) seven times, followed by the calculation of mean recovery and the standard deviation

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multiplied by the Student’s t-value (3.143). The calculated LOD for iodide in DBS sample

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extracts was 0.15 ng/mL. The lower limit of quantification (LLOQ) was three times the LOD

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value, which matched the lowest concentration of the calibration curve at 0.5 ng/mL. A


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chromatogram showing the signal of iodide at 0.5 ng/mL is presented in the supporting

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information (Fig. S1). The signal-to-noise (S/N) ratio of iodide at 0.5 ng/mL was between 8 and

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10.

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Background Contamination. A major issue associated with the use of DBS for trace level

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quantitative analysis of environmental chemicals is background levels of contamination.21,22,29,34-

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contamination by iodide in laboratory supplies and apparatus used in extraction and purification

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of DBS samples. Trace levels of iodide (below 0.997. In comparison to iodide

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standard prepared in reagent water, the calibration curves prepared with matrix-matched DBS

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cards had a slope 2.5 times lower, suggesting the existence of ion suppression from DBS-based

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sample matrix (Fig. 3). Thus, the quantification of iodide in DBS was based on the slope of the

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matrix-matched calibration curve, which would eliminate the bias introduced by ion suppression

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of the sample matrix.

I are radioactive, the isotopic dilution method of quantification is not practical.

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O-labeled perchlorate was also spiked into samples to

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The reagent water injected between calibration standards showed no carryover of iodide

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between samples. A mid-point calibration standard (40 ng/mL) was injected after every ten h to

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monitor for drift in instrumental sensitivity, and the observed coefficient of variation (CV) was

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less than 15% for the standard injected throughout the analysis.

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Fig. 3. Slopes of responses of iodide from matrix-matched calibration curves (solid line)

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prepared using dried blood spots and those prepared in reagent water (dotted line).

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perchlorate was used as the internal standard.

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O-labeled

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Recovery (Accuracy).

Percent recoveries of iodide spiked onto DBS filter card were

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determined as the measured concentrations divided by the nominal spiked concentrations at 5

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ng/mL (MS-L; n=5), 40 ng/mL (MS-M; n=5) and 100 ng/mL (MS-H; n=5). These concentrations

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cover the range of those found in human blood.10 The mean (RSD) recovery of iodide spiked into

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sample matrix was 97.7 (6.2) % (Table 1).

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The accuracy of the analytical method was further validated by spiking whole blood samples

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certified for iodine levels (two samples certified at different levels) on DBS filter cards and

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passing them through the entire procedure (QC-A and QC-B; certified values are 53.8 and 40.5

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ng/mL, respectively) (Table 1). Repeated analysis (n=5 each) of two certified reference samples

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spiked on the DBS cards showed a recovery of 99-102% with an RSD of 8.74%.

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Table 1. Recoveries of iodide from dried blood spots (DBS) spiked at different concentrations

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(in percent, mean ± SD) and those of certified reference whole blood samples spiked on DBS

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filter card. Overall MS-L

MS-M

MS-H

QC-A

QC-B

RSD mean (%)

(n=5)

(n=5)

(n=5)

(n=5)

(n=5)

(%) (n=25)

Nominal concentration

5

40

100

53.8

40.5

95.3±5.96

96.4±5.35

95.6±4.74

102±8.74

98.7±6.10

(ng/mL) Iodide recovery 97.7

6.18

(%, mean±SD)

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(Note. MS-L: matrix spike (low), MS-M: matrix spike (medium), MS-H: matrix spike (high), QC: quality control samples with certified values for iodide)

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Precision.

Intra- and inter-day variations in responses of iodide were assessed by the

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calculation of precision as percent relative standard deviation [% RSD] and percent relative error

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[% RE]) from replicate analysis at three different levels of matrix spiked samples (MS-L, MS-M,

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MS-H and QC-A) (Table 2). RSD (%) was calculated as the standard deviation of observed

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iodide concentration from each matrix spike divided by the mean measured concentration,

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whereas RE (9%) was calculated as the absolute difference between measured and nominal

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concentration of iodide (i.e., |measured conc. – nominal conc.|) divided by the nominal

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concentration of iodide in each matrix spike. Intra-day variations were calculated on a single day,

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and inter-day variations were calculated for five separate days. Overall, the inter-day variations

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were below 12% RSD and below 5% RE.


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Previous studies have shown that hematocrit levels of whole blood can influence the diffusion

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of blood on the DBS filter card, which can contribute to intra-disc variability if only a portion of

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the DBS filter card is used in the analysis.15,37,38 Although our original goal was to use the entire

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16-mm disc for the analysis (which would alleviate filter paper effect or intra-disc variability

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introduced by punching a portion of the disc), because validation samples were available to

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calculate intra-disc variability, we analyzed (n=5 each) 4-mm punches of spiked DBS specimen

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(QC-A and QC-B) by taking samples from the central and peripheral area of the blood spot, as

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well as the full circle (Table S2). No significant intra-disc variability in iodide concentration was

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observed with RSD values

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