Cyanide and Thiocyanate in Human Saliva by Gas - Journal of ...

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CA) was 8-10 psi. The instrument was operated in splitless and temperature program modes. To avoid peak tailing, the purge valve was turned on at 0.50 rain after sample injection. The MSD was operated in electron impact mode at 70 eV. The detector was initially off and was turned on after a strong reagent peak of PFB-Br (approximately 3.0 rain). Preparation of standards and reagents Stock solutions of CN and SCN were prepared at a concentration of 10 mmol/L in water. Working solutions of CN/SCN at concentrations of 0, 25, 50, and 100 IJmol/L in water were used for specimen analysis. An SCN control (200 IJmol/L in water) was used to monitor artifact formation of CN. The phase-transfer catalyst (TBAS, 10 mmol/L) was prepared in a saturated solution of sodium borate (pH 9.2). Derivatizing agent (PFB-Br) and internal standard (2,5-DBT) were prepared in ethyl acetate at concentrations of 20 mmol/L and 50 ]Jmol/L, respectively. All solutions were stored at 2-5~ except the TBAS solution, which was left at room temperature. The reagents were stable for at least six months. For K6nig color reaction, 0.6 g of barbituric acid was dissolved in 10 mL of a mixture of pyridine and 1M hydrochloric acid (3:7, v/v). The solution had a short shelf-life and was prepared before the analysis. Preparation of saliva samples Ten unstimulated saliva (oral fluid) specimens were collected from three male (M1, M2, M3) and two female (F1, F2) nonsmoking subjects who expectorated directly into polypropylene collection tubes. The waiting time was at least a week when collected from the same subject. Approximately 3 mL of saliva was centrifuged at RCF 7300 xg for 30 rain. For analysis, 0.5 mL of the clear solution was diluted to 5 mL with water for a 10-fold dilution. Extractive alkylation procedure Solutions of 0.3 mL of PFB-Br (20 mmol/L), 0.2 mL of 2,5- DBT (50 lJmol/L), and 0.8 mL of TBAS (10 mmol/L) were added to 0.2 mL of standards (CN/SCN 0, 25, 50, 100 ]Jmol/L), control (SCN 200 IJmol/L), and saliva specimens in 5-mL glass centrifuge tubes. The tubes were capped, vortex mixed for 1 min, and heated at 55~ for 30 rain in a water bath. The solutions were centrifuged at RCF 1700 x g for 5 rain, and approximately 100 IJL of the clear ethyl acetate solutions was transferred into screw-capped injection vials. GC-MS analysis The oven temperature was increased from 60~ (held for 1.0 rain) to 90~ at 10~ and then to 150~ at 30~ Both injector and detector were set at 200~ Ions monitored were as follows: m/z 207, 188, and 157 for PFB-CN; m/z 239, 181, and 161 for PFB-SCN; m/z 250 and 169 for 2,5-DBT (IS). Electron multiplier was set 200 V above autotune. To enhance sensitivity, PFB-CN in one group and PFB-SCN and IS in another group were monitored. Approximately 1-3 IJL of the ethyl acetate solution was injected onto the instrument. The retention times for PFB-CN, PFB-SCN, and 2,5-DBT were 3.37, 5.16, and 5.41 rain, respectively. 512 Journal of Analytical Toxicology, Vol. 30, October 2006 K~nig color test for saliva specimens The procedure was the same as that used to test CN and SCN in blood with minor modifications (18). A solution of 0.8 mL of perchloric acid (0.33 tool/L) was added to 0.2 mL of standards (SCN 0, 25, 50, 100, 150 tJmol/L) and saliva specimens. After 2 rain of oxidation of SCN to CN, 0.2 mL of sodium acetate (1M) and 60 IJL of sodium hypochlorite (50 mmol/L) were added to the solutions. The so]ution was mixed, and within a minute, 0.2 mL of barbituric acid-pyridine reagent was added. After 5 rain, the absorbance was recorded at 583 nm. The instrument was set to zero using a water blank prior to analysis of specimens. Results and Discussion In extractive alkylation, CN and SCN in an aqueous solution reacted poorly with PFB-Br in ethyl acetate. But with the use of TBAS as a phase-transfer catalyst the reaction began im- mediately (CN ~ PFB-CN, SCN ~ PFB-SCN). After 30 min of reaction, the total ion chromatogram (TIC) areas were greater than 177,000 and 25,000 after an on-column injection of 5 picomol (0.29 ng) of SCN and 12.5 picomol (0.33 ng) of CN, respectively. In both cases the background was less than 1%. The reactions were studied over a period of 120 rain. When monitored by ion/IS ratios, the reactions were almost complete after 90 rain for both CN and SCN. Because of the excellent on- column sensitivity, we ran the reaction for only 30 rain. At this reaction time, the yield was 55-65% for both CN and SCN. In GC-MS analysis, three ions from PFB-CN in one group and Table I. Cyanide and Thiocyanate (pmol/L) in Ten Saliva Specimens Collected from Three Male (M) and Two Female (F) Nonsmoking Subjects* Colorimetric GC-MS Method Method (CN + SCN) CN/SCN (CN + SCN) Saliva CN SCN pmol/L % pmol/L M1-1 29 794 823 3.7 929 M1-2 11 442 453 2.5 555 M1-3 12 741 753 1.6 825 M1-4 8.2 1029 1037 0.8 1056 M1-5 5.7 595 601 1.0 634 M2-1 4.8 293 298 i .6 323 M3-1 8.8 463 472 1.9 494 F1-1 6.9 726 733 1.0 797 F1-2 15 771 786 1.9 815 F2-1 17 573 590 3.0 615 Min 4.8 293 298 0.8 323 Max 29 1029 1037 3.7 1056 Median 9.9 661 667 1.8 716 Average 11.8 643 655 1.9 704 Std 7.2 213 215 0.9 220 * Method comparison for (CN + SCN), intercept = -26.7, slope = 0.9673, r = 0.9882.
Journal of Analytical Toxicology, Vol. 30, October 2006 three ions from PFB-SCN and two ions from IS in another group were monitored. The identification of the compounds was based on comparing retention times ( 2%) and relative ion abundances ( 20%) with the reference compounds. Ex- cellent linearities were observed over the concentration range of i to 100 lamol/L (0.026-2.60 pg/mL) for CN and 5 to 200 lamol/L (0.29-11.6 lag/mL) for SCN. The intercept, slope, and correlation coefficient were -0.0073, 0.9933, and 0.9978 for CN and 0.0695, 0.0.9989, and 0.9996 for SCN, respectively. In- terrun variations of ion/IS ratios were less than 11% (N = 5). The procedure was used to examine 10 saliva specimens collected from 3 male (M) and 2 female (F) subjects. Saliva in our study is used synonymously with oral fluid to remain con- sistent with terminology in past publications. The specimens were initially centrifuged, and the clear solutions diluted 10- Table II. Literature Reported Thiocyanate Concentrations in Saliva from Nonsmokers SCN mmol/L Reference Methods Average -+ SD Range N 4 Colorimetric* 0.54 + 0.41 20 30 IC 0.51 + 0.31 10 31 FT-IR 0.83 + 0.42 0.41-1.25 25 32 CZE 0,23-1.20 48 Present method GC-MS 0.66 + 0.22 0,30-1.04 10 Present method Colorimetric 0.70 + 0.22 0.32-1,06 10 * For colorimetric method, the amounts represent (CN + SCN). l,ge+0 1.6e+0 1.4r 1.2e+0 lc+07 ~c+06 6c+06 4e+06 2e+05 ! 3+§ to.~9.~ / l+Fo 4,+ 1o. tlt~ +i .......... I ....... 9 . .~. +, ,.+..*++ . .... t t + . . . . . 72 ...... ~lxii t 1+4 n.,,r % 3.1s i';-/ '~s ' '3':C L~i 5.16 PFB-SCN 5.41 I5 3.37 PFB-CN + A 3.20 3.40 3.60 3.80 4.00 4.20 4,40 4.60 4.80 5.00 5.20 5.40 5.60 5,80 Time (min) Figure 1. Total ion chromatogram (TIC) and selected ion monitoring (SIM) of CN as PFB-CN (RT 3.374 rain, 5.7 +stool/L) and SCN as PFB-SCN (RT 5.16 rain, 595 tJmol/L) from a saliva specimen (M1-5). 2,5-Dibromotoluene was used as internal standard (IS, RT 5.41 rain). fold before testing. The results are recorded in Table I. The method was verified by testing the same specimens by a colorimetric method used to test SCN and CN in blood (18). In the procedure the SCN was oxidized to CN and tested by the Kgnig procedure. Therefore, the result indicated total of CN and SCN. The total CN and SCN values between the two methods compared favorably (r = 0.9882). In all cases, the values in GC-MS were less than those of the colorimetric method (intercept = -26.7 pmol/L). This difference may be due to an increase in background absorbance from the saliva ma- trix. The SCN concentrations found in our experiments are similar to those published in the literature (Table II). Consid- erable variation was observed in specimens collected from the same subject (M1, 442-1029 pmol/L). Generally, the amount of CN in all specimens was low (average 11.8 pmol/L) and varied in the range of 0.8-3.7% of SCN. The ion chromatogram of specimen M1-5 is presented in Figure 1. Linearity and precision of CN and SCN were verified in saliva. A sample (F2-1) was tested at 5, 10, 15, 20-fold dilutions. The corresponding final concentrations were 20.0, 16.5, 15.4, and 15 t~mol/L (average 16.7 _+ 14%) for CN and 600, 573, 597, and 607 lJmol/L (average 594 2.5%) for SCN. Two more specimens also gave similar results (M3-1, CN 7.9 + 11%, SCN 477 + 3.9%, and F1-2, CN 11.4 + 1.5%, SCN 827 ___ 9.2%). For precision, M1-5 was tested in five replicates. The average + CV% were 5.7 11.6% for CN and 595 4.3% for SCN. It ap- peared that salivary concentrations of CN and SCN determined by this method were linear and reproducible. Cyanide was reported as an artifact when SCN was derivatized under a basic condition (35). A basic condition was also used to determine CN and SCN in blood (25). In this method, SCN was derivatized in pres- ence of benzyldimethyltetradecylammonium chloride (BDM-TDA) as a phase-transfer catalyst. We studied the BDM-TDA method and monitored the artifact formation of CN from SCN. Solutions of SCN (0.8 mL, 25-200 pmol/L) were mixed with 0.3 mL of PFB-Br (20 retool/L), 0.2 mL of 2,5-DBT (IS, 50 1Jmol/L), and 0.8 mL of BDM-TDA (5 mmol/L in saturate sodium borate in water, pH 9.2) and heated at 55~ for 30 min. In the GC-MS chromatogram, a peak of CN was observed. The artifact CN concentrations were 7.2-9.2% of SCN (average 8.5 _+ 9%), compared to only 0-0.34% by the TBAS method. Moreover, when tested by the BDM-TDA method, the amount of CN increased with increasing time of reaction. No significant change was observed with the TBAS method. It indicated that the small amount of CN detected by the TBAS was not an artifact but an impurity present in the commercial SeN. Artifact formation of CN from SeN was also monitored in specimen analysis. When specimen F2-1 was tested by the BMD-TDA method, the CN and SeN concentrations were 86 and 514 pmol/L, respectively, compared to 17 and 573 pmol/L, 513
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