5.7 5.7 © Springer-Verlag Berlin Heidelberg 2005 II.5.7 p-Dichlorobenzene by Kanako Watanabe Introduction Nowadays, p-dichlorobenzene ( paradichlorobenzene, 1,4-dichlorobenzene) is becoming more popular than naphthalene as a moth repellant (insecticide) worldwide. e discrimination be- tween p-dichlorobenzene and naphthalene is usually di cult only by their smells and their appearances. However, speci c gravities of their crystals are di erent; they are 1.152 and 1.5 for naphthalene and p-dichlorobenzene, respectively. erefore, when the crystals sink in NaCl-saturated water, it is estimated to be p-dichlorobenzene; while when they oat in the same solution, it may be naphthalene [1]. Camphor also gives a similar aromatic smell and appearance, but its discrimination is even easier, because its speci c gravity is only 0.99; it oats in tap water. e weight of p-dichlorobenzene being sold as a moth repellant is about 4 g; the crystal mass of the same compound being used for a toilet mothball ranges from 40 to 200 g. erefore, when a large amount of this compound is ingested, it is dangerous for life. Like naphthalene, p-dichlorobenzene easily vaporizes from its solid into gas; in this chapter, a method for headspace GC/MS analysis of p-dichlorobenzene is presented utilizing the vola- tile property. Reagents and their preparation i. Reagents p-Dichlorobenzene and p-dichlorobenzene-d 4 (product No. 32,933-9) a can be purchased from Aldrich (Milwaukee, WI, USA). Other common chemicals used were of the highest purity commercially available. ii. Preparation p-Dichlorobenzene and p-dichlorobenzene-d 4 solutions: 2-mg aliquot each is dissolved in 1 mL methanol to prepare stock solutions. A 1-µL volume of each stock solution is injected into GC/MS to record each mass spectrum. For spiked tests, the above each stock solution is diluted 10-fold with methanol; a di erent volume of the p-dichlorobenzene solution (1–10 µL containing 0.2–2 µg) and 10 µL (contain- ing 2 µg) of p-dichlorobenzene-d 4 (IS) are placed in each glass vial with a Te on-septum cap containing 0.2 mL of blank whole blood. 444 p-Dichlorobenzene GC/MS conditions GC column b : an Rtx-1 fused silica medium-bore capillary column (30 m × 0.32 mm i. d., lm thickness 0.25 µm, Restek, Bellefonte, PA, USA). GC conditions; instrument: a GC-17A gas chromatograph (Shimadzu Corp., Kyoto, Japan); column (oven) temperature: 50 °C (1 min) → 10 °C/min → 150 °C → 20 °C/min → 280 °C; injection temperature: 250 °C; carrier gas: He; its 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-5050 quadrupole mass spectrometer c (con- nected with the above GC); ionization: positive ion EI; electron energy: 70 eV; emission cur- rent: 60 µA; ion source temperature: 280 °C; accelerating voltage: 1.5 kV. Procedure i. A 0.2-mL volume of a whole blood specimen d , 10 µL (containing 2 µg) of IS solution and 0.8 mL distilled water are placed in a 7-mL volume glass vial with a Te on-septum screw cap, capped and mixed gently. ii. e vial is heated at 80 °C for 30 min on a heat block or in a water bath. At the same time, the 5-mL volume syringe e is also heated on the block. e needle f size for the syringe is 23 G. A er heating, a 1-mL volume of the headspace vapor is withdrawn into the heated syringe, and carefully injected into GC/MS not to in uence the vacuum degree of the MS instrument. iii. Detection is made using ions at m/z 146 and at m/z 152 for IS in the SIM mode. iv. Construction of a calibration curve: to 0.2 mL each of blank whole blood (not less than 3 vials) obtained from healthy subjects, 2 µg of IS and a di erent amount of p-dichloroben- zene are added. e calibration curve consists of peak area ratio of p-dichlorobenzene to IS on the vertical axis and p-dichlorobenzene concentration on the horizontal axis. e peak area ratio obtained from a blood specimen is applied to the calibration curve to obtain its concentration g . It is essential that the concentration to be analyzed is within the concentra- tion range of the calibration curve. Assessment of the method > Figure 7.1 shows mass spectra of p-dichlorobenzene and IS. p-Dichlorobenzene contains two chlorine atoms, which give characteristic isotopic peaks at m/z M + 2 and M + 4. ere- fore, the molecular base peak of p-dichlorobenzene found at m/z 146 is not interfered with by any peak of other compounds, while that of p-dichlorobenzene-d 4 found at m/z 150 is inter- fered with by a minor peak of non-labelled p-dichlorobenzene ( > Figure 7.1). However, the relatively intense isotopic peak at m/z 152 appearing for p-dichlorobenzene-d 4 is usable, because it is not interfered with by any peak. us we have decided to use peaks at m/z 146 and 152 for p-dichlorobenzene and IS, respectively. > Figure 7.2 shows SIM chromatograms a er headspace extraction of whole blood, to which p-dichlorobenzene and IS had been added. e detection limit of p-dichlorobenzene in whole blood was about 50 ng/mL. 445p-Dichlorobenzene Mass spectra of p-dichlorobenzene and p-dichlorobenzene-d 4 (IS). ⊡ Figure 7.1 SIM chromatograms for p-dichlorobenzene and IS after their headspace extraction from a whole blood sample. The amounts of IS and p-dichlorobenzene spiked into 0.2 mL whole blood were 2 µg and 200 ng, respectively. ⊡ Figure 7.2 446 p-Dichlorobenzene e extraction e ciency of headspace extraction of p-dichlorobenzene was tested; 10–15 % of total compound spiked into blood was distributed in the gaseous phase in the vial under heating at 80 °C for 30 min. In this method, in spite of the use of a medium-bore capillary column in the splitless mode, as much as 1 mL gas is injected into GC/MS. Usually, such conditions cause remarkable broad- ening of a peak to be detected, but it was not the case for p-dichlorobenzene at 50 °C of the oven temperature ( > Figure 7.2). e author et al. examined various initial oven temperatures by using a trapping device (see Chapter 5 of general nature of this book); it was con rmed that an entire amount of p-dichlorobenzene contained in the headspace gas injected was completely trapped inside the column at 50 °C. Poisoning cases and fatal concentrations Case 1 [2]: a 74-year-old female ingested a mothball (p-dichlorobenzene ingested, 160–200 g) and fell into clouding of her consciousness and severe constipation. When an enema was given to her, strongly aromatic-smelled feces were excreted. Neither gastrolavage, intestinal lavage nor administration of an adsorbent was performed. On day 13, she died of dysfunctions of the liver and kidney. Case 2 [3]: a 85-year-old female ingested 40 pieces of Neoparasol ® (p-dichlorobenzene 140 g) due to senile dementia. At a clinic nearby, gastrolavage was performed, but she fell into respiratory suppression, bradycardia and a shock state, and was sent to a general hospital. She was treated with gastrolavage, purgative administration, enforced diuresis, oxygen inhalation and administration of Alotec ® (metaproterenol sulfate) and Inovan ® (dopamine hydrochlo- ride). On day 4, hemoperfusion was performed and she was discharged on day 34. Case 3 [4]: a 73-year-old male kept Neoparaace ® (p-dichlorobenzene) in a warm kotatsu (Japanese quilt-covered frame with a heat source inside) to dry it up for 3 days, because it had gotten wet with water. erefore, a strongly irritable smell took place inside the kotatsu and around it. When he was resting with his legs inside the kotatsu, he fell into dyspnea suddenly and was sent to a hospital. Upon his arrival, there was a mild clouding of his consciousness, but no orientation disturbance. He showed tachypnea, cyanosis in every part of his body, and crep- itations audible for both lungs by auscultation. PaO 2 was as low as 34.1 mmHg even under oxygen inhalation; PaCO 2 was 58.7 mmHg. Severe lung edema was observed for both lungs by X-ray photography. Under arti cial respiration, diuretic and steroid drugs were administered. By these treatments, his respiratory conditions were rapidly improved, and he could get out of the arti cial respiration 25 h a er the entrance into ICU. Although there were slight increases of liver transminases and high values of blood sedimentation and CRP, he was discharged without any severe sequela about 1 month a er admission. ere is no literature on toxic and fatal blood concentrations of p-dichlorobenzene. Human oral lethal dose is estimated to be 0.5–5 g/kg; the minimal lethal dose for adults estimated to be 25 g. ere was a case, in which a 2-month-old baby had died a er ingestion of 3–6 g of p-dichlorobenzene [3]. 447p-Dichlorobenzene Notes a) Stable-isotopic p-dichlorobenzene is commercially available from Aldrich. Its price is not expensive. b) Any type of non-polar dimethylsilicone capillary columns can be used, irrespective of their manufacturers. c) Any type of GC/MS, including sector, quadrupole and ion-trap types, can be used. d) Urine specimens seem also analyzable with the same procedure, although the author has not tested it yet. e) Either a usual 5-mL volume glass syringe or a gas-tight syringe can be used. f) e author et al. are using a special type of 23 G needles, which has a tip being cut coni- cally; this shape of the tip prevents the needle from being clogged by septum debris. g) e quantitation using a stable-isotopic IS is most desirable. When it is not available, the external calibration method can be used by spiking known amounts of p-dichlorobenzene into 0.2 mL each of blank whole blood, followed by treatments according to the same pro- cedure. References 1) Ukai T (ed) (1999) Manual of Treatments in Acute Poisoning. 3rd edn. Jiho Inc., Tokyo, pp 154–155 (in Japanese) 2) Japan Poison Information Center (1995) Poisoning data card No. 56, p-dichlorobenzene. Jpn J Toxicol 8:209– 210 (in Japanese) 3) Nishi K (ed) (1999) Emergency Manual for Poisoning. Iyaku Journal, Osaka, pp 246–247 (in Japanese) 4) Tamagawa R, Ito H, Kamihira A et al. (1995) A case of lung edema caused by inhalation of p-dichlorobenzene. Jpn J Toxicol 8:450–451 (in Japanese) . µL containing 0.2–2 µg) and 10 µL (contain- ing 2 µg) of p-dichlorobenzene-d 4 (IS) are placed in each glass vial with a Te on-septum cap containing 0.2. bradycardia and a shock state, and was sent to a general hospital. She was treated with gastrolavage, purgative administration, enforced diuresis, oxygen inhalation