Mercury speciation by differential photochemical

Spectrochimica Acta Part B 137 (2017) 1–7

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Mercury speciation by differential photochemical vapor generation at UV-B vs. UV-C wavelength☆ Guoying Chen a,⁎, Bunhong Lai a, Ni Mei b, Jixin Liu c, Xuefei Mao c a b c

U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, 600 E. Mermaid Lane, Wyndmoor, PA 19038, USA Shanghai Institute for Food and Drug Control, 1500 Zhangheng Road, Shanghai 201203, China Institute of Quality Standard and Testing Technology for Agro-Products, Chinese Academy of Agricultural Sciences, Beijing 100081, China

a r t i c l e

i n f o

Article history: Received 24 January 2017 Received in revised form 28 July 2017 Accepted 11 September 2017 Available online 12 September 2017 Keywords: Mercury Speciation UV-C UV-B Photochemical vapor generation Fish oil

a b s t r a c t Photochemical vapor generation (PVG) is an effective sample introduction scheme for volatile mercury (Hg). Speciation of Hg++ and MeHg+ was fulfilled for the first time by differential PVG under UV-B vs. UV-C wavelength and applied to fish oil supplements. After liquid-liquid extraction, the aqueous extract was mixed with 0.4% anthranilic acid (AA)-20% formic acid (FA) in a quartz coil, and exposed sequentially to 311 nm or 254 nm UV light. The resulting Hg0 vapor was detected by atomic fluorescence spectrometry (AFS). At each wavelength, the AFS intensity was a linear function of Hg++ and MeHg+ concentrations, which were solvable from a set of two equations. This method achieved ultrahigh sensitivity with 0.50 and 0.63 ng mL−1 limits of detection for Hg++ and MeHg+, respectively, and 73% recovery for MeHg+ at 10 ng mL−1. Validation was performed by ICP-MS on total Hg. Obviation of chemical or chromatographic separation rendered this method rapid, green, and cost-effective. © 2017 Published by Elsevier B.V.

1. Introduction Mercury (Hg) is a notorious toxic element that cycles between the atmosphere and aquatic systems. Inorganic mercury (iHg) in water and sediment is converted by microbial actions to methylmercury (MeHg+), which readily bioaccumulates along the food chain. Seafood consumption is the main pathway of human exposure to mercury [1]. Once inside human body, MeHg+ manifests toxicity by multiple means affecting cardiovascular, renal, immune, and neural systems, collectively known as Minamata disease [2]. The Food and Agriculture Organization/World Health Organization (FAO/WHO) Joint Expert Committee on Food Additives (JECFA) has established provisional tolerable weekly intakes (PTWI) for the general population at 1.6 μg MeHg+/kgbw and 4 μg iHg/kgbw, but warned a greater risk for pregnant and breast-feeding women. The FAO/WHO Codex Alimentarius Commission has adopted recommendations on guideline levels of 1 mg kg−1 MeHg+ for predatory fish, and 0.5 mg kg−1 for non-predatory fish. On the other hand, fish is an important source of beneficial Ω-3 unsaturated fatty acids [3]. Mackerel,

☆ Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture (USDA). USDA is an equal opportunity employer. The authors declare no financial interest. ⁎ Corresponding author. E-mail address: [email protected] (G. Chen).

http://dx.doi.org/10.1016/j.sab.2017.09.007 0584-8547/© 2017 Published by Elsevier B.V.

herring, tuna, salmon, cod, etc. are commonly used to produce fish oil supplements; thus widespread concerns are raised on possible presence of Hg [3,4]. To protect public health, presence of Hg in foods and supplements must be closely monitored. Total mercury (tHg) can be determined by thermal decomposition–atomic absorption spectrometry (AAS) at low-cost [3,5]. MeHg+ is much more toxic than Hg++ and usually dominates in foods, so it is the focus of most regulations. Conventional speciation methods include gas chromatography (GC) with electron capture [6], mass spectrometric (MS) [7], or optical emission spectrometric (OES) [8] detection; and high performance liquid chromatography (HPLC)–inductively coupled plasma (ICP)–MS [9]. Based on the fact that seafood contains only detectable MeHg+ and Hg++ [10,11], non-chromatographic methods offer effective alternatives at low cost [12]. Conversion of dissolved Hg species to elementary vapor (Hg0) or other volatile species is an effective sample introduction scheme [13] that allows gaseous analytes to thoroughly separate from interfering matrix components in sample solutions. As a result, sensitivity and specificity are greatly enhanced leading to extensive implementation in AAS [14], atomic fluorescence spectrometry (AFS) [15], microwaveinduced plasma (MIP)–atomic emission spectrometry (AES) [16], ICP– AES [17], etc. Vapor generation can be carried out by stepwise reduction, or first oxidization of MeHg+ to Hg++, which was later reduced to Hg0 vapor [18]. In the presence of certain low-molecular-weight organic compound (LMWOC), such as alkyl carboxylic acids [19,20], reduction of Hg++ and

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G. Chen et al. / Spectrochimica Acta Part B 137 (2017) 1–7

MeHg+ can occur photochemically producing Hg0 vapor. Photochemical vapor generation (PVG) can be fulfilled simply and quickly with green chemistry, and so far has been applied to water [19], fish [20, 21], and hair matrices [22]. To rely solely upon PVG to fulfil speciation of two Hg species without prior chromatographic or chemical separation, two sets of PVG conditions are needed using different photoreductants or light sources of different wavelengths. UV-C radiation, such as the 254 nm line from a low-pressure Hg discharge lamp, has long proven effective for reduction of both Hg++ and MeHg+ [20]. Natural room light [19] and 365 nm light emitting diode (LED) [23] were used as complementary sources for PVG of Hg++. Nevertheless, further studies failed to observe PVG signal from Hg++ or MeHg+ under visible (400–700 nm) or UV-A (320–400 nm) wavelength [24, 25]. In this work, more energetic UV-B (280–320 nm) region was explored for the first time to complement a 254 nm low-pressure Hg lamp. Differential behaviors at these wavelengths enable a mathematical approach to speciate Hg++ vs. MeHg+, similar to speciation of four toxicologically relevant As species based on four linear equations [26]. Performance of this method was demonstrated using fish oil supplement as a model matrix. 2. Experimental 2.1. Reagents and solutions ACS reagent grade anthranilic acid (AA), ≥96% formic acid (FA), and nitric acid were purchased from Sigma-Aldrich (Milwaukee, WI, USA); a 0.4% (w/v) AA–20% (v/v) FA reductant solution was prepared daily. Solid MeHgCl (99.9%) and Hg(NO3)2 standard in 12% HNO3 at 1000 μg mL−1 were purchased from Fluka (Milwaukee, WI, USA). MeHgCl standard in water at 1000 μg mL− 1 was purchased from Alfa Aesar (Ward Hill, MA, USA). Stock standards at 0.5 μg mL−1 were made weekly by serial dilution in deionized water (DIW) and stored at 4 °C prior to use; all working standard solutions at 0–5 ng mL−1 were prepared daily. Used glassware was soaked in 15% nitric acid overnight and rinsed thoroughly with DIW. DIW was made using a Barnstead E-pure system (Dubuque, IA). 2.2. Sample preparation Fish oil (liquid or capsule) was purchased online or from stores in Philadelphia, PA, USA. Capsules were punctured using a clean needle

and oil was squeezed first to a glass vial; then 2 mL of fish oil was mixed with 40 mL of DIW in a 50 mL polypropylene centrifuge tube. The tubes were capped tightly, arranged vertically in tube racks which were tightened securely. Vigorous mixing was provided by a LC1012 vortexer (Glas-Col, Terre Haute, IN, USA) for 10 min with motor speed set at 80 with pulsing. After centrifugation at 4000 rpm for 10 min, the upper oil layer was discarded with a disposable pipette; the aqueous layer was used for analysis. For ICP-MS determination of total Hg (tHg), 0.5 mL of fish oil was pipetted into a 55 mL Perfluoroalkoxy alkane (PFA) Xpress vessel. After addition of 5 mL concentrated HNO3, the vessel was sealed and placed in a 40-position carousal. With power set at 600 W, temperature was programmed in 3 steps: 120 °C for 2 min, 160 °C for 8 min, and 190 °C for 30 min with 2 min ramps. After digestion, samples were allowed to cool down to room temperature and filled with DIW to a final volume of 50 mL. 2.3. Photochemical reactor The key part of the photochemical reactor assembly (Fig. 1) was a 19 × 200 mm (od × l) reaction coil made of 6 × 4 mm (od × id) synthetic silica tubing. The capacity of the reactor was measured at 16.2 mL. Both coil inlet and outlet were drawn down to 3 mm od to allow connection to 1/8″ od polytetrafluoroethylene (PTFE) tubings using PTFE heatshrink tubes. Inserted loosely inside the coil was the 9.5 × 229 mm (od × l) lighted portion of a Pen-Ray® low-pressure Hg lamp (3SC-9, UVP, Upland, CA, USA.) with output at 254 nm rated at 6.9 mW cm−2 at a distance of 19 mm. Installed at one side of the coil were two narrowband UV-B fluorescent lamps (PL-S 9W/01, Philips Lighting, Sommerset, NJ, USA), each rated at 1 W output at 311 nm (Fig. 2). To protect users from UV exposure, the assembly was installed on an aluminum base and covered by a light-tight aluminum enclosure. When the UV-C lamp was on, the UV-B lamps were shielded. After daily operation, the reactor coil must be rinsed with ethanol and hexane, 20 mL each, to remove buildup on the inner wall. 2.4. Photochemical vapor generation As shown in Fig. 3, the photoreactor assembly was connected to a Millennium Merlin atomic fluorescence spectrometer (AFS) (P S Analytical, Kent, UK) between a mixing valve and a gas/liquid separator (G/L). The sample solution was mixed with 0.4% AA–20% FA reductant for 10 s

Fig. 1. Interior of the photoreactor assembly.


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Standard curves were constructed daily to compensate both short- and long-term variations in experimental conditions. 3. Results and discussion 3.1. Hg extraction from fish oil

Fig. 2. Output spectrum of Philips PS-S 9W/12 narrow-band UV-B fluorescent lamp.

in flow injection (FI) mode with PVG parameters listed in Table 1. Under 72 s UV exposure, the resulting gas was swept by high-purity argon to the G/L where the liquid was drained. The gas flew upward to a 12″ Perma Pure dryer (Farmingdale, NJ, USA) through which most moisture permeated into a counter flowing nitrogen stream; dry Hg0 vapor finally reached the AFS detector. 2.5. Atomic fluorescence spectrometry Inside the detector assembly, the Hg0 vapor at the exit of a chimney was excited by a high-intensity Hg hollow cathode lamp; the resulting 254 nm resonance fluorescence was collected at 90° and detected by a photomultiplier tube. Both AFS operation and data processing were controlled by Millennium software (P S Analytical) with parameters also listed in Table 1. Data were acquired for 250 s; quantitation was based on peak height. In the last 120 s of the cycle, the FI system was washed with DIW. 2.6. ICP-MS An Agilent 7900 ICP-MS (Santa Clara, CA, USA) was used for method validation based on tHg. The major operation parameters were shown in Table 2. The operation was controlled by MassHunter software 4.3 (Agilent). The operation was set to autotune before sample acquisition. A series of Hg++-in-10% (v/v)-HNO3 standards (0.00–1.00 ng mL−1) were prepared by serial dilution. 2.7. Speciation of Hg in fish oil Sample solutions were analyzed in triplicate. A series of Hg(NO3)2 or MeHgCl standards in water, both expressed as Hg in ng mL−1, was used to obtain four calibration curves (1–100 ng mL−1) under UV-B or UV-C.

Extraction induced by emulsion breaking (EIEB) is a common approach to extract analytes from oil matrix [27]. A surfactant turns oil into an emulsion that enables rapid mass transfer; heating or centrifugation follows to break down the emulsion leaving analytes in a surfactant phase. However, surfactant would cause foaming in the G/L. In the worst scenario, liquid enters the membrane dryer defeating its function allowing moisture to quench AFS signal. Fortunately, both MeHg+ and Hg++ are polar enough to render liquid-liquid extraction (LLE) effective. The partition coefficients, Kow, of HgCl2 and MeHgCl, are favorable at 0.61 [28], and 1.6 ± 0.2 [29], respectively. With LLE, foaming was no longer an issue. With a water-to-oil volume ratio of 20, MeHgCl recovery at 10 ng mL−1 maintained constant at ~73% under 5–60 min agitation. HgCl2, with a Kow at 0.61, is more polar than MeHgCl, so its recovery was expected to be much higher. Microwave-aided digestion must solubilize target Hg analyte in aqueous phase. Concentrated HNO3-H2O2 is effective for this goal [30]. For tHg quantification by ICP-MS, it is unnecessary to oxidize MeHg+ to Hg++, so H2O2 was excluded resulting in less foaming. After 46 min heating mostly at 190 °C, this protocol successfully solubilized fish oil extracting Hg to aqueous phase, and achieved a 90.4% recovery from a MeHgCl spiked fish oil sample at 80 ng mL−1 Hg. 3.2. Selection of actinic light sources Photoreduction of both Hg++ and MeHg+ can be reliably fulfilled at 254 nm using a low-pressure Hg discharge lamp [20]. Medium-pressure Hg discharge lamps emits multiple lines from UV to green; these lines broaden at high pressure and a continuum appears [31]. To fulfil Hg speciation solely by PVG, a second light source must renders different PVG responses vs. UV-C. Visible room light [19] and 365 nm LED [23] were reported to serve this purpose. However, visible light was later found ineffective using fluorescent lights or a 50 W tungsten lamp [25]; so was UV-A using an array of ten 0.8 mW 355 nm LEDs, or a 1 W 365 nm black light [24]. Such controversies prompted an investigation on Hg PVG efficiency at visible and UV-A. The AFS responses using an RGB or 365 nm LED array were hardly distinguishable from that under total darkness. The first array consisted of 54 LEDs (SMD5050RGB, TaoTronics San Jose, CA, USA); each was made up of 3 dice rated at 100, 400, and 100 mcd at 620, 515, and 460 nm, respectively. The second array consisted of 108 LEDs (XL5050UVC3C/365, TaoTronics) with a total 0.76 W output. It was likely that the reported Hg++ signals at

Fig. 3. The PVG-AFS experimental setup.

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G. Chen et al. / Spectrochimica Acta Part B 137 (2017) 1–7 Table 1 PVG-AFS conditions. Parameters

Values

Sample flow rate (mL min−1) Reductant flow rate (mL min−1) Reductant concentration (v/v) Argon flow rate (mL min−1) Nitrogen flow rate (mL min−1) HCL current (mA) HCL voltage (V) Measurement mode UV lamp wavelength (nm) Detection wavelength (nm)

9 4.5 20% FA-0.4% AA 300 2500 30 250 Peak height 311 or 254 254

visible and UV-A were of thermal origin [24]; but small slopes with good linearity made such response easily mistaken as of PVG origin. More energetic UV-B region (280–320 nm) was thus explored. Discharge and arc lamps emit mostly visible and infrared, with only few weak lines in the UV-A and UV-B regions. Their high power consumption also renders thermal management difficult. On the other hand, semiconductor devices in the UV-B region suffer from weak output (mW level), short life, and high price, in part due to high defect density. Fortunately, a 9 W UV-B fluorescent lamp (Philips PL-S 9W/01) was identified that generated 1 W output at 311 nm (Fig. 2) with a 2.6 nm full width half maximum (FWHM). Two such lamps were installed 1 cm away from the quartz coil (Fig. 1) that yielded an order of magnitude Hg++ signal enhancement vs. visible or UV-A LEDs. The MeHg+ slope was ~20 fold smaller (Table 3). Satisfactory (R2 N 0.99) linearity (Tables 3–5) enabled a mathematical approach to quantify both Hg++ and MeHg+, vide infra. 3.3. Selection of photochemical reductants Reductants play a key role in PVG of Hg species. At 254 nm, both Hg++ and MeHg+ can be effectively reduced using 6–20% FA [32]. With FA at 20%, unfortunately, the yields at 311 nm were 75% and 95% lower for Hg++ and MeHg+, respectively (Fig. 4). An effective reductant at 311 nm was needed for adequate sensitivity. Among LMWOC candidates, dicarboxylic acids (DCA) play a role in geochemical Hg cycling [33], of which oxalic acid (OA) has the highest yield at sea level where UV-B is the most energetic. In comparison to 20% FA, 9% OA improved Hg++ signal at 311 nm only slightly with poor linearity (Table 3); but failed to generate any response from MeHg+ at both wavelengths (Fig. 4). Under solar radiation, benzoic acids (BAs) play an important role in photolysis of certain metallic compounds in natural waters [34,35], among which 2-aminobenzoic acid (2-ABA), also known as AA, has the highest kinetic constant in part due to its absorption at 313 nm. AA was thus tested for its PVG performance at 0.4% (w/v), just below its 0.572% solubility. High yields for both species at 254 nm and a reasonable yield for Hg++ at 311 nm were obtained (Fig. 4) with excellent Table 2 ICP-MS operation parameters. ICP-MS instrument

Agilent 7900

Uptake time Stabilization time Rinse time Monitored mass Peristaltic pump flow rate ICP parameter RF power Plasma mode Carrier gas flow rate Aerosol dilution Use gas He flow rate

45 s 20 s 80 s Hg200 1.4–1.5 mL min−1 ≤1600 W High matrix introduction (HMI-4) 0.6–0.8 L min−1 0.6–0.8 L min−1 On 1.0 mL min−1

Table 3 Comparison of correlation coefficients (R2) of Hg++ and MeHg+ calibration curves (0– 100 ng mL−1) using formic (FA), oxalic (OA), and anthranilic acid (AA) reductants at 311 vs. 254 nm. Reductant

20% FA 9% OA 0.4% AA 0.4% AA-20%FA

Hg++

MeHg+

311 nm

254 nm

311 nm

254 nm

0.993 0.955 0.999 0.994

0.997 0.994 0.994 0.991

0.991 0.971 0.995 0.995

1.000 0.975 0.997 0.998

linearity (Table 3), but the yield for MeHg+ at 311 nm was very low. Fortunately, during optimization process (Figs. 5–7), a mixture of 0.4% AA and 20% FA was found to achieve adequate yields for Hg++ at 311 nm and for both species at 254 nm (Fig. 4) while maintaining good linearity in all cases (Tables 3–5). A single reductant was desirable for its simplicity, as long as Hg++ and MeHg+ responded well at one or both wavelengths. The lack of exclusive response at each wavelength necessitates a set of two linear equations, similar to the 4-equation approach for As speciation [26]. 3.4. Design of photochemical reactor Thus far, PTFE or quartz is used to construct photochemical reactors (PCRs). PTFE, albeit convenient due to its flexibility, is inferior to quartz in UV transmission. Performances of PCRs made of various grades of quartz have been compared [36]; synthetic quartz rendered superior PVG performance. In this work, a 6 × 4 mm (od × id) synthetic fused silica tubing was used to construct a reactor in tight coil geometry (Fig. 1) to match the dimensions of a 3SC-9 Hg lamp. The 16.2 mL coil volume was a key parameter that allowed the content 72 s exposure at 13.5 mL min−1 combined flow rate, the highest for this spectrometer. Because of high absorbance of AA in the UV region, the bulk of the mixture was basically opaque to both UV wavelengths. The penetration depth, defined as the distance where light attenuates to 1/e of its original intensity, were calculated to be merely 0.17 and 0.06 mm in the sample-reductant mixture at 311 and 254 nm, respectively. In the presence of LMWOC, non-thiolated Hg compounds follow photoinitiated reduction pathway [37]. Light absorption results in reactive intermediates, such as radicals, that reduce Hg++ to Hg0 [24]. Upon absorption of a photon, excited ABA− produces triplet-state ABA− 3 − (3ABA−), ABA• radical, and hydrated electron (e− eq) [37]. Both ABA − and eeq decay quickly by recombination or reaction with oxidants with t1/2 at 160 ns and 50 ns, respectively. Long-life ABA• (t1/2 = 50 μs) radical could move to the bulk to reduce Hg++ and MeHg+ [35]. Diffusion is a slow process at room temperature; mixing must play a key role for the reduction to go to completion before reaching the G/L. In this design, both the tight-coil geometry and the drag on the quartz inner surface promoted turbulent flow and mixing. The same signal intensity was observed when one UV-B lamp was turned off, indicating reduction was fast and 72 s exposure was sufficient. Longer exposure would adversely affect sample throughput, and increase the odds of Hg0–to–Hg++ photooxidation [38].

Table 4 Correlation coefficients (R2) of Hg++ and MeHg+ calibration curves (0–100 ng mL−1) at 311 nm using reductants that contained 20% FA and 0–0.4% AA. Reductant

20% FA-0% AA 20% FA-0.05% AA 20% FA-0.10% AA 20% FA-0.20% AA 20% FA-0.40% AA

Hg++

MeHg+

311 nm

311 nm

0.993 0.976 0.962 0.988 0.994

0.991 0.999 0.999 0.999 0.995


5

Table 5 Correlation coefficients (R2) of Hg++ and MeHg+ calibration curves (0–100 ng mL−1) at 311 and 254 nm using reductants that contained 0–20% FA and 0.4% AA. Reductant

0% FA-0.40% AA 4% FA-0.40% AA 8% FA-0.40% AA 12% FA-0.40% AA 20% FA-0.40% AA

Hg++

MeHg+

311 nm

254 nm

311 nm

254 nm

0.999 0.973 0.953 0.975 0.994

0.994 0.959 0.961 0.982 0.991

0.995 0.981 0.992 0.984 0.995

0.997 1.000 1.000 1.000 0.998

3.5. Photochemical vapor generation Calibration curves must be obtained daily to compensate day-to-day variations in experimental conditions such as batches of chemicals and solutions especially FA; as well as long-term variations such as degradation and deformation of peristaltic pump tubings, and output changes of aging UV sources. The slopes are then used in the calculation. In comparison, the dark AFS intensity revealed a linear (R2 N 0.99) dependence on concentration that cannot be accounted for by background noise alone. The Hg++ slope using the above mentioned RGB LED array was the same as that in the dark, and the slope using a 365 nm UV LED array was only ~ 70% higher. The ineffectiveness of both visible and UV-A light in PVG of Hg++ was thus confirmed despite relatively high intensity of both sources. 3.6. Mathematical approach for Hg++ vs. MeHg+ speciation Using 0.4%AA-20% FA, both Hg++ and MeHg+ responded well to 254 nm; the response of Hg++ at 311 nm was ~ 1/3 lower while that of MeHg+ was extremely low (Fig. 4). Lack of reaction exclusivity at either wavelength necessitated a mathematical approach that correlated combined AFS intensity, IB or IC, to Hg++ and MeHg+ concentrations: IB ¼ mB Hgþ þ nB MeHgþ

ð1Þ

IC ¼ mC Hgþ þ nC MeHgþ

ð2Þ

where m and n are slopes of Hg++ and MeHg+ calibration curves, respectively. Four prerequisites of this approach are: (1) Hg++ and MeHg+ are the only detectable Hg species in products of fish origin; (2) AFS responses are linear; (3) AFS responses are additive; and (4) The Hg++ to MeHg+ slope ratios at two wavelengths should not be identical. The first prerequisite is observed in a global scale [10,11]. Theoretically, the second prerequisite holds with a wide (N 5 orders of

Fig. 4. Slopes of Hg++ and MeHg+ calibration curves by PVG at 311 or 254 nm using four reductants: 20% FA, 9% OA, 0.4% AA, and 0.4% AA-20% FA.

Fig. 5. Slopes of Hg++ and MeHg+ calibration curves by PVG at 311 nm using reductants that contained 20% FA and 0–0.4% AA.

magnitude) linear dynamic range; in practice, correlation coefficients were typically adequate (Tables 3–5). The third prerequisite also holds as long as MeHg+ concentration is expressed as Hg. The fourth prerequisite is necessary to render the equation set solvable. 3.7. ICP-MS validation based on tHg In elemental analysis, spike-recovery study must be performed when no CRM is available [39]. For MeHg+ a 73% recovery was obtained. However, despite over-night agitation, the effort to prepare a stable and homogeneous Hg++-in-fish-oil standard failed, due to instability of iHg in organic media [40] as well as adsorption on container wall. Commercial Hg-in-oil standards, on the other hand, are of no use because added stabilizing ligand disables PVG. ICP-MS is extremely sensitive thus qualified as a validation technique for trace-level elements. In this work, one MeHgCl spiked fish oil and four other fish oil samples with relatively high (2.18– 6.06 ng mL−1) total Hg (tHg) were measured by ICP-MS; and the results (1.52–3.88 ng mL−1) were compared vs. PVG-AFS in Fig. 8. Experimentally, 5 mL of concentrated (~70%) HNO3 was added to digest 0.5 mL of fish oil. To prevent corrosion to the nickel cones, the digest was diluted by 10-fold to render HNO3 below 10%. As a result, Hg concentration was reduced by two orders of magnitude; accordingly the resulting method LOD becomes higher despite a much lower instrument LOD expected from ICP-MS. Because these data are very close to method LODs for MeHg+: 0.63 and 1.6 ng mL−1 for PVG-AFS and ICP-MS, respectively. Overlap of error bars in Fig. 8 indicated reasonable agreement between these data sets. The LOQs for MeHg+ are 3.3 times higher at 1.4 and 5.4 ng mL−1 for PVG-AFS and ICP-MS, respectively.

Fig. 6. Slopes of Hg++ and MeHg+ calibration curves by PVG at 311 nm using reductants that contained 0.4% AA and 0–20% FA.

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G. Chen et al. / Spectrochimica Acta Part B 137 (2017) 1–7 Table 6 Hg++ and MeHg+ concentrations in 25 fish oil (FO) samples.a #

Fig. 7. Slopes of Hg++ and MeHg+ calibration curves by PVG at 254 nm using reductants that contained 0.4% AA and 0–20% FA.

3.8. Results of fish oil analysis Limits of detection (LODs) of both methods were calculated based on 3σ. LOD of Hg++, 0.50 ng mL−1, was obtained at 311 nm; whereas LOD of MeHg+, 0.63 ng mL−1, was obtained at 254 nm. The MeHg+ data were reported after correction for incomplete (73%) recovery by PVG-AFS. Among 25 fish oil samples tested so far (Table 6), seven contained MeHg+ above LOD; of which only three exceeded 1.4 ng mL− 1 LOQ. Meanwhile, 11 samples contained Hg++ above LOD, of which only one exceeded 1.1 ng mL−1 LOQ. Up to now, overthe-counter omega-3 fatty acid supplements are not regulated by the US FDA; the fish oil industry follows the 100 parts per billion (ppb) total Hg safe level set by the Global Organization for EPA and DHA Omega-3s (GOED), the European Pharmacopoeia, Norwegian Medicinal Standard, and the Council for Responsible Nutrition. The Hg contents in this sample set were well below 100 ppb, so fish oil in general is much safer than fish muscle or organs in terms of Hg impurities. These results are in line with independent test results on current generation of products [41], but lower than the test data a decade ago [3,4] as a result of continuous technology advancement. From manufacturing point of view, species lower in the food chain are now used as raw material for fish oil, such as herring, sardine, anchovy, and even krill. Furthermore, advancements in purification practices such as distillation, water washing, and bleaching with sorbent (activated carbon, synthetic earths, or silicates) are especially effective to exclude polar and hydrophilic components such as phospholipids, proteins, and minerals. In comparison, cold vapor atomic absorption spectrometry (AAS) achieved only 6 ng mL−1 LOD for total Hg [3]; amalgamation enrichment further

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 a

PVG-AFS

PVG-AFS

MeHg+ (ng mL−1)

Hg++ (ng mL−1)

bLOD bLOD bLOD 0.73 bLOD bLOD 0.73 2.75 bLOD bLOD bLOD bLOD bLOD 1.13 bLOD bLOD bLOD bLOD 0.90 bLOD bLOD bLOD bLOD 5.07 2.13

0.70 bLOD 3.18 bLOD 0.78 bLOD bLOD bLOD bLOD bLOD 0.63 0.76 0.87 0.63 0.75 0.94 bLOD bLOD bLOD bLOD bLOD bLOD bLOD 0.99 0.84

The LODs are 0.50 and 0.63 ng mL−1 for Hg++ and MeHg+, respectively.

improved LOQ to 1.5 ng g−1 [4]. The ability to speciate Hg at trace level was fulfilled in this work that provided extra insight and guidance to quality control and assurance on this toxic impurity. In fish, MeHg+ accounts for 70–95% of Hg content, and is known to preferentially bind to cysteine rather than fat, leading to formation of MeHg-L-cysteine [42]. Cysteine is a thiol-containing amino acid that exists in all animals and plants. Strong Hg\\S bond interferes with photochemical reduction mechanisms, rendering this PVG method ineffective for fish muscle or organ. 4. Conclusions A UV-B fluorescent light was identified that enhanced PVG efficiency of Hg++ by an order of magnitude over visible and UV-A sources. Differential PVG behaviors of Hg++ and MeHg+ under UV-B vs. UV-C wavelength enabled a mathematical approach for sensitive Hg speciation in fish oil supplement. Elimination of chemical or chromatographic separation gained simplicity, throughput, cost, and green chemistry advantages. References

Fig. 8. Comparison of tHg data by PVG-AFS vs. ICP-MS.

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