5.8 5.8 © Springer-Verlag Berlin Heidelberg 2005 II.5.8 Ethylene glycol by Kanako Watanabe Introduction Ethylene glycol is being widely used as solvents, lubricants and surfactants in industries; for daily necessities, it is being used as a component of cold-reserving materials and of nonfreezing coolants of automobiles.  ere were cases of accidental and suicidal ingestion of such  uids [1–3]. As toxic actions of ethylene glycol, suppression of central nervous system activities similar to that of alcohol and metabolic acidosis caused by glycolic acid produced from ethylene glycol can be mentioned.  e glycolic acid is further metabolized into oxalic acid, which is bound with calcium ions to form the insoluble salt; the salt precipitates in various tissues. Especially, such precipitation sometimes causes renal dysfunction (failure) [4]. For analysis of ethylene glycol, methods by GC [5–8], GC/MS [9–11] and HPLC [12–14] were reported. In this chapter, a method of GC/MS analysis of ethylene glycol in human plas- ma is presented. Reagents and their preparation i. Reagents Ethylene glycol and phenylboronic acid can be purchased from Sigma (St. Louis, MO, USA) and other manufacturers; ethylene glycol-d 4 from Aldrich (Milwaukee, WI, USA, No. 34,744-2) a . Other common chemicals used were of the highest purity commercially available. ii. Preparation • A er sampling venous blood with the vacuum tubes containing an anticoagulant, they are centrifuged at 3,000 rpm for 5 min to obtain blood plasma b . • Ethylene glycol-d 4 (IS) solution: a 8-µL aliquot of ethylene glycol-d 4 (speci c gravity, 1.189) is dissolved in 50 mL acetonitrile. • Ethylene glycol standard solution: a 9-µL of ethylene glycol (speci c gravity, 1.135) is dis- solved in 1 mL acetonitrile (100 µg/10 µL). • Derivatization solution: a 11.8-mg aliquot of phenylboronic acid is dissolved in 1 mL ace- tonitrile. GC/MS conditions GC column c : an Rtx-17 fused silica medium-bore capillary column (30 m × 0.32 mm i. d.,  lm thickness 0.25 µm, Restek, Bellefonte, PA, USA). GC conditions: a GC-17A gas chromatograph d (Shimadzu Corp., Kyoto, Japan); column (oven) temperature: 70 °C (1 min) → 10 °C/min → 120 °C → 5 °C/min → 150 °C → 20 °C/min → 450 Ethylene glycol 280 °C; injection temperature: 250 °C; carrier gas: He;  ow rate: 3 mL/min; injection mode: splitless for 1 min a er injection, followed by the split mode. MS conditions; instrument: a Shimadzu QP-5050A quadrupole mass spectrometer d (com- bined with the above GC); ionization: positive ion EI; electron energy: 70 eV; emission current: 60 µA; ion source temperature: 280 °C; accelerating voltage: 1.5 kV. Procedure i. A 0.5-mL volume of a plasma specimen e and 0.5 mL of ethylene glycol-d 4 acetonitrile solu- tion (100 µg IS) are placed in a small test tube, sealed with Para lm, vortex-mixed for 5 min and centrifuged at 3,000 rpm for 5 min. ii. A 0.5-mL volume of the supernatant solution is mixed well with 125 µL of the phenylboronic acid derivatization solution and le at room temperature for 15 min for complete reaction. iii. To the above solution, 1 g anhydrous sodium sulfate is added, vortex-mixed and le for several minutes for dehydration. iv.  e above supernatant solution is transferred to a 4-mL volume glass vial with a screw cap using a Pasteur pipette and evaporated to dryness under a stream of nitrogen.  e residue is dissolved in 100 µL acetonitrile. A 1-µL of it is injected into GC/MS for measurements in the SIM mode using ions at m/z 148 and 152. v. Construction of a calibration curve: to 0.5 mL each of blank plasma (not less than 3 vials) obtained from healthy subjects, 0.5 mL of ethylene glycol-d 4 (IS) solution and a di erent amount (1–50 µL) of the ethylene glycol standard solution are added.  e mixture is treat- ed according to the above procedure.  e calibration curve consists of peak area ratio of ethylene glycol to IS on the vertical axis, and ethylene glycol concentration on the horizon- tal axis.  e peak area ratio obtained from a plasma specimen is applied to the calibration curve to obtain an ethylene glycol concentration f . Assessment of the method > Figure 8.1 shows mass spectra of the derivatives of ethylene glycol and IS. Although many fragment ions appeared, the molecular ions were relatively intense to be used for SIM.  e peaks at m/z 148 and 152 were not interfered with by each other. > Figure 8.2 shows SIM chromatograms for the derivatives of ethylene glycol and IS, which had been spiked into blood plasma.  e detection limit of ethylene glycol obtained by this method was about 1 µg/mL in blood plasma. Poisoning case, and toxic and fatal concentrations A 20-year-old male [2] attempted suicide by ingesting a large amount of a nonfreezing coolant at about noon, vomited at 1:00 p. m. and was sent to a critical care medical center at 2:25 a. m. on the next day by an ambulance car. Upon arrival at the center, he showed the respiration rate at 40/min, consciousness level at 200 (Japan Coma Scale), macroscopic hematuria, whitish turbid urine suggesting calcium oxalate crystals, severe metabolic acidosis, anion gap of 451Ethylene glycol Mass spectra of the derivatives of ethylene glycol and ethylene glycol-d 4 (IS). ⊡ Figure 8.1 SIM chromatograms for the derivatives of ethylene glycol and IS, which had been spiked into human plasma. The amounts of ethylene glycol and IS spiked into 0.5 mL plasma were 25 and 100 µg, respectively. ⊡ Figure 8.2 452 Ethylene glycol 38.3 mEq/L, and hypocalcinemia (7.0 mg/dL).  e treatments with hemodialysis and ethanol administration were started. A er the  rst hemodialysis, his metabolic acidosis and conscious- ness level were improved; he could answer that he had ingested about a quarter of 2 L of the coolant solution at about noon. Since general tonic convulsion appeared in the morning of the 2nd day of admission, an arti cial respiratory control was achieved until the 6th day. Hemo- dialysis was continued until the 10th day to cope with the acute renal dysfunction; he could get out of the hemodialysis system on day 15, and could move to a general ward on day 17. Although diplopia caused by central gaze palsy remained, he was discharged on day 34, be- cause of general improvement.  e blood ethylene glycol concentrations, at which toxic symptoms appear, are about 500 µg/mL; the oral fatal doses for adults were estimated to be about 100 mL [15]. Notes a) Stable-isotopic ethylene glycol is commercially available from Aldrich. Its price is not ex- pensive. b) In these experiments, the author tried to use whole blood, but it was not successful, because erythrocytes aggregated to form masses. It was essential to separate plasma to achieve e - cient extraction of ethylene glycol. c) Any type of intermediately polar 50 % phenylsilicone/50 % dimethylsilicone capillary columns can be used, irrespective of their manufacturers. d) Any type of GC/MS instruments of the sector, quadrupole and ion-trap types can be used. e) Urine seems also analyzable with the same procedure. f) It is possible to make quantitative analysis without IS using an external calibration method. A 0.5-mL volume of a blood plasma specimen is mixed well with 0.5 mL acetonitrile for deproteinization, followed by the same procedure as described above.  e analysis of ethylene glycol by GC-FID is also possible according to the same procedure, because of the high contents of the compound in blood. References 1) Iguchi Y, Miyakaya Y, Ikuoka T et al. (1994) A case of ethylene glycol poisoning, in which urine precipitates were useful for its diagnosis. Jpn J Toxicol 7:430 (in Japanese) 2) Kirita A, Hagiwara T, Yoshikawa O (1997) A case of acute ethylene glycol (non-freezing coolant) poisoning. Jpn J Toxicol 10:220 (in Japanese) 3) Gotoh H, Miyakaya Y, Ota S et al. (1999) Two cases of ethylene glycol poisoning. Jpn J Toxicol 12:469 (in Japanese) 4) Naito H (2001) Poisoning of Industrial Products, Gases, Pesticides, Drugs, and Natural toxins – Cases, Pathoge- nesis and its Treatment – 2nd edn. Nankodo Co., Ltd., Tokyo, pp 48–49 (in Japanese) 5) Houze P, Chaussard J, Harry P et al. (1993) Simultaneous determination of ethylene glycol, propylene glycol, 1,3-butylene glycol and 2,3-butylene glycol in human serum and urine by wide-bore column gas chromatogra- phy. J Chromatogr 619:251–257 6) Aarstad K, Dale O, Aakervik O et al. (1993) A rapid gas chromatographic method for determination of ethylene glycol in serum and urine. J Anal Toxicol 17:218–221 7) Livesey JF, Perkins SL, Tokessy NE et al. (1995) Simultaneous determination of alcohols and ethylene glycol in serum by packed-or capillary-column gas chromatography. Clin Chem 41:300–305 453Ethylene glycol 8) Yao HH, Porter WH (1996) Simultaneous determination of ethylene glycol and its major toxic metabolite, glycolic acid, in serum by gas chromatography. Clin Chem 42:292–297 9) Maurer H, Kessler C (1998) Identification and quantification of ethylene glycol and diethylene glycol in plasma using gas chromatography-mass spectrometry. Arch Toxicol 62:66–69 10) Dasgupta A, Macauley R (1995) A novel derivatization of ethylene glycol from human serum using 4-carbetho- xyhexafluorobutyryl chloride for unambiguous gas chromatography-chemical ionization mass spectrometric identification and quantification. Am J Clin Pathol 104:283–288 11) Dasgupta A, Blackwell W, Griego J et al. (1995) Gas chromatographic-mass spectrometric identification and quantitation of ethylene glycol in serum after derivatization with perfluorooctanoyl chloride: a novel deriva- tive. J Chromatogr B 666:63–70 12) Gupta RN, Eng F, Gupta ML (1982) Liquid-chromatographic determination of ethylene glycol in plasma. Clin Chem 28:32–33 13) Wu NM, Malinin TI (1987) High performance liquid chromatography determination of ethylene glycol and ethylene chlorohydrin in tissues. J Anal Toxicol 11:63–66 14) Vollmer PA, Harty DC, Erickson NB et al. (1996) Serum ethylene glycol by high-performance liquid chromato- graphy. Chromatogr B 685:370–374 15) Ukai T (ed) (1999) Manual of Treatments in Acute Poisoning, 3rd edn. Jiho Inc., Tokyo, pp 232–233 (in Japanese) . was about 1 µg/mL in blood plasma. Poisoning case, and toxic and fatal concentrations A 20-year-old male [2] attempted suicide by ingesting a large amount. lubricants and surfactants in industries; for daily necessities, it is being used as a component of cold-reserving materials and of nonfreezing coolants of automobiles.