TY - JOUR
AU1 - Lech,, Teresa
AU2 - Turek,, Wioletta
AB - Abstract Mercury is a heavy metal with high toxicity, the level of which depends on the form of the metal. One of the newer techniques for determining trace amounts of total mercury in various materials, including biological samples, is thermal decomposition, amalgamation and atomic absorption spectrometry (TDA AAS). The TDA AAS method was optimized and validated using a mercury analyzer (DMA-80). The limits of detection for mercury were 0.10 and 0.20 μg/L (nickel and quartz boats, respectively). The working range of the calibration curve was at least from 0.6 to 200 ng Hg/mL; the intra-day precision in samples (RSD)—in the range of: 1.66–6.86% (blood), 0.82–1.47% (urine) and 2.01–3.44% (hair); the inter-day precision (over 8 days): 2.51%, and 2.5% (blood spiked with 2.5 and 10 ng Hg, respectively), 5.10% and 3.16% (urine spiked with 2.0 and 6.0 ng Hg, respectively). The accuracy (as relative error, mean value) determined on the basis of the study of reference materials of blood (Seronorm Trace Elements Whole Blood L-1, L-2, L-3), urine (Seronorm Trace Elements Urine, Urine L-2), and hair (Human Hair NIES CRM No. 13) was: 2.00% (blood), 0.50% (urine) and 0.86% (hair); recovery of 2.5 ng Hg (blood): 93–97%. The method was used for the determination of mercury in 76 samples of various biological matrices, including samples of whole blood, urine, hair, bile and vitreous humor. Mercury concentrations in postmortem blood (n = 24) were in the range: 0.61–12.4 μg/L (median 3.02 μg/L); urine (n = 12): 0.16–2.19 μg/L (median 0.81 μg/L); hair (n = 14): 0.08–0.53 μg/g (median 0.22 μg/g); bile (n = 12): 1.15–7.11 μg/L (median 2.41 μg/L and vitreous humor (n = 13): 0.22–1.01 μg/L (median 0.47 μg/L). The method is suitable for the purposes of forensic toxicology analysis. Introduction Mercury and its compounds have been classified as one of most hazardous substances for living organisms. This is above all connected with the emission of mercury into the environment, the processes of redistribution, and the influence of mercury on human health. Moreover, mercury salts and organic mercury compounds can cause the most often acute mercury poisonings, whereas elemental mercury is responsible for chronic ones. It depends on the dose and the route of administration and the time of exposure to mercury (1). Mercury may be introduced into the human body by different ways: the respiratory tract (mercury vapor), the gastrointestinal tract (alkyl- and inorganic compounds) and also through the skin (vapor, organic compounds). In the case of occupational exposure, it is assumed that the main route of introduction for this metal into the organism is inhalation (80% of mercury is absorbed in this way). In the process of inhalation, elemental mercury vapor is transported to the systemic circulation of blood, where it is partially catalytically oxidized. An important role in the metabolism of mercury is played by sulfhydryl ligands (RSH) and amino-acids (cysteine, glycine), which form complexes with mercury. Inorganic compounds of mercury accumulate mainly in the liver and kidneys. Some organic compounds (phenylmercury) metabolize in the human body into inorganic compounds. Methylmercury collects in the central nervous system (and is very dangereous for the fetus). In the case of food consumption, organic compounds of mercury are almost totally absorbed (95%), whereas inorganic ones are scarcely (7%) absorbed. The elimination of mercury from the body (via kidneys) is rather poor: the biological half-life ranges between 40 and 70 days, depending on the form of mercury (2). The analytical methods currently used for mercury determination in different biological materials include various techniques (3, 4): the cold vapor technique in atomic absorption or atomic fluorescence spectrometry (CV AAS//CV AFS) (5, 6), inductively-coupled plasma optical emission spectrometry (ICP OES) (7) or inductively-coupled plasma mass spectrometry (ICP-MS) (6, 8–11). In order to evaluate the kind and severity of poisoning with mercury in living persons, the best materials for analysis include whole blood, urine and hair; however, in expert opinions concerning death of unknown cause, postmortem material obtained at the autopsy is analyzed: besides whole blood, urine and sometimes hair, internal organs, particularly liver, kidneys, lung or brain (accumulating organs) are also sampled. The CV AAS technique has been used most commonly up to now for different matrices. Samples of examined material should usually be pre-prepared (in a separate apparatus or an instrument) before analysis through a digestion process by concentrated acids in a high temperature and under pressure, which is relatively time-consuming and requires some expensive reagents, and, moreover, can also be linked with some losses of analyte. Currently, an examination of environmental and biological materials for total mercury can be performed by thermal decomposition, amalgamation and atomic absorption spectrometry (TDA AAS) (3, 4, 8, 12–14). Moreover, as has been done in some experiments, it is possible to use sequential atomic absorption and atomic fluorescence spectrometry after sample combustion and concentration of mercury by gold amalgamation (16). This sensitive method (TDA AAS) does not require any separate preparation of a sample before analysis, and, in addition, it is characterized by the possibility of analyzing samples with different, even very difficult matrices. The fact that only small amounts of samples need to be analyzed and that it is unnecessary to carry out separate decomposition of the examined material, significantly reduces the time required and the use of corrosive and high purity reagents, which are in accordance with “green chemistry” principles (13). The total costs of analysis per one sample are also reduced. A solid or liquid sample is weighed (measured) out into a metal or quartz boats, and loaded onto the instrument autosampler. A sample is first dried and then thermally decomposed in an oxygen-rich furnace. Mercury and other combustion products are released from the sample, where interfering compounds (nitrogen and sulfur oxides, halogens), are eliminated. Mercury is selectively trapped, in a separate furnace, through gold amalgamation. After heating, mercury is rapidly released and flown via the carrier gas into a unique block with a dual-cell arrangement, where is quantitatively measured by atomic absorption at 253.65 nm. TDA AAS has already been applied mainly to environmental samples (soil, sediments, water and air) (14, 15, 17), and food, particularly fish and seafood (3, 8, 12, 18–26). There are a few articles on the determination of mercury in blood (13, 27), urine (27) and hair (6, 27, 28). The method has not been used in forensic toxicology for the investigation of postmortem material until now. The aim of the study was to evaluate use of the TDA AAS method for total mercury determination in postmortem samples of biological material (blood, urine, hair, bile and vitreous humor) in forensic examinations directly, without time-consuming and potentially hazardous hot-acid digestion. Bile and vitreous humor have been rather rarely applied in forensic toxicology up to now; however, in some cases they are the only available material—for example, when blood and urine samples are collected in insufficient amounts to carry out complete toxicological analysis. Experimental Samples Samples of whole blood (n = 24), urine (n = 12), hair (14), bile (n = 12) and vitreous humor (n = 13) were obtained from autopsy cases relating to inhabitants of Southern Poland who had not been poisoned and had not been exposed environmentally or occupationally to mercury (exposure to mercury was unknown) or other heavy metals. The samples of biological material (blood, urine, vitreous humor pipetted, head hair samples cut from the body) were collected into 30-mL polypropylene vessels (Sarstedt, Germany) and stored frozen (at a temperature below −15°C) until analysis. Some samples of blood, urine and hair were used to validate the procedure for mercury and all samples were analyzed to estimate levels of total mercury: such data might serve as a contribution to general reference levels in the Polish population. The levels can be preliminary data obtained by the TDA AAS method. Characteristics of the examined material are given in Table I. Table I. Characteristics of the examined samples Material Men Women Blood 20 4 Urine 14 3 Hair 12 2 Bile 12 2 Vitreous humor 13 2 Material Men Women Blood 20 4 Urine 14 3 Hair 12 2 Bile 12 2 Vitreous humor 13 2 Table I. Characteristics of the examined samples Material Men Women Blood 20 4 Urine 14 3 Hair 12 2 Bile 12 2 Vitreous humor 13 2 Material Men Women Blood 20 4 Urine 14 3 Hair 12 2 Bile 12 2 Vitreous humor 13 2 The mean age of the examined people was: 49.3 ± 14.3 years (men), 75.0 ± 9.9 years (women). Reagents All reagents used in the study were analytical grade: 65% (v/v) nitric acid (V), 30% (v/v) chloric acid and standard stock solution of mercury (II) (1,000 mg/L, Merck, Darmstadt, Germany). A working standard of mercury (10 μg/mL) was prepared by appropriate dilution of the stock solution by deionized water, with addition of concentrated nitric acid (V) in amounts of 0.5 mL/100 mL of the final volume, and stored in a refrigerator. From this standard solution, a control solution of mercury: 100 or 50 μg/L, was made fresh daily. Method, instrumentation Analysis of biological material for total mercury was carried out by TDA AAS using a Direct Mercury Analyzer DMA-80 Dual Cell, Milestone (Sorisole, Italy), supplied with compressed oxygen in a bottle (purity at least 99.5%). Quartz boats were used as recommended by the manufacturer for the analysis of liquids (urine, vitreous humor), and nickel boats (Milestone, Sorisole, Italy) were used for whole blood, bile and hair samples. In order to completely eliminate mercury from the quartz boats after use, they were soaked in 10% (v/v) nitric acid for 24 h, and then carefully rinsed with deionized water and dried in a dryer and stored in an exsiccator until analysis. Metal (nickel) boats were roasted in a muffle oven (Griffin, Great Britain) at a temperature of 600°C for 20 min (2 min used previously appeared insufficient for good cleaning), and then stored in an exsiccator (like the quartz boats). Validation procedure The main validation parameters evaluated in this study were: calibration model, detection and quantification limits, accuracy, recovery, and precision as repeatability (one-day) and reproducibility (intra-day) of measurements. The expanded uncertainty of measurement, using a coverage factor of two which gives a level of confidence of ~95% (U= 2 u), was also estimated. Optimization of the method encompassed: the influence of the volume of a sample on the results of analysis (using the example of blood analysis), the influence of the kind of method (taken from the Application Book assigned to the instrument) on the results of analysis (by the example of blood analysis—Whole Blood L-3), the influence of the kind of boat (quartz, nickel) on the results of blood analysis. The instrument was calibrated by the manufacturer, and it had two calibration curves inserted into the instrument: “Ultratrace”—up to 7 ng Hg/sample, and another calibration curve– up to 1,800 ng Hg/sample. To control the stability of these two calibration curves, every day before an analysis, both curves were controlled by the measurement of a standard of 100 ng/mL of Hg. However, when only the “Ultratrace” curve (up to 7 ng/sample) was chosen as the calibration curve for a given method (e.g., for reference levels), a standard of 50 ng/mL of Hg was used. As recommended by the manufacturer, Cal-Factor (which illustrates current deviation from the calibration curve) should be in the range of 0.9–1.1; if it is not contained within this range, a new calibration has to be carried out by the user. For validation purposes, calibration was checked with the use of three different matrices: blood, urine and hair, taking samples of 0.2 mL (blood), 0.1 mL (urine) and 10 mg (hair) for each point of the calibration curve from 1 to 200 ng/mL of Hg for urine and blood, and from 0.5 to 5.0 ng/mg for hair samples. Bearing in mind that the TDA AAS method does not require any separate mineralization, and that determination of mercury is carried out by indirect analysis of the examined material, the limit of detection (LOD) and limit of quantification (LOQ) were evaluated by measurement (10 times) of empty clean quartz and nickel boats used to introduce a sample into the analyzer. LOD, according to the formula LOD = 3 SD (in ng/L) for a “blank” (empty boat) for 10 measurements, and LOQ, as 10 SD (in ng/L) for a “blank” (empty boat) for 10 measurements. We also tested LOD and LOQ, using deionized water and diluting nitric acid (0.3%). Precision was evaluated by parameters such as repeatability (one-day) and intermediate precision (inter-day). Repeatability was determined by the use of certified samples of blood, urine and hair: Whole Blood L-1, Whole Blood L-2, Whole Blood L-3, Urine, Urine L-2, Human Hair GBW 09101 No. 13, and Human Hair NIES CRM No. 13. Before analysis, samples of materials were prepared in accordance with instructions enclosed in the standards. Precision was determined by the measurement of each sample on the same day 10 times. Inter-day precision was evaluated using two samples of blood (each 0.2 mL), one fortified at 2.5 ng Hg, the second fortified at 10 ng Hg and two samples of urine (each 0.1 mL) fortified at 2.0 and 6.0 ng/sample, respectively—and double measuring of each sample on eight successive days. In the accuracy study of the validation procedure, CRM materials (SERO AS, Norwegian): SeronormTM Trace Elements Whole Blood L-1, SeronormTM Trace Elements Whole Blood L-2, SeronormTM Trace Elements Whole Blood L-3, SeronormTM Trace Elements Urine, and SeronormTM Trace Elements Urine L-2, and also Human Hair from NIES CRM No. 13, Japan. The Human Hair No. 13 (China) was, valid to 2008, used for test of precision only. Samples of hair (taken from CRM stored in its original bottle at −20°C) were dried at 85°C in an electric oven, and then cooled in a silica gel desiccator at room temperature. The accuracy (as relative error) of the TDA AAS method was evaluated on the basis of study of certified samples of blood (0.2 mL), urine (0.1 mL) and hair (30 mg). Additionally, the recovery was tested using samples of blood without any additions (baseline blood, background value) and samples fortified with 2.5 ng of Hg/sample (0.2 mL). Reference levels of Hg in whole blood, urine, bile, vitreous humor and hair The optimized and validated method was applied to evaluate concentrations of Hg in body fluids (urine, blood, bile and vitreous humor) and hair. Samples were taken from autopsy cases, and were stored in a refrigerator (−15°C) until examination. The samples of liquids were unfrozen and mixed using a Vortex (for at least 30 s) before analysis. Hair samples were washed with acetone and deionized water, according to the procedure of the International Agency of Atomic Energy in Vienna (IAEA) (29), and then dried at a temperature of 80°C. Deionised water, 18.2 MΩ (NANOpure DIamond, Barnstead, Dubuque, IA, USA), was used for dilution of the standard solutions and CRM materials. Glass and polypropylene vessels were soaked for 24 h in 5% (v/v) nitric acid solution and rinsed before use with deionised water. Results and Discussion On the basis of results of the optimization study (Table II), it was ascertained that the volume of a blood sample taken for analysis is not of great importance, however, RSD and relative error (in %) were somewhat lower (0.60–4.09% vs. 1.83–4.55%) using larger volume of blood (0.2 mL vs. 0.1 mL). In the next determinations of mercury, volumes of 0.2 mL of blood samples (and 0.1 mL of urine), for which RSD was usually <1.5%, were applied. Table II. Influence of the volume of a sample on the results of analysis of blood (n = 3) for mercury Material Volume of sample of blood (mL) Concentration of Hg found Mean ± SD (μg/L) Certified value Mean ± SD (μg/L) Analytical uncertainty (acceptable range) (μg/L) RSDa (%) Relative error (%) Whole blood L-1b 0.1 2.35 ± 0.11 1.97 ± 0.10 1.77–2.17 4.55 19.3 0.2 2.25 ± 0.06 2.62 14.2 Whole blood L-2b 0.1 16.1 ± 0.30 15.2 ± 0.8 13.6–16.8 1.83 5.92 0.2 16.3 ± 0.10 0.60 7.24 Whole blood L-3 0.1 34.2 ± 0.94 37.1 ± 7.5 29.6–44.8 2.74 7.82 0.2 37.0 ± 1.51 4.09 0.27 Material Volume of sample of blood (mL) Concentration of Hg found Mean ± SD (μg/L) Certified value Mean ± SD (μg/L) Analytical uncertainty (acceptable range) (μg/L) RSDa (%) Relative error (%) Whole blood L-1b 0.1 2.35 ± 0.11 1.97 ± 0.10 1.77–2.17 4.55 19.3 0.2 2.25 ± 0.06 2.62 14.2 Whole blood L-2b 0.1 16.1 ± 0.30 15.2 ± 0.8 13.6–16.8 1.83 5.92 0.2 16.3 ± 0.10 0.60 7.24 Whole blood L-3 0.1 34.2 ± 0.94 37.1 ± 7.5 29.6–44.8 2.74 7.82 0.2 37.0 ± 1.51 4.09 0.27 aRSD, relative standard deviation. bStandard valid until 2016. Table II. Influence of the volume of a sample on the results of analysis of blood (n = 3) for mercury Material Volume of sample of blood (mL) Concentration of Hg found Mean ± SD (μg/L) Certified value Mean ± SD (μg/L) Analytical uncertainty (acceptable range) (μg/L) RSDa (%) Relative error (%) Whole blood L-1b 0.1 2.35 ± 0.11 1.97 ± 0.10 1.77–2.17 4.55 19.3 0.2 2.25 ± 0.06 2.62 14.2 Whole blood L-2b 0.1 16.1 ± 0.30 15.2 ± 0.8 13.6–16.8 1.83 5.92 0.2 16.3 ± 0.10 0.60 7.24 Whole blood L-3 0.1 34.2 ± 0.94 37.1 ± 7.5 29.6–44.8 2.74 7.82 0.2 37.0 ± 1.51 4.09 0.27 Material Volume of sample of blood (mL) Concentration of Hg found Mean ± SD (μg/L) Certified value Mean ± SD (μg/L) Analytical uncertainty (acceptable range) (μg/L) RSDa (%) Relative error (%) Whole blood L-1b 0.1 2.35 ± 0.11 1.97 ± 0.10 1.77–2.17 4.55 19.3 0.2 2.25 ± 0.06 2.62 14.2 Whole blood L-2b 0.1 16.1 ± 0.30 15.2 ± 0.8 13.6–16.8 1.83 5.92 0.2 16.3 ± 0.10 0.60 7.24 Whole blood L-3 0.1 34.2 ± 0.94 37.1 ± 7.5 29.6–44.8 2.74 7.82 0.2 37.0 ± 1.51 4.09 0.27 aRSD, relative standard deviation. bStandard valid until 2016. The results obtained by the use of whole blood and standard—liquids methods were comparable (Table III). It was decided that, in next examinations, the whole blood method would be used for determination of mercury in blood samples. Table III. Influence of the kind of method on the results of analysis of blood (n = 3) for mercury Method (from Application Book) Volume of sample of blood (mL) Concentration of Hg found Mean ± SD (μg/L) Certified value Mean ± SD (μg/L) RSDa (%) Relative error (%) “Blood” 0.1 34.8 ± 0.74 37.1 ± 7.5 2.13 6.20 0.2 36.2 ± 0.94 2.60 2.42 “Standard—liquids” 0.1 35.0 ± 0.49 1.39 5.66 0.2 35.1 ± 1.00 2.85 5.40 Method (from Application Book) Volume of sample of blood (mL) Concentration of Hg found Mean ± SD (μg/L) Certified value Mean ± SD (μg/L) RSDa (%) Relative error (%) “Blood” 0.1 34.8 ± 0.74 37.1 ± 7.5 2.13 6.20 0.2 36.2 ± 0.94 2.60 2.42 “Standard—liquids” 0.1 35.0 ± 0.49 1.39 5.66 0.2 35.1 ± 1.00 2.85 5.40 aRSD, relative standard deviation. Table III. Influence of the kind of method on the results of analysis of blood (n = 3) for mercury Method (from Application Book) Volume of sample of blood (mL) Concentration of Hg found Mean ± SD (μg/L) Certified value Mean ± SD (μg/L) RSDa (%) Relative error (%) “Blood” 0.1 34.8 ± 0.74 37.1 ± 7.5 2.13 6.20 0.2 36.2 ± 0.94 2.60 2.42 “Standard—liquids” 0.1 35.0 ± 0.49 1.39 5.66 0.2 35.1 ± 1.00 2.85 5.40 Method (from Application Book) Volume of sample of blood (mL) Concentration of Hg found Mean ± SD (μg/L) Certified value Mean ± SD (μg/L) RSDa (%) Relative error (%) “Blood” 0.1 34.8 ± 0.74 37.1 ± 7.5 2.13 6.20 0.2 36.2 ± 0.94 2.60 2.42 “Standard—liquids” 0.1 35.0 ± 0.49 1.39 5.66 0.2 35.1 ± 1.00 2.85 5.40 aRSD, relative standard deviation. It was also stated that the influence of the kind of boat on the results of the determination in blood is inconsiderable (Table IV), but the relative error was lower for the nickel boat (the small difference is probably due to the material—quartz versus nickel. In further analyses, this boat was used). Table IV. Influence of kind of boat on the results of analysis of blood (n = 5) for mercury Kind of boat Volume of sample of blood (mL) Concentration of Hg found Mean ± SD (μg/L) Certified value Mean ± SD (μg/L) RSDa (%) Relative error (%) Nickel 0.2 37.0 ± 1.51 37.1 ± 7.5 4.09 0.27 Quartz 0.2 35.4 ± 0.77 2.16 4.58 Kind of boat Volume of sample of blood (mL) Concentration of Hg found Mean ± SD (μg/L) Certified value Mean ± SD (μg/L) RSDa (%) Relative error (%) Nickel 0.2 37.0 ± 1.51 37.1 ± 7.5 4.09 0.27 Quartz 0.2 35.4 ± 0.77 2.16 4.58 aRSD, relative standard deviation. Table IV. Influence of kind of boat on the results of analysis of blood (n = 5) for mercury Kind of boat Volume of sample of blood (mL) Concentration of Hg found Mean ± SD (μg/L) Certified value Mean ± SD (μg/L) RSDa (%) Relative error (%) Nickel 0.2 37.0 ± 1.51 37.1 ± 7.5 4.09 0.27 Quartz 0.2 35.4 ± 0.77 2.16 4.58 Kind of boat Volume of sample of blood (mL) Concentration of Hg found Mean ± SD (μg/L) Certified value Mean ± SD (μg/L) RSDa (%) Relative error (%) Nickel 0.2 37.0 ± 1.51 37.1 ± 7.5 4.09 0.27 Quartz 0.2 35.4 ± 0.77 2.16 4.58 aRSD, relative standard deviation. On the basis of the performed calibration curves, presented in Figures 1–3, the working range of the calibration curve was established as being at least from 0.6 to 200 μg Hg/L (blood and urine), and 0.3–5.0 μg/g (hair). Figure 1. View largeDownload slide Calibration curve of Hg with the use of blood matrix. Figure 1. View largeDownload slide Calibration curve of Hg with the use of blood matrix. Figure 2. View largeDownload slide Calibration curve of Hg with the use of urine matrix. Figure 2. View largeDownload slide Calibration curve of Hg with the use of urine matrix. Figure 3. View largeDownload slide Calibration curve of Hg with the use of hair matrix. Figure 3. View largeDownload slide Calibration curve of Hg with the use of hair matrix. Limit of detection (LOD) and quantification (LOQ) were the following: LOD − 0.10 ng/g or 0.10 μg/L (which correspond to the amount of 0.01 ng of Hg in blood and a solid sample) for the empty nickel boat, − 0.20 μg/L (0.02 ng of Hg in urine and a liquid sample) for the quartz boat, LOQ − 0.30 ng/g (nickel boat), 0.60 μg/L (empty quartz boat), respectively. In comparison, the values of LOD and LOQ, established for deionized water samples and 0.3% nitric acid were: 0.14 and 0.46 μg/L (water), and 0.5 and 1.5 μg/L (diluted acid). The results for both kinds of precision, evaluated as repeatability (one-day) and intermediate (intra-day), are summarized in Tables V–VI. Table V. Precision as repeatability (one-day), n = 10 Biological material Reference material Concentration of Hg Mean ± SD (μg/L) Precision (repeatability) RSDa (%) Blood Whole Blood L-1b 2.28 ± 0.16 6.86 Whole Blood L-2b 16.9 ± 0.37 2.17 Whole Blood L-3 35.8 ± 0.60 1.66 Urine Urine 40.6 ± 0.60 1.47 Urine L-2 40.1 ± 0.30 0.82 Hair Human Hair No. 13c (China)a 1803 ± 62.0d 3.44 Human Hair No. 13 (Japan) 4458 ± 89.4d 2.01 Biological material Reference material Concentration of Hg Mean ± SD (μg/L) Precision (repeatability) RSDa (%) Blood Whole Blood L-1b 2.28 ± 0.16 6.86 Whole Blood L-2b 16.9 ± 0.37 2.17 Whole Blood L-3 35.8 ± 0.60 1.66 Urine Urine 40.6 ± 0.60 1.47 Urine L-2 40.1 ± 0.30 0.82 Hair Human Hair No. 13c (China)a 1803 ± 62.0d 3.44 Human Hair No. 13 (Japan) 4458 ± 89.4d 2.01 aRSD, relative standard deviation. bValid until 2016. cThis standard (valid until 2008) of hair used for precision test only. dμg/kg dry weight. Table V. Precision as repeatability (one-day), n = 10 Biological material Reference material Concentration of Hg Mean ± SD (μg/L) Precision (repeatability) RSDa (%) Blood Whole Blood L-1b 2.28 ± 0.16 6.86 Whole Blood L-2b 16.9 ± 0.37 2.17 Whole Blood L-3 35.8 ± 0.60 1.66 Urine Urine 40.6 ± 0.60 1.47 Urine L-2 40.1 ± 0.30 0.82 Hair Human Hair No. 13c (China)a 1803 ± 62.0d 3.44 Human Hair No. 13 (Japan) 4458 ± 89.4d 2.01 Biological material Reference material Concentration of Hg Mean ± SD (μg/L) Precision (repeatability) RSDa (%) Blood Whole Blood L-1b 2.28 ± 0.16 6.86 Whole Blood L-2b 16.9 ± 0.37 2.17 Whole Blood L-3 35.8 ± 0.60 1.66 Urine Urine 40.6 ± 0.60 1.47 Urine L-2 40.1 ± 0.30 0.82 Hair Human Hair No. 13c (China)a 1803 ± 62.0d 3.44 Human Hair No. 13 (Japan) 4458 ± 89.4d 2.01 aRSD, relative standard deviation. bValid until 2016. cThis standard (valid until 2008) of hair used for precision test only. dμg/kg dry weight. Table VI. Precision as intermediate precision (inter-day), n = 2, on 8 days Day of determination of Hg Concentration of Hg (mean ± SD, μg/L) Blood Urine Blood 1 (+2.5 ng Hg) Blood 2 (+10 ng Hg) Urine 1 (+2.0 ng Hg) Urine 2 (+6.0 ng Hg) 1 13.0 ± 0.11 53.7 ± 0.36 20.6 ± 0.02 67.2 ± 0.57 2 13.4 ± 0.63 52.2 ± 0.70 19.9 ± 0.29 65.8 ± 0.90 3 12.7 ± 0.42 52.2 ± 0.68 18.7 ± 0.11 65.6 ± 0.06 4 13.0 ± 0.26 51.4 ± 0.23 18.9 ± 0.41 63.2 ± 1.14 5 12.7 ± 0.20 52.0 ± 1.07 18.3 ± 0.41 64.6 ± 0.08 6 12.5 ± 0.20 51.2 ± 0.19 17.8 ± 0.44 62.7 ± 1.60 7 12.9 ± 0.87 50.7 ± 1.25 17.9 ± 0.26 61.1 ± 0.47 8 13.4 ± 0.18 54.6 ± 0.37 18.8 ± 0.58 62.7 ± 1.54 Total mean (μg/L) 13.0 52.3 18.9 64.1 SD (μg/L) 0.33 1.30 0.96 2.02 RSD (%) 2.51 2.50 5.10 3.16 Day of determination of Hg Concentration of Hg (mean ± SD, μg/L) Blood Urine Blood 1 (+2.5 ng Hg) Blood 2 (+10 ng Hg) Urine 1 (+2.0 ng Hg) Urine 2 (+6.0 ng Hg) 1 13.0 ± 0.11 53.7 ± 0.36 20.6 ± 0.02 67.2 ± 0.57 2 13.4 ± 0.63 52.2 ± 0.70 19.9 ± 0.29 65.8 ± 0.90 3 12.7 ± 0.42 52.2 ± 0.68 18.7 ± 0.11 65.6 ± 0.06 4 13.0 ± 0.26 51.4 ± 0.23 18.9 ± 0.41 63.2 ± 1.14 5 12.7 ± 0.20 52.0 ± 1.07 18.3 ± 0.41 64.6 ± 0.08 6 12.5 ± 0.20 51.2 ± 0.19 17.8 ± 0.44 62.7 ± 1.60 7 12.9 ± 0.87 50.7 ± 1.25 17.9 ± 0.26 61.1 ± 0.47 8 13.4 ± 0.18 54.6 ± 0.37 18.8 ± 0.58 62.7 ± 1.54 Total mean (μg/L) 13.0 52.3 18.9 64.1 SD (μg/L) 0.33 1.30 0.96 2.02 RSD (%) 2.51 2.50 5.10 3.16 Background levels: 0.5 μg/L Hg in blood, 0.2 μg/L Hg in urine. Table VI. Precision as intermediate precision (inter-day), n = 2, on 8 days Day of determination of Hg Concentration of Hg (mean ± SD, μg/L) Blood Urine Blood 1 (+2.5 ng Hg) Blood 2 (+10 ng Hg) Urine 1 (+2.0 ng Hg) Urine 2 (+6.0 ng Hg) 1 13.0 ± 0.11 53.7 ± 0.36 20.6 ± 0.02 67.2 ± 0.57 2 13.4 ± 0.63 52.2 ± 0.70 19.9 ± 0.29 65.8 ± 0.90 3 12.7 ± 0.42 52.2 ± 0.68 18.7 ± 0.11 65.6 ± 0.06 4 13.0 ± 0.26 51.4 ± 0.23 18.9 ± 0.41 63.2 ± 1.14 5 12.7 ± 0.20 52.0 ± 1.07 18.3 ± 0.41 64.6 ± 0.08 6 12.5 ± 0.20 51.2 ± 0.19 17.8 ± 0.44 62.7 ± 1.60 7 12.9 ± 0.87 50.7 ± 1.25 17.9 ± 0.26 61.1 ± 0.47 8 13.4 ± 0.18 54.6 ± 0.37 18.8 ± 0.58 62.7 ± 1.54 Total mean (μg/L) 13.0 52.3 18.9 64.1 SD (μg/L) 0.33 1.30 0.96 2.02 RSD (%) 2.51 2.50 5.10 3.16 Day of determination of Hg Concentration of Hg (mean ± SD, μg/L) Blood Urine Blood 1 (+2.5 ng Hg) Blood 2 (+10 ng Hg) Urine 1 (+2.0 ng Hg) Urine 2 (+6.0 ng Hg) 1 13.0 ± 0.11 53.7 ± 0.36 20.6 ± 0.02 67.2 ± 0.57 2 13.4 ± 0.63 52.2 ± 0.70 19.9 ± 0.29 65.8 ± 0.90 3 12.7 ± 0.42 52.2 ± 0.68 18.7 ± 0.11 65.6 ± 0.06 4 13.0 ± 0.26 51.4 ± 0.23 18.9 ± 0.41 63.2 ± 1.14 5 12.7 ± 0.20 52.0 ± 1.07 18.3 ± 0.41 64.6 ± 0.08 6 12.5 ± 0.20 51.2 ± 0.19 17.8 ± 0.44 62.7 ± 1.60 7 12.9 ± 0.87 50.7 ± 1.25 17.9 ± 0.26 61.1 ± 0.47 8 13.4 ± 0.18 54.6 ± 0.37 18.8 ± 0.58 62.7 ± 1.54 Total mean (μg/L) 13.0 52.3 18.9 64.1 SD (μg/L) 0.33 1.30 0.96 2.02 RSD (%) 2.51 2.50 5.10 3.16 Background levels: 0.5 μg/L Hg in blood, 0.2 μg/L Hg in urine. Generally, it was ascertained that precision in both these cases—as repeatability and intermediate precision—did not exceed 10%. Accuracy of determination (as relative error, presented in Table VII) did not exceed 5%. Recovery of Hg (added to a sample in trace amounts, e.g., 2.5 ng Hg) in blood was satisfactory—over 93% (Table VIII). Table VII. Accuracy of determination of mercury estimated on the basis of analysis of certified materials (blood, urine, n = 10; hair, n = 6) Biological material Reference material Concentration of Hg (μg/L) Relative error (%) Found Certified value Acceptable range Blood Whole Blood L-1a 1.55 ± 0.06 1.57 ± 0.32 1.25 ± 1.88 1.3 Whole Blood L-2a 16.8 ± 0.11 16.6 ± 3.3 13.3 ± 20.0 1.2 Whole Blood L-3 35.8 ± 0.60 37.1 ± 7.5 29.6–44.8 3.50 Urine Urine 40.6 ± 0.60 40.7 ± 2.3 36.1–45.3 0.25 Urine L-2 40.1 ± 0.30 39.8 ± 8.0 23.8–55.8 0.75 Hair Human Hair No. 13 (Japan) 4458 ± 89.4b 4420 ± 200b 4020–4820b 0.86 Biological material Reference material Concentration of Hg (μg/L) Relative error (%) Found Certified value Acceptable range Blood Whole Blood L-1a 1.55 ± 0.06 1.57 ± 0.32 1.25 ± 1.88 1.3 Whole Blood L-2a 16.8 ± 0.11 16.6 ± 3.3 13.3 ± 20.0 1.2 Whole Blood L-3 35.8 ± 0.60 37.1 ± 7.5 29.6–44.8 3.50 Urine Urine 40.6 ± 0.60 40.7 ± 2.3 36.1–45.3 0.25 Urine L-2 40.1 ± 0.30 39.8 ± 8.0 23.8–55.8 0.75 Hair Human Hair No. 13 (Japan) 4458 ± 89.4b 4420 ± 200b 4020–4820b 0.86 aStandard valid until 2022. bμg/kg dry weight. Table VII. Accuracy of determination of mercury estimated on the basis of analysis of certified materials (blood, urine, n = 10; hair, n = 6) Biological material Reference material Concentration of Hg (μg/L) Relative error (%) Found Certified value Acceptable range Blood Whole Blood L-1a 1.55 ± 0.06 1.57 ± 0.32 1.25 ± 1.88 1.3 Whole Blood L-2a 16.8 ± 0.11 16.6 ± 3.3 13.3 ± 20.0 1.2 Whole Blood L-3 35.8 ± 0.60 37.1 ± 7.5 29.6–44.8 3.50 Urine Urine 40.6 ± 0.60 40.7 ± 2.3 36.1–45.3 0.25 Urine L-2 40.1 ± 0.30 39.8 ± 8.0 23.8–55.8 0.75 Hair Human Hair No. 13 (Japan) 4458 ± 89.4b 4420 ± 200b 4020–4820b 0.86 Biological material Reference material Concentration of Hg (μg/L) Relative error (%) Found Certified value Acceptable range Blood Whole Blood L-1a 1.55 ± 0.06 1.57 ± 0.32 1.25 ± 1.88 1.3 Whole Blood L-2a 16.8 ± 0.11 16.6 ± 3.3 13.3 ± 20.0 1.2 Whole Blood L-3 35.8 ± 0.60 37.1 ± 7.5 29.6–44.8 3.50 Urine Urine 40.6 ± 0.60 40.7 ± 2.3 36.1–45.3 0.25 Urine L-2 40.1 ± 0.30 39.8 ± 8.0 23.8–55.8 0.75 Hair Human Hair No. 13 (Japan) 4458 ± 89.4b 4420 ± 200b 4020–4820b 0.86 aStandard valid until 2022. bμg/kg dry weight. Table VIII. Recovery of determination of mercury estimated on the basis of examination of recovery of analyte (2.5 ng Hg) added to blood samples (0.2 mL), n = 3, on 5 days Biological material Day of determination Amounts of Hg in a sample (ng) Mean ± SD Recovery (%) Blood (background value) Blood + 2.5 ng Hg Blood 1 0.26 ± 0.04 2.61 ± 0.00 94 2 2.68 ± 0.13 97 3 2.61 ± 0.05 94 4 2.68 ± 0.04 97 5 2.58 ± 0.17 93 Biological material Day of determination Amounts of Hg in a sample (ng) Mean ± SD Recovery (%) Blood (background value) Blood + 2.5 ng Hg Blood 1 0.26 ± 0.04 2.61 ± 0.00 94 2 2.68 ± 0.13 97 3 2.61 ± 0.05 94 4 2.68 ± 0.04 97 5 2.58 ± 0.17 93 Table VIII. Recovery of determination of mercury estimated on the basis of examination of recovery of analyte (2.5 ng Hg) added to blood samples (0.2 mL), n = 3, on 5 days Biological material Day of determination Amounts of Hg in a sample (ng) Mean ± SD Recovery (%) Blood (background value) Blood + 2.5 ng Hg Blood 1 0.26 ± 0.04 2.61 ± 0.00 94 2 2.68 ± 0.13 97 3 2.61 ± 0.05 94 4 2.68 ± 0.04 97 5 2.58 ± 0.17 93 Biological material Day of determination Amounts of Hg in a sample (ng) Mean ± SD Recovery (%) Blood (background value) Blood + 2.5 ng Hg Blood 1 0.26 ± 0.04 2.61 ± 0.00 94 2 2.68 ± 0.13 97 3 2.61 ± 0.05 94 4 2.68 ± 0.04 97 5 2.58 ± 0.17 93 The optimized and validated TDA AAS method was applied to estimation of levels of Hg in whole blood, urine, bile, vitreous humor and hair samples obtained from autopsy cases involving individuals who had not been exposed to mercury (without known environmental or occupational exposure to mercury). The results obtained are summarized in Table IX. The values can be considered as preliminary data obtained by the TDA AAS method. Table IX. Concentration of mercury in postmortem material from the adult non-exposed population (n = 3, each sample) Material Number of samples Concentration of Hg Mean ± SD (μg/L) Median (μg/L) Range (μg/L) Blood 24 3.72 ± 3.37 3.02 0.61–12.4 Urine 11 0.85 ± 0.63 0.81 0.16–2.19 Hair 11 0.272 ± 0.163a 0.215a 0.082–0.527a Vitreous humor 13 0.54 ± 0.26 0.47 0.22–1.01 Bile 11 3.21 ± 1.80 2.41 1.15–7.11 Material Number of samples Concentration of Hg Mean ± SD (μg/L) Median (μg/L) Range (μg/L) Blood 24 3.72 ± 3.37 3.02 0.61–12.4 Urine 11 0.85 ± 0.63 0.81 0.16–2.19 Hair 11 0.272 ± 0.163a 0.215a 0.082–0.527a Vitreous humor 13 0.54 ± 0.26 0.47 0.22–1.01 Bile 11 3.21 ± 1.80 2.41 1.15–7.11 aμg/g dry weight. Table IX. Concentration of mercury in postmortem material from the adult non-exposed population (n = 3, each sample) Material Number of samples Concentration of Hg Mean ± SD (μg/L) Median (μg/L) Range (μg/L) Blood 24 3.72 ± 3.37 3.02 0.61–12.4 Urine 11 0.85 ± 0.63 0.81 0.16–2.19 Hair 11 0.272 ± 0.163a 0.215a 0.082–0.527a Vitreous humor 13 0.54 ± 0.26 0.47 0.22–1.01 Bile 11 3.21 ± 1.80 2.41 1.15–7.11 Material Number of samples Concentration of Hg Mean ± SD (μg/L) Median (μg/L) Range (μg/L) Blood 24 3.72 ± 3.37 3.02 0.61–12.4 Urine 11 0.85 ± 0.63 0.81 0.16–2.19 Hair 11 0.272 ± 0.163a 0.215a 0.082–0.527a Vitreous humor 13 0.54 ± 0.26 0.47 0.22–1.01 Bile 11 3.21 ± 1.80 2.41 1.15–7.11 aμg/g dry weight. On the basis of the results of examination, it was ascertained that the arithmetic mean of the concentration of Hg in human blood of adults was 3.72 μg/L, median: 3.02 μg/L; and in urine: 0.85 μg/L, median: 0.81 μg/L. The concentrations of mercury in blood were similar to those measured using TDA AAS by Stube et al. (13) for the American population, which ranged from 0.74 to 14 μg/L, with a mean of 3.36 μg/L, but somewhat higher than the results obtained for blood in human autopsy material from the non-exposed adult Polish population by CV AAS (30): 1.6 ± 1.2 μg/L (mean), 1.4 μg/L (median). The concentrations of mercury in urine were generally lower than in blood. In the Polish population in 2004, the urine mercury levels (mean, median) could not be established by the use of CV AAS—it was only found that the concentrations of mercury in urine were below 2.6 μg/L (LOD was 1 μg/L). The content of mercury found in hair (mean: 0.272 ± 0.163 μg/g, median: 0.215 μg/g) was similar to the levels given in an international comparison published by Takagi et al. (31): 0.28 μg/g (mean for Polish people, data obtained using CV AAS). It is probable that the digestion process in a separate instrument does not affect the level of mercury in hair (concentrations are in μg/g, not in μg/L). The median of mercury in hair obtained for Polish people was lower than that in the hair of the Italian population examined by TDA AAS—0.475 μg/g (6). The mean levels of mercury in bile established with the new method are comparable to those in blood: 3.21 ± 1.80 μg/L; however, the median is a little lower: 2.41 μg/L. There is only a little information on hepatic biliary mercury concentrations (32–34), and there is none on vitreous humor concentrations in humans (33–35). Urinary concentrations of inorganic mercury are lower than hepatic biliary levels. Additionally, in general, mercury urine concentrations are lower, not only in our study, but also in other authors’ work (9, 10, 27, 30), independent on the technique used in examinations (ICP-MS, CV AAS, TDA AAS). What is important to stress is that the CV AAS technique requires preliminary treatment—a sample should be mineralized in a separate apparatus, and then transferred into an instrument to analyze. The TDA AAS is free from this stage. For this reason, the concentrations obtained by TDA AAS can be somewhat greater (less losses of analyte) than those obtained by CV AAS. The volume of blood or urine samples required for analysis by CV AAS using an AAS spectrometer is usually greater; for example, for MQZe Solaar Thermo, it is necessary to mineralize at least 3–10 mL of sample (blood, urine) before analysis. Analysis for mercury with the use of a TDA AAS direct analyser requires small samples of urine and blood (0.1–0.2 mL). Hair sample size subjected to the test for mercury is also small: maximum 0.05 g. Summarizing, the described method is selective, sensitive and precise enough to evaluate the concentration of mercury in different biological materials from non-exposed people (and cadavers), even when only small amounts of examined material are at the analyst’s disposal. Conclusion The TDA AAS technique requires little sample preparation, which is usually time-consuming in other methods. It is characterized by good analytical parameters. It is selective, sensitive, precise and reliable enough to evaluate the concentration of mercury in different biological materials of non-exposed people, such as blood, urine, hair, vitreous humor and bile. The method is suitable for the purposes of forensic toxicological analysis of autopsy material. Acknowledgments The authors would like thank to Dr hab. Tomasz Konopka from the Department of Forensic Medicine, Medical College, Jagiellonian University, Kraków, for providing samples of biological material used in this study. 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TI - Application of TDA AAS to Direct Mercury Determination in Postmortem Material in Forensic Toxicology Examinations
JF - Journal of Analytical Toxicology
DO - 10.1093/jat/bky107
DA - 2019-06-01
UR - https://www.deepdyve.com/lp/oxford-university-press/application-of-tda-aas-to-direct-mercury-determination-in-postmortem-6vZIb0yeN1
SP - 385
VL - 43
IS - 5
DP - DeepDyve
ER -