**2.2 Reagents and standards**

All chemicals used were of analytical reagent grade unless stated otherwise. Tetramethylammonium hydroxide (TMAH, 25% w/w in water) and ethylmercury chloride were purchased from Alfa Aesar (UK). Methanol, sodium acetate, and acetic acid glacial (super grade) were purchased from VWR (BDH, UK). Sodium tera-npropylborate (NaBpR4, ≥98% purity) was purchased from Chemos GmbH (Germany). 2, 2, 4 trimethylpentane (isooctane, spectrophotometric grade, ≥99% purity) and methylmercury (II) chloride were purchased from Sigma Aldrich (UK). Inorganic mercury (In-Hg) standard solution for ICP (934 3.0 mg/kg) was purchased from Fluka (UK).

The derivatization solution was prepared by dissolving 1 g of sodium tetrapropylborate (NaBpR4) in 100 ml of deionized water. The solution was stored at 20°C in a refrigerator and protected from light. Buffer acetate (0.1 M) in deionized water was prepared by mixing 0.1 M sodium acetate solution (90 ml) with 0.1 M acetic acid


### **Table 2.**

*GC and ICP-MS operating parameters.*

*A Study on the Methyl and Ethylmercury Artifacts in Biological Samples Using Sodium… DOI: http://dx.doi.org/10.5772/intechopen.110050*

solution (410 ml) and adjusted the final volume to 1000 ml (1 L) with deionized water and adjusting to pH 3.9. Inorganic mercury working standard solutions (1.0 and 10 mg/kg as Hg) were prepared from appropriate dilution of inorganic mercury standard stock solution (934 � 3.0 mg/kg). Enriched inorganic mercury (1.0 mg/kg 199In-Hg199 as Hg) and enriched methylmercury (1.0 mg/kg 201MeHg as Hg) working solutions were prepared from appropriate dilution of their standard stock solutions. Milli-Q quality water (Millipore) was used throughout.

### **2.3 Derivatization by sodium tetrapropylborate and analytical procedures**

Blank (TMAH) and hair samples with a low level (0.98 mg/kg) of In-Hg and one hair sample from ICL with a high level of In-Hg (1000 mg/kg) were spiked with the same amount of 201MeHg and 199InHg (double spike, 70 μl from 1.0 ppm of each enriched Hg standard). The spiked and un-spiked hair samples were digested using a microwave device. The spiked sample solution (blank and digested spiked hairs) and un-spiked hair solution samples in cleaned and dried glass vials (1 ml of each) were then adjusted to pH 3.9 with acetate buffer, and then, 1 ml of 1% NaBPr4 was added in the glass vials for derivatization to form the corresponding peralkylated mercury (Hg) species such as.

$$\text{Hg}^{2+} + 2\text{NaB} \left( \text{C}\_{3}\text{H}\_{5} \right)\_{4} \rightarrow \text{Hg} \left( \text{C}\_{3}\text{H}\_{5} \right)\_{2} + 2\text{Na}^{+} + 2\text{B} \left( \text{C}\_{3}\text{H}\_{5} \right)\_{3} \tag{3}$$

$$\text{CH}\_3\text{Hg}^+ + \text{NaB} \left( \text{C}\_3\text{H}\_5 \right)\_4 \rightarrow \text{CH}\_2\text{HgC}\_3\text{H}\_5 + \text{Na}^+ + \text{B} \left( \text{C}\_3\text{H}\_5 \right)\_3 \tag{4}$$

Extraction of derivatized Hg species (peralkylated Hg) was done by vigorous shaking for 5 min with 1 ml isooctane, the isooctane extract was afterwards centrifuged for 10 min at 3000 rpm, and then, the extracted Hg species into isooctane layer were transferred to GC vials and analyzed with a coupling of GC-ICP-MS. In addition, the conc. Normal abundance In-Hg, conc. Enriched In-Hg199, and hair sample from chlor alkali plant with similar Hg conc. to both In-Hg conc. (about 1000 mg/ kg) and different concentrations of normal abundance In-Hg standard solutions (20, 40, 60, 80, and 100 mg/kg) were derivatized and extracted as described above. The samples were diluted after derivatization and extraction steps (D.F 1:10 for the concentrated Hg standards and chlor alkali hair sample).

### **3. Results and discussion**

### **3.1 Hg isotope ratio calculation for spiked and un-spiked samples**

The mercury isotope ratios (IR) were calculated for four measured Hg isotopes (199, 200, 201, 202) in spiked, un-spiked blank (TMAH), hair samples, and Hg standard solution (normal abundance In-Hg and enriched 199In-Hg) to compare the formation of mercury artifact in spiked and un-spiked samples during propylation with NaBPr4. The artifact formation of monomethy lmercury (Me-Hg) and ethylmercury (EtHg) from inorganic mercury (In-Hg) was observed during propylation with NaBPr4 for spiked blank with enriched 199InHg and enriched 199In-Hg standard solution comparing with un-spiked blank (**Figures 1** and **2**). The extent of artifact formation for the organomercury compounds was in the order: Et-Hg > - Me-Hg > Hg(0). Moreover, the artifact formation of monomethyl–ethylmercury

**Figure 1.** *Typical chromatogram of un-spiked blank (1.0 ml TMAH) obtained during derivatization using NaBPr4.*

### **Figure 2.**

*Typical chromatogram of spiked blank (TMAH) with mixed enriched 201MeHg & 199In-Hg standard solutions obtained during derivatization using NaBPr4.*

(MeEt-Hg) from methylmercury was observed also during propylation with NaBPr4 for **a** spiked blank (TMAH) with mixed enriched mercury standards (201Me-Hg and 199In-Hg) as shown in **Figure 2**.

For mercury isotope ratio (IR) calculation results for un-spiked blank (TMAH) compared with spiked blank with the same amount of mixed enriched mercury standards (201MeHg and 199InHg, 70 μl from 1.0 ppm of each into 1 ml of TMAH) after *A Study on the Methyl and Ethylmercury Artifacts in Biological Samples Using Sodium… DOI: http://dx.doi.org/10.5772/intechopen.110050*

### **Figure 3.**

*Compression of calculated mercury isotope ratios (Hg-IR) in un- spiked blank with spiked blank with mixed enriched 201MeHg & 199In-Hg standard solutions during the derivatization using NaBPr4.*

propylation with NaBPr4 as shown in **Figure 3**. It can be seen that the signal in each of the mercury isotopic ratios (Hg199/200, Hg199/201, Hg199/202, Hg200/201, Hg200/ 202, and Hg201/200) for spiked blank is increased in the order: In-Hg > Et-Hg > - MeEt-Hg > Me-Hg, but for un-spiked blank was observed only the similar ratio for all Hg isotope ratios for In-Hg only. This means that the artifact formation of an organomercury compound is increased with increasing amounts of inorganic mercury (InHg) when spiked the blank with enriched mercury standards (201Me-Hg and 199In-Hg) and propylated with NaBPr4. However, this is indicating that the artifact formation of MeHg and EtHg from a high concentration of inorganic mercury is caused by NaBPr4.

In addition, it can be seen from **Figure 4** that the results of IR calculation for spiked blank (TMAH) with enriched 199InHg are similar to those calculated in

### **Figure 4.**

*Compression of calculated mercury isotope ratios (Hg-IR) in spiked blank with enriched 199InHg spiked blank versus enriched 199In-Hg standard solution during derivatization using NaBPr4.*

**Figure 5.**

*Typical chromatogram of un- spiked chlor alkali hair sample (CA hair sample with T-Hg conc. = 1000 mg/kg) obtained during derivatization using NaBPr4.*

enriched 199InHg standard solution after propylation by NaBPr4. Moreover, it can be deduced from IR calculations that MeHg and EtHg artifact creation originate exclusively from InHg, but MeEtHg artefact formation is not detected. This indicates that MeEtHg artefacts only originate from enriched 201MeHg when the blank or sample is spiked with high levels of enriched 201MeHg (greater than 50 l of 1.0 ppm to 1.0 ml of blank or 0.01.

For un-spiked hair samples from one chlor alkali plant with a high concentration of inorganic mercury (In-Hg) and normal abundance inorganic mercury (In-Hg) with similar Hg concentration to those found in selected chlor alkali plants hair sample, the mercury isotope ratios (IR) were calculated for four measured Hg isotopes (199, 200, 201, and 202) same as in spiked, un-spiked blank (TMAH), and enriched 199InHg. The observation of artifact formation of methylmercury (MeHg) and ethylmercury (EtHg) from inorganic mercury (InHg) was similar to those found in the spiked blank with enriched 199InHg and enriched 199InHg standard solution during propylation with NaBPr4 (**Figures 5** and **6**). However, as shown in **Figures 5** and **6**, the transformation of MeHg and EtHg from In-Hg in both chlor alkali hair and normal abundance InHg is similar and the extent of artifact formation for the organomercury compounds was in the order: EtHg>MeHg>Hg(0).

Mercury isotope ratio (IR) calculation results for un-spiked chlor alkali hair compared with spiked same hair sample with mixed enriched mercury standards ( 201MeHg and 199InHg, 70 ul from 1.0 ppm of each into 1 ml of TMAH) and normal abundance InHg after propylation with NaBPr4 as shown in **Figures 7** and **8** below showed similar amounts of all mercury isotope ratio (IR) in both spiked and un-spiked hair samples. This indicates that the methylmercury (MeHg) and ethylmercury (EtHg) compounds are artifacts of high amounts of inorganic mercury in hair samples owing to the propylation with NaBPr4 and are not reliant on the spiking quantity of

*A Study on the Methyl and Ethylmercury Artifacts in Biological Samples Using Sodium… DOI: http://dx.doi.org/10.5772/intechopen.110050*

**Figure 6.**

*Typical chromatogram of normal abundance In-Hg (1000 mg/kg as Hg2+) obtained during derivatization using NaBPr4.*

enhanced mercury standards, as well as depending on the purity of the propylation reagent.

Moreover, when Hg isotope ratios (IR) calculating results in spiked normal hair the sample (control hair sample) is compared to spiked chlor alkali hair (both hair samples were spiked with the same quantity of mixed enriched 201MeHg and 199InHg standard solutions), as shown in **Figure 9** below, it can be noted that the Hg isotope ratio calculation findings are identical in both spiked hair samples except for In-Hg isotope ratios (IR).

### **Figure 7.**

*Compression of calculated mercury isotope ratios (Hg-IR) in un- spiked CA hair spiked CA hair mixed enriched 201MeHg & 199In-Hg standards during derivatization using NaBPr4.*

### **Figure 8.**

*Compression of calculated mercury isotope ratios (Hg-IR) in un- spiked CA hair verses similar In-Hg concentration of normal abundance In-Hg standard solution during derivatization using NaBPr4.*

**Figure 9.**

*Comparing the compression of (Hg-IR) in spiked normal hair (N-hair) with mixed enriched 199In-Hg and 201MeHg standards to spiked CA-hair mixed standard solutions utilizing NaBPr4.*
