Letter pubs.acs.org/ac
Micro Ion Extractor for Single Drop Whole Blood Analysis Yukihide Nakamura,† Shiori Maeda,† Hiroka Nishiyama,† Shin-Ichi Ohira,† Purnendu K. Dasgupta,‡ and Kei Toda*,† †
Department of Chemistry, Kumamoto University, 2-39-1 Kurokami, Kumamoto 860-8555, Japan Department of Chemistry and Biochemistry, University of Texas at Arlington, Arlington, Texas 76019-0065, United States
‡
S Supporting Information *
ABSTRACT: A micro ion extractor (MIE) was developed for trace anion determination by ion chromatography−mass spectrometry from a single drop (25 μL) of whole blood without pretreatment. Target analytes were iodide and thiocyanate, which play key roles in thyroid hormone production. Whole blood (25 μL) was pipetted from an earlobe or finger prick and placed in the 16 μL cavity of the device. A reproducible fraction of iodide and thiocyanate was transferred through a membrane to an acceptor solution layer by electromigration for 60 s. An isolator solution layer and a cation exchange membrane is provided between the acceptor and the anode to prevent gas formation or redox processes in the acceptor. The acceptor contents are transferred online to the ion chromatograph. Isolator solution composition and applied voltage were optimized. Recoveries from samples from 16 different volunteers of both sexes and differing ages were the same within ±10% relative standard deviation. Dietary effects on blood iodide and thiocyanate levels are reported. The very low sample requirement permitted multiple sample collections per day. The MIE device is expected to be useful for clinical studies that require several/many temporally spaced blood samples by keeping the invasive nature of blood collection as minimal as possible. race ionic analysis in biological fluids and other complex matrices is a continuing challenge, especially if one has limited sample. Examples include perchlorate in serum, plasma, and saliva,1 organic acids in saliva causing tooth decay,2 etc. In most cases, chromatography requires sample pretreatment; conventional procedures3 are simply inapplicable with sub-100 μL samples. Direct analysis of whole blood is essentially impossible. The ability to carry out macroscale chromatography with a single drop of whole blood would greatly simplify sample collection; a lancet prick of the fingertips or earlobes will suffice and be minimally invasive. Microelectrodialysis 4 and electromembrane extraction (EME)5−7 are recent entries for the matrix isolation of charged analytes utilizing an electric field; sample requirement can be as little as 50 μL. A supported liquid membrane is used in EME; whole blood8 and serum9 have also been processed. Extraction recoveries from whole blood were between 9% and 51% for various drugs with 5−10 min extraction time;10,11 the limitation is not the size/mobility of the analyte, and formate recovery was only 19%.12 EME is typically performed off-line in a batch mode and provides sufficient sample for analysis by CE. We previously demonstrated an online continuous flow ion extractor that successfully extracted cations and anions into individual ion chromatograph (IC) analysis channels with extraction efficiencies of >95% in seconds and demonstrated its use for milk and urine samples.13 Even weakly dissociated organic acids
T
© 2015 American Chemical Society
were successfully extracted from real samples.14 However, neither the μL-scale nor viscous samples like blood will be easy to adapt. Herein, we report a micro ion extraction (MIE) device and demonstrate its in-line use for the extraction of the trace inorganic anions iodide and thiocyanate from a single 25 μL drop of whole blood coupled to in-line analysis by ion chromatography−electrospray ionization mass spectrometry.
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EXPERIMENTAL SECTION Further experimental details are given in the Supporting Information. Device and Analytical Procedure. From the bottom, the MIE device is comprised of a clear polyvinyl chloride (cPVC) plate (t = 10 mm), anode plate (stainless steel, t = 0.5 mm, sputter-coated with ∼50 nm of Ti and ∼200 nm of Pt atop), isolator channel gasket (silicone, t = 0.5 mm), cation exchange membrane (Nafion, t = 0.05 mm, NRE-212, Sigma-Aldrich, www.sial.com), extraction channel gasket (silicone, t = 0.5 mm), dialysis membrane (Spectra/Por Dialysis Membrane; MWCO: 6000−8000; www.spectrumlabs.com), sample holder plate (cPVC, t = 2 mm), and top plate (PEEK, t = 5 mm); the plate stack (30 × 30 mm area) was held by four stainless steel Received: May 3, 2015 Accepted: June 18, 2015 Published: June 18, 2015 6483
DOI: 10.1021/acs.analchem.5b01681 Anal. Chem. 2015, 87, 6483−6486
Letter
Analytical Chemistry
interelectrode distance of 3.2 mm and as a first approximation, the field is assumed to be uniform, on the basis of the known equivalent conductance and hence ionic mobilities,21 the migration velocities of I− and SCN− will be 0.25 and 0.21 mm s−1. In whole blood or 50% glycerol (these have comparable viscosities,22 ∼6.0× more viscous than water), these velocities will be a sixth as much, but 1 min should therefore still be enough to move both these ions across the well depth of ∼2 mm. With standards containing 50% v/v glycerol, no significant difference in transfer dependence on applied voltage was observed compared to standards in water (Figure 2). However,
screws. The gaskets contained the liquid channel (4 × 9 mm, w × l, Figure 1), with a cross-flow arrangement. The holder plate contained a well (ϕ3.2 mm × 2 mm, 16 μL) for the sample.
Figure 1. Illustration of MIE principle (left) and schematic structure of the device (right). a, cathode carbon electrode; b, cathode support plate (PEEK); c, sample well plate (clear PVC); d, dialysis membrane; e, acceptor channel spacer (silicone sheet); f, Nafion membrane; g, isolator channel spacer (silicone sheet); h, anode plate (Pt plated stainless steel); and i, bottom plate (clear PVC). Figure 2. MIE extraction efficiencies for 1 mg/L iodide and 1 mg/L thiocyanate in water and 50% glycerin solutions.
Acceptor solution (pure water) and isolator solution (25 mM KOH) were continuously introduced to the respective channels at flow rates of 0.1 and 0.7 mL min−1. Fresh whole blood sample (25 μL) was pipetted into the sample well, and a glassy carbon cathode (6 mm o.d.) was lowered to contact the sample. After carrier flow was stopped, voltage (typ. 12 V) was applied for 60 s. The acceptor flow was restarted without turning off the voltage until the acceptor plug was fully introduced in the sample loop (300 μL) of the ion chromatograph. After the electrodialytic extraction, the blood analyzed was washed out with water. The dialytic membrane and the cathode were rinsed with water three times, and the acceptor and isolator solutions were continuously flowed to be ready for the next analysis (memory effect was less than 1%). The membrane could be used repeatedly when the membrane was cleaned appropriately, and the device was stored in a 100 mL beaker of water to prevent drying of the membrane after daily use.
75% extraction of I− and SCN− from spiked saliva required 40 V over 1 min with water as the isolator. There are two reasons for this: (a) unlike standards, there are many other ions in saliva and transport will be competitive with these, with the overall amount transported being current limited; (b) the sample/ receiver/isolator solutions represent a three component series resistance. With the last two being pure water, only a small fraction of the field will appear across a conductive sample, limiting transport. Similar situations are encountered in EME.23 Blood is more saline (0.095−0.105 M chloride24) and viscous than saliva; the best case extraction efficiency was 30 μg/L. Seaweeds are known to concentrate iodide from seawater and contain high concentrations of iodide.25 A clear effect of smoking was noted on [SCN−]blood, as is well-known.26 Nonsmokers and smokers averaged 1.2 ± 0.6 and 4.5 ± 1.8 mg/L, respectively. Among nonsmokers, the two lowest [SCN−]blood values were from subjects who reported eating no vegetables; many vegetables are known to contain significant levels of thiocyanate.27,28 Diurnal Variation of Blood Iodide and Thiocyanate and Diet. As the procedure is minimally invasive, diurnal variation of the target anions could be followed without undue burden. The results (Figure 4) for one of authors, a nonsmoker, indicate that [SCN−]blood showed the same diurnal pattern over both days, increasing in the morning and decreasing in the afternoon with small jumps after dinner, spanning a range of 0.73−1.74 mg/L. In contrast, the span for [I−]blood was 4−140 μg/L. The subject refrained from seafood/seaweed consumption prior to the first sampling, and the initial [I−]blood was ∼4
Figure 3. (a) Effect of applied voltage and isolator composition (ultrapure water (UPW) to 100 mM KOH) on the recoveries of 1 mg/L I− spiked in human whole blood; extraction time, 1 min. (b) Effect of extraction time and isolator solution composition for 50 nM (6.4 μg/L) I− added to equine whole blood, Vapp = 12 V; the temporal current profile and extraction efficiencies are indicated.
current is far higher with the KOH isolator as K+ migrates into the acceptor and the overall resistance drops. At least with the KOH isolator, the progression of current, however, does not equate to continued analyte transfer: the peak area exhibits little increase beyond ∼0.7 min, suggesting that little is to be gained beyond a 1 min extraction time. Extraction continues over a longer period with a water isolator, but overall efficiency remains lower. Standard Addition and Recovery from Whole Blood. Chromatographic peak areas increased linearly with the added anion concentrations (See Figure S2 in the Supporting Information). The good linearity (see eq 1 and 2 below) and excellent reproducibility illustrates method robustness. AI − = 4.24 ± 0.03 × 102 [I−, μg/L] + 4.40 ± 0.14 × 103,
r 2 = 0.9992
(1)
ASCN − = 6.25 ± 0.11 × 107 [SCN−, μg /L] + 1.71 ± 0.15 × 105,
r 2 = 0.9978
(2)
Figure 4. Variation of blood iodide and thiocyanate in one subject over 2 days. 6485
DOI: 10.1021/acs.analchem.5b01681 Anal. Chem. 2015, 87, 6483−6486
Letter
Analytical Chemistry μg/L. Breakfast on both days consisted of steamed white rice with konbu (kelp boiled in soy), miso soup with seaweed (wakame), and tofu; [I−]blood increased to ∼10.3 μg/L by noon on day 1. Day 1 lunch consisted of white rice, wakame soup, chicken, soybean-mixed hijiki salad, and coleslaw. Hijiki (Sargassum f usiforme), a brown seaweed growing in shallow seawater is a common constituent of Japanese salad in sunlightdried form that is soaked before consumption. The subject had 110 g of soaked hijiki for lunch on both days. [I−]blood increased to 16.8, 45.8, and 59.5 μg/L in hourly samples before gradually decreasing. The decrease continued after dinner (rice, chicken, and potato salad) before retiring for the night. On day 2, [I−]blood showed a similar temporal profile as the first day, except starting from a much higher morning baseline. The maximum was at 3 pm as on day 1 but at 139 μg/L, 2.3× that of the first day. Intake thus has both an immediate and a longer term effect. Only such simple minimally invasive measurements can conveniently provide this type of information: to our knowledge, the appearance time scale of iodide in blood from consumption in real foods and its disappearance half-life as well as reservoir behavior in a human has never previously been documented. The MIE device represents a simple interface for trace ionic analysis applicable to very small sample volumes. Although dried blood spot (DBS) analysis was introduced some 50 years ago,29 it is increasingly popular today due to its ease of use, high throughput, and low cost.30 The present approach will allow the introduction of a DBS punch into the sample well followed by the introduction of an extractant spiked with an isotopically labeled marker if desired. Such approaches have the potential to be fully automated for rapid and convenient analysis. In principle, as in the original macroscale ion transfer device,13 the positively and negatively charged species can be moved to individual chromatographic systems; because the substrate itself may act as a retentive filter, one can envision that the remaining neutrals can be analyzed by a third chromatographic system.
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in text. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b01681.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +81-96-342-3389. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by Development of System and Technology for Advanced Measurement and Analysis program from JST and KAKENHI (Basic Research B, no. 25288068) from JSPS. The participation of P.K.D. was made possible by the Hamish Small Endowment for Ion Analysis by Thermo Fisher Corp at the University of Texas at Arlington
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DOI: 10.1021/acs.analchem.5b01681 Anal. Chem. 2015, 87, 6483−6486
