Forensic Toxicol (2008) 26:23–26 DOI 10.1007/s11419-008-0043-0
SHORT COMMUNICATION
Determination of fluoride in human whole blood and urine by gas chromatography-mass spectrometry Shigetoshi Kage · Keiko Kudo · Naoki Nishida Hideaki Ikeda · Naofumi Yoshioka · Noriaki Ikeda
Received: 7 January 2008 / Accepted: 4 March 2008 / Published online: 29 April 2008 © Japanese Association of Forensic Toxicology and Springer 2008
Abstract We developed a simple and sensitive method for determination of fluoride in human whole blood and urine using gas chromatography-mass spectrometry (GC-MS). Fluoride was alkylated with pentafluorobenzyl bromide in a mixture of acetone and phosphate buffer (pH 6.8). The derivative obtained was analyzed by GC-MS in the positive-ion electron-impact mode. The lower limit of detection for the compound was 0.5 mg/l for both matrices. The calibration curve for fluoride was linear over the concentration range of 1– 100 mg/l. The precision and accuracy of the method were evaluated, and relative standard deviation was within 10%. Using this method, levels of fluoride in whole blood and urine were determined in a case of poisoning caused by hydrofluoric acid exposure. Keywords Fluoride · Hydrofluoric acid · Gas poisoning · GC-MS · Pentafluorobenzyl · Whole blood
S. Kage · H. Ikeda Forensic Science Laboratory, Fukuoka Prefectural Police Headquarters, Fukuoka, Japan K. Kudo · N. Ikeda (*) Department of Forensic Pathology and Sciences, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan e-mail:
[email protected] N. Nishida Department of Legal Medicine, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan
Introduction Fluoride in whole blood and urine is occasionally analyzed in cases of poisoning due to contact [1–5] with or ingestion [6–8] of hydrofluoric acid. Fluoride poisoning also occurs by taking inorganic fluoride salts during long-term hemodialysis [9] or in public water systems [10,11], by ingestion of roach powder containing fluoride [12], and by the excessive ingestion of fluoride in dental products [13,14]. Exposure to fluoridated hydrocarbons such as sarin [15] is another cause of fluoride poisoning, because fluoride is a metabolite of fluoridated hydrocarbons. Many methods have been reported for the determination of fluoride in blood and/or urine using a colorimetric method [16], an ion-selective electrode [17,18], ion chromatography [19], gas chromatography (GC) [15,20], and capillary electrophoresis [21,22]. However, because the majority of these methods detect fluoride only on the basis of its absorbance and/or retention time, they sometimes lack specificity. Gas chromatography-mass spectrometry (GC-MS) can identify drugs and poisons using both their retention times and mass spectra; therefore, it is very common in forensic toxicological examinations. However, no methods are reported to determine fluoride in biological materials by GC-MS. We have developed a simple procedure to determine formate, acetate [23], and bromide [24] in whole blood and urine as pentafluorobenzyl derivatives. In this report, we describe a simple GC-MS method to determine fluoride in whole blood and urine developed by improving the above derivatization techniques [23,24].
N. Yoshioka Division of Forensic Sciences, Department of Social Medicine, Akita University School of Medicine, Akita, Japan
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Materials and methods Materials A standard solution of fluoride (1000 mg/l) was purchased from Wako (Osaka, Japan), and was further diluted with distilled water to prepare a series of solutions in the concentration range of 1–200 mg/l. An internal standard (IS), chlorobenzene (CB) was also purchased from Wako. A solution of IS was prepared by dissolving CB in n-hexane to give a concentration of 0.1 mM. An alkylating agent, pentafluorobenzyl bromide (PFBBr, Aldrich, Milwaukee, WI, USA), was dissolved in acetone at a concentration of 300 mM. Other reagents used were of analytical grade. Blank whole blood and urine were collected from a healthy volunteer. Analytical procedure A 0.5-ml volume of 300 mM PFBBr solution in acetone was put into a 3-ml micro glass tube with 0.2 ml 0.5 M phosphate buffer (pH 6.8). Then, 0.1 ml of the sample solution, in the presence and absence of fluoride, was added and the mixture was vortexed for 1 min at room temperature. The mixture was heated at 80°C in a block heater for 60 min. After cooling the solution to room temperature, 1.0 ml of 0.1 mM CB solution in n-hexane was added. It was vortexed for 1 min at room temperature and centrifuged at 2500 rpm for 15 min. The organic phase was placed in another test tube and a 1.0-μl aliquot of the solution was injected into the GC-MS instrument.
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75, and 100 mg/l. These samples were derivatized and extracted as described above. Calibration curves were constructed by plotting the peak-area ratio of the molecular peak of the fluoride derivative at m/z 200 to the base peak of CB (IS) at m/z 112 against the concentration of fluoride using mass chromatography.
Results and discussion The mass spectrum of the fluoride derivative is shown in Fig. 1A. The molecular ion of the derivative appeared as the base peak at m/z 200, and the fragment ion peaks were observed at m/z 181 [M − F]+ and m/z 150. The molecular ion of IS also appeared as the base peak at m/z 112 (Fig. 1B). Mass chromatograms of the derivatized extracts from whole blood spiked with 10 mg/l fluoride are shown in Fig. 2. Sharp and symmetrical peaks of the fluoride derivative and IS were observed with retention times of 3.1 and 4.1 min, respectively. The calibration curves for fluoride were linear over the range from 1 to 100 mg/l in whole blood and urine. The equations for the curves were y = 0.0020x + 0.0117 (r = 0.999) for whole blood and y = 0.0018x + 0.0060 (r = 0.998) for urine. Relative recoveries of fluoride from whole blood and urine at 10, 50, and 100 mg/l were determined by comparing the peak-area ratios of the fluoride derivative to IS in the
GC-MS conditions The instrument used was an HP 5790A gas chromatograph (Agilent, Palo Alto, CA, USA) interfaced with a JEOL AX505A mass spectrometer (Tokyo, Japan). The column was a fused-silica DB-5 capillary (Agilent, 30 m × 0.32 mm i.d., 0.25 μm film thickness), and splitless injection mode was selected with a valve-off time of 1.5 min. The initial temperature of the column was held at 40°C for 5 min, and was programmed to rise to 220°C at 20°C/min. The injection port, separator, and ion source were kept at 220°, 200°, and 220°C, respectively. Helium was used as carrier gas at a flow rate of 2 ml/min. The ionization energy under positive-ion electron-impact ionization (EI) conditions was 70 eV. Scan mode was selected for the determination of fluoride. Preparation of calibration curves Fresh whole blood and urine samples were prepared to contain fluoride at concentrations of 1, 2.5, 5, 10, 25, 50,
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Fig. 1A, B Mass spectra of the derivative of fluoride (A) and chlorobenzene (internal standard, IS) (B)
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samples with those in the aqueous sample using mass chromatography; such values obtained from whole blood and urine were about 75% and 70%, respectively. The lower detection limit [signal-to-noise ratio (S/N) = 3] for fluoride in whole blood and urine was 0.5 mg/l. The lower quantification limit (S/N = 6) for fluoride in both matrices was 1 mg/l. Table 1 shows the validation data of the present method. The intraday and interday relative standard deviations were less than 10%, and the accuracy ranged from 98.6% to 109% for all spiked blood and urine samples.
Sakayanagi et al. [25] attempted to determine 10 inorganic anions, including fluoride, in beverages (not human samples) with pentafluorobenzyl p-toluenesulfonate (PFB-TsO) in the presence of a counter ion–crown ether complex by GC-MS. However, their method did not give enough sensitivity for fluoride with a detection limit of 4.75 mg/l. The present method showed sensitivity much higher than theirs [25]. PFBBr is much superior to PFBTsO as a fluoride alkylating agent. Our derivative of fluoride was stable for at least 1 month at room temperature. We applied this method to the blood and urine samples collected from a 59-year-old man, who had been exposed to 60% hydrofluoric acid due to the explosion of an absorption tank located outside a chemical plant. He had fallen into cardiopulmonary arrest about 30 min after the accident. He suffered burns to 30%–40% of his body due to exposure to the 60% hydrofluoric acid. The blood and urine levels of fluoride in the victim by this method were 47 mg/l and less than 1 mg/l, respectively. The blood fluoride concentration observed in this case was higher than the levels reported in similar skin burn cases (4.17 mg/l [1] and 15.2 mg/l [2]). The low level of fluoride in the urine sample in this case can be explained by the short time between the accident and death of the victim.
Conclusions Fig. 2 Mass chromatograms of the derivatized extract obtained from whole blood spiked with 10 mg/l fluoride. Numbers at the right of the chromatograms indicate the degree of magnification used to obtain similar-sized peaks in the chromatograms
In this study, we developed a simple and sensitive method for analysis of fluoride in human whole blood and urine by GC-MS, and applied it to samples obtained in an
Table 1 Precision and accuracy of the present determination of fluoride in human whole blood and urine by gas chromatography-mass spectrometry Sample
Added fluoride (mg/l)
n
Detected fluoride (mg/l) Mean
Blood Intraday
Interday
Urine Intraday
Interday
0 10 50 0 10 50
5 5 5 5 5 5
NQ 10.0 53.1 NQ 9.86 54.5
0 10 50 0 10 50
5 5 5 5 5 5
NQ 10.7 54.4 NQ 10.5 54.5
RSD (%)
Accuracy (%)
0.85 4.22
8.42 7.94
100 106
0.90 4.83
9.15 8.87
98.6 109
0.93 4.54
8.73 8.34
107 109
0.96 4.78
9.22 8.79
105 109
SD
SD, Standard deviation; RSD, relative standard deviation; NQ, not quantified
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actual case. Because this method gives specificity and reliability higher than those of the previously published methods, such as ion chromatography and gas chromatography, it is likely to be very useful in forensic, occupational, and clinical toxicology.
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