Drugs and Poisons in Humans - A Handbook of Practical Analysis (Part 63)

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Drugs and Poisons in Humans - A Handbook of Practical Analysis (Part 63)

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Introduction: Cresol is being used for an antiseptic, disinfectant, maggot-killing agent and cresol soap solution. Since various kinds of more powerful and odorless disinfectants have nowadays become available in practical use, the frequency in the use of cresol seems decreasing. However, the cases of acute poisoning by cresol are still being reported at the present time. The toxic effects of cresol are due to its corrosive actions, resulting in the destruction of cell membranes and coagulation of proteins, and its suppressive action on the central nervous system [1]. There are three isomeric forms of cresol, vis., o-, m- and p-cresols;...

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  1. 7.6 II.7.6 Cresol by Chiaki Fuke Introduction Cresol is being used for an antiseptic, disinfectant, maggot-killing agent and cresol soap solu- tion. Since various kinds of more powerful and odorless disinfectants have nowadays become available in practical use, the frequency in the use of cresol seems decreasing. However, the cases of acute poisoning by cresol are still being reported at the present time. The toxic effects of cresol are due to its corrosive actions, resulting in the destruction of cell membranes and coagulation of proteins, and its suppressive action on the central nervous sys- tem [1]. There are three isomeric forms of cresol, vis., o-, m- and p-cresols; the toxicity of each isomer is somewhat different [2]. The composition ratios of cresol isomers are different accord- ing to cresol-containing products; it, therefore, seems very important to measure the concentra- tions of each isomer of cresol to identify a causative cresol product used in its poisoning case. Cresol, after being absorbed into human bodies, is metabolized into glucuronide- and/or sulfate-conjugated forms and excreted into urine. The half-life of unchanged cresol in blood is as short as about 1.5 h [3]; this means that it becomes undetectable several hours after emer- gency treatments. However, the metabolites (conjugated forms) remain in the body for rela- tively a long time [4–6]; the detection of the conjugated form(s) sometimes becomes necessary. As methods for analysis of cresol, GC [4, 7–9], HPLC [5, 6, 10–14] and capillary electro- phoreisis [15] were reported. In this chapter, procedures for HPLC and GC/MS analysis of cresol isomers and their conjugates are presented. HPLC analysis Reagents and their preparation • A 10-mg aliquot each of o-, m- and p-cresols (Aldrich, Milwaukee, WI, USA and other manufacturers) is dissolved in 10 mL methanol separately (1 mg/mL). • A 10-mg aliquot of 4-ethylphenola (internal standard, IS, Aldrich and other manufacturers) is dissolved in 10 mL methanol (1 mg/mL). • β-Glucuronidase: 10 mg of bovine liver glucuronidase (EC 3.2.1.31, type B-10, 11,000 units/mg solid, Sigma, St. Louis, MO, USA) is dissolved in 1 mL distilled water. • Sulfatase: Aerobacter aerogenes sulfatase (EC 3.1.6.1, 19 units/mL, Sigma). HPLC conditions Instruments; pump: LC-10A; detectors: SPD-10A and RF-10A (all from Shimadzu Corp., Kyo- to, Japan); column: a Nova-Pak C18 stainless cartridge column (150 × 3.9 mm i.d., particle size © Springer-Verlag Berlin Heidelberg 2005
  2. 582 Cresol 4 µm, Waters, Milford, MA, USA); guard column: Guard-Pak Nova-Pak C18 (Waters); mobile phaseb: acetonitrile/20 mM potassium dihydrogenphosphate buffer solution (pH 3.0, to be ad- justed with phosphoric acid) (1:4, v/v), containing 20 mM β-cyclodextrin (Sigma and other manufacturers); its flow rate: 1.0 mL/min; detection wavelength: 270 nm for the UV detector; fluorescence detector: Ex 270 nm and Em 305 nm; injection volume: 20 µL. Procedures i. Analysis of unconjugated forms i. A 100-µL volume of a specimenc is mixed with 10 µL of IS solution. ii. A 100-µL volume of acetonitrile is added to the above mixture with stirringd. iii. It is centrifuged at 12,000 g for 10 min. iv. A 20-µL aliquot of the supernatant solution is injected into HPLC. v. Various concentrations (not less than 4 plots) of a cresol isomer plus 10 µL of IS solution are added to blank specimens and processed in the same way to construct a calibration curve. The concentration of a cresol isomer in a test specimen is calculated with the curve. ii. Analysis of the glucuronide-conjugated forms i. A 100-µL volume of a specimenc is mixed with 10 µL of IS solution. ii. A 5-µL volume of 4 M sodium acetate buffer solution (pH 5.0) and 5 µL of β-glucuroni- dase solution are added to the above mixture and incubated at 37 °C for 2 h. iii. After cooling to room temperature, 100 µL acetonitrile is placed in the above mixture with stirring. iv. The following procedure is achieved according to the iii–v steps of the above section. iii. Analysis of the sulfate-conjugated formse i. A 100-µL volume of a specimenc is mixed with 10 µL of the IS solution. ii. A 5-µL volume of 2.5 M Tris-HCl buffer solution (pH 7.5) and 5 µL of sulfatase are added to the above mixture and incubated at 37 °C for 2 h. iii. After cooling to room temperature, 100 µL acetonitrile is added to the above mixture with stirring. iv. The following procedure is achieved according to the iii–v steps of the above section for ana- lysis of unconjugated forms. Assessment of the method In this method, the pretreatment procedures are very simple and thus enable rapid analysis of cresol isomers and their conjugates. It does not include no condensation step; it means that there is no concern about low recovery rates due to loss of a test compound caused by evapora- tion. However, a great difference in composition ratio of acetonitrile in the supernatant solu- tion may affect the peak area ratio of cresol to IS; it is preferable to fix the composition ratio of acetonitrile before injection into HPLC. > Figure 6.1 shows HPLC chromatograms for the authentic cresol isomers and related compounds and for extracts of plasma or urine of a poisoning case. The cresol isomers are
  3. HPLC analysis 583 ⊡ Figure 6.1 HPLC chromatograms for the authentic standard cresol isomers and related compounds (10 µg/mL each) and for extracts of plasma and urine of a poisoning case. 1: phenol; 2: p-cresol; 3: m-cresol; 4: o-cresol; 5: 4-ethylphenol; 6: 2,4-xylenol. completely separated from each other; in addition, the test peaks are not interfered with by phenol or xylenol being contained in the cresol soap solution commercially available. With the UV detector, the quantitative range for each cresol isomer is 1–100 µg/mL; the detection limit is 0.1 µg/mL. For the urine specimen, the impurity peaks interfere with that of p-cresol; it is difficult to measure p-cresol at low concentration (not higher than 1 µg/mL) by HPLC-UV detection. By using a fluorescence detector, the specificity and sensitivity are increased; the detection limit of cresol isomers by HPLC-fluorescence detection is 0.01 µg/mL. The concentration of a conjugated form can be calculated by subtracting the amount of a free form of a cresol isomer from its total amount obtained after enzymatic hydrolysis.
  4. 584 Cresol GC/MS analysis Reagents and their preparation o-, m- and p-Cresols and IS are prepared according to the section of reagents and preparation of the HPLC analysis. GC/MS conditions Instrument: an HP 6890 Series GC/MS instrument (Agilent Technologies, Palo Alto, CA, USA). Condition 1: Column: HP-5 Trace Analysis (30 × 0.25 mm i.d., film thickness 0.25 µm, Agilent Technologies); carrier gas: He (1.0 mL/min); column (oven) temperature: 50 °C (4 min) → 20 °C/min → 300 °C (3.5 min); injection volume: 1 µL (splitless); injection temperature: 300 °C; detector temperature: 280 °C. Condition 2: Column: DB-WAX (60 m × 0.32 mm i. d., film thickness 0.5 µm, J & W Scientific, Folsom, CA, USA); carrier gas: He (1.0 mL/min); column temperature: 200 °C; injection volume: 1 µL (splitless); injection temperature: 250 °C; detector temperature: 280 °C. Procedure i. An Oasis HLB (3 cc, 60 mg) cartridge (Waters, Milford, MA, USA) is activated by passing 3 mL methanol and 3 mL distilled water. ii. A 0.1-mL volume of a specimenf is mixed with 0.9 mL distilled water and 10 µL IS solution, and poured into the activated cartridge. iii. The test tube, which had contained the specimen, is rinsed with 1 mL distilled water; the rinsed water is also poured into the cartridge. iv. The cartridge is washed with 1mL distilled water, and the water inside the cartridge is re- moved by aspiration under reduced pressure. v. The target compound(s) and IS are eluted with 1 mL ethyl acetate. vi. The organic eluate is condensedg into about 100 µL under a stream of nitrogen with warm- ing at 50 °C. vii. A 1-µL aliquot of it is injected into GC/MS. Assessment of the method > Figure 6.2 shows total ion chromatograms (TICs) for the authentic cresol isomers and related compounds (10 µg/mL each). When non-polar and slightly polar columns (HP-1 or HP-5) are used, p-cresol cannot be separated from m-cresol. With use of a DB-WAX column, such separation can be achieved ( > Figure 6.2, lower panal). The relative recovery rate of cresol isomers as compared with that of IS was 98 %; their detection limit in the scan mode is about 1 ng on-column.
  5. Poisoning cases, and toxic and fatal concentrations 585 ⊡ Figure 6.2 TICs by GC/MS for the authentic cresol isomers and related compounds (10 µg/mL each in ethyl acetate) using different GC columns. Poisoning cases, and toxic and fatal concentrations Cresol poisoning case due to its percutaneous absorption: a male child was playing on a slide in a park, and slid into a puddle with his buttocks getting wet with water probably containing a large amount of cresol. After 30 min, he fell into a disturbance of consciousness, underwent treatments at an emergency hospital and was discharged 24 days after, because of improvement of his conditions. The time courses of plasma concentrations of cresol isomers and their conju- gates, measured by HPLC, are shown in > Figure 6.3. The plasma concentrations of free, sul- fate-conjugated and glucuronide-conjugated forms for p-cresol 2 h after the accident were 15.7, 21.3 and 38.6 µg/mL, respectively; those for m-cresol 31.4, 17.0 and 82.9 µg/mL, respec- tively. After 8 h, the concentrations of sulfate-conjugated forms were higher than those of the glucuronide-conjugated forms, and detectable for a long time. The urinary concentrations at an early stage of admission were 17.4, 102 and 709 µg/mL for the free, sulfate-conjugated and glucuronide-conjugated forms of p-cresol, respectively; 12.0, 151 and 1,510 µg/mL for those of m-cresol, respectively.
  6. 586 Cresol ⊡ Figure 6.3 Time courses of plasma concentrations of cresol isomers and their conjugates in a cresol- poisoned patient after percutaneous absorption. Cresol poisoning case due to its oral intake: a female ingested about 80 mL of cresol soap solution for suicidal purpose, underwent treatments, such as gastrolavage and hemophoresis and was remitted. The time courses of plasma concentrations of cresol isomers and their con- jugates, measured by HPLC, are shown in > Figure 6.4. The plasma concentrations 3.5 h after ingestion were 16.3, 19.6 and 78.5 µg/mL for the free, sulfate-conjugated and glucuronide-con- jugated forms of p-cresol, respectively; 37.4, 18.7 and 147 µg/mL for those of m-cresol, respec- tively. The concentrations of glucuronide-conjugated forms were higher than those of sulfate- conjugated forms until several hours after ingestion; but the former concentrations become lower than the latter after 26 h ( > Figure 6.4). Phenol and p-cresol endogenously exist in humans, because they are produced during metabolic decomposition of tyrosine by enteric bacteria [11]. When plasma and urine speci- mens from 5 healthy subjects were analyzed, p-cresol sulfate-conjugate was found in plasma and urine at concentrations of 0.4 ± 0.3 and 31.0 ± 14.4 µg/mL, respectively; the concentration of p-cresol glucuronide-conjugate in urine was 1.3 ± 0.9 µg/mL. The endogenous p-cresol con- centrations in plasma are relatively low and give no problems upon analysis in acute poisoning; but with urine specimens, appreciable amounts of the endogenous p-cresol sulfate-conjugate should be taken into consideration. Although there are numerous reports dealing with cresol poisoning, the reports describing cresol concentrations are not so many; they are listed in > Table 6.1 [3–7, 14, 16–21]. Case 3 shows a high blood cresol concentration; but her cause of death was exsanguina- tions due to being stabbed in her abdomen. Case 5 died after treatments for 4 days; cresols were measured for the serum, which had been sampled about 24 h after ingestion, and were ex- pressed as a total amount of phenols, but free phenol could not be detected. The victim in Case 6 with blood cresol concentration at only 10 ng/mL was suffering from severe liver cirrhosis,
  7. Poisoning cases, and toxic and fatal concentrations 587 ⊡ Figure 6.4 Time courses of plasma concentrations of cresol isomers and their conjugates in a cresol- poisoned patient after its oral intake. ⊡ Table 6.1 Cresol poisoning cases Case Age Sex Amount Route Blood or plasma Time Presence/ Out- Ref. No. of intake concentration* after absence come (mL) unconju- conju- intake of therapy gated gated (h) form form 1 74 F – oral 190 – – – dead [7] 2 76 F – oral 71 – 2 + dead [7] 3 52 M – oral 99 87 – – dead [14] 4 1 M – percut. 120 – 4 + dead [16] 5 32 M 30 oral 0 90** 24 + dead [17] 6 48 F – oral 10 – – – dead [18] 7 46 M 100 oral 25 – 3 + alive [3] 8 46 M 100 oral 29 88 2 + alive [4] 9 7 M – percut. 47 160 2 + alive [5] 10 – F 80 oral 54 264 3.5 + alive [6] 11 62 F 150 oral 9.5** – 2 + alive [19] 12 37 F 100 oral 30 – – + alive [20] 13 19 M 500 percut. 30 – 11 + alive [21] 14 48 M – percut. 58** – 1 + alive [21] * : cresol concentration, ** : cresol + phenol concentration, –: data not available, percut.: percutaneous.
  8. 588 Cresol and was thus considered exceptional as a fatal case. The blood concentrations of unconjugated cresol in fatal poisoning cases are 71–190 µg/mL. In the survived Cases 7–14, the blood specimens were sampled at the first medical exami- nation; the plasma concentrations of unconjugated cresol were 9.5–58 µg/mL. Notes a) 4-Ethylphenol to be used as IS may contain phenol and p-cresol as impurities. The contents of the impurities should be carefully checked before use. b) By adding β-cyclodextrin to the mobile phase, the separation of p-cresol from m-cresol can be realized. c) As a specimen, blood, plasma or urine can be used. When organ tissue is used, 1 g of it is put in 4 mL of cold distilled water, minced into small pieces with surgical scissors and homogenized with cooling with ice. The homogenate can be used as a specimen; but the cresol glucuronide-conjugates may be hydrolyzed by the coexisting glucuronidase, result- ing in a higher concentration of the unconjugated cresols during the procedure. d) Without stirring, the surface layer of the specimen solution may be coagulated, hindering the solution from well-mixing. e) To analyze the sulfate-conjugated forms of cresol isomers in organ tissues, the effect of endogenous glucuronidase should be excluded by adding saccharolactone as an inhibitor of the enzyme. f) As a specimen, blood, plasma or urine can be used. g) The organic eluate should not be evaporated to dryness, because it causes very low recovery rates due to evaporation of free cresol isomers. References 1) Naito H (1991) Poisoning of Industrial Products, Gases, Pesticides, Drugs, and Natural Toxins – Cases, Pathogen- esis and Its Treatment. 2nd edn. Nankodo, Tokyo, pp 65–67 (in Japanese) 2) Budavari S (1996) The Merck Index. 12th edn. Merck & Co., Whitehouse Station, pp 436–437 3) Kumano H, Kuroki H, Tsutsumi H et al. (1986) Cresol poisoning. The Pharmaceuticals Monthly 28:1697–1701 (in Japanese) 4) Yashiki M, Kojima T, Miyazaki T et al. (1990) Gas chromatographic determination of cresols in the biological fluids of a non-fatal case of cresol intoxication. Forensic Sci Int 47:21–29 5) Fuke C, Sakai Y, Yagita K et al. (1998) The quantitative analysis of cresols in a case of cresol poisoning following percutaneous absorption. Jpn J Toxicol 11:55–60 (in Japanese with an English abstract) 6) Fuke C, Morinaga Y, Arao T et al. (1999) Time course changes of cresols and their conjugates in plasma from two case of cresol poisoning. In:Tatsuno Y (ed) Proceedings of the 6th Indo Pacific Congress on Legal Medicine and Forensic Sciences. INPALMS-1998-KOBE, pp 808–811 7) Bruce AM, Smith H, Watson AA (1976) Cresol poisoning. Med Sci Law 16:171–176 8) Niwa T, Maeda K, Ohki T et al. (1981) A gas chromatographic-mass spectrometric analysis for phenols in uremic serum. Clin Chim Acta 110:51–57 9) Pendergrass SM (1994) An alternative method for the analysis of phenol and o-, m-, and p-cresol by capillary GC/FID. Am Ind Hyg Assoc J 55:1051–1054 10) Brega A, Prandini P, Amaglio C et al. (1990) Determination of phenol, m-, o- and p-cresol, p-aminophenol and p-nitrophenol in urine by high-performance liquid chromatography. J Chromatogr 535:311–316 11) Niwa T (1993) Phenol and p-cresol accumulated in uremic serum measured by HPLC with fluorescence detec- tion. Clin Chem 39:108–111
  9. Poisoning cases, and toxic and fatal concentrations 589 12) Taguchi T, Horiie T, Ogata M (1993) Sensitive simultaneous analysis of cresol isomers in urine by high-perfor- mance liquid chromatography. Medicine and Biology 126:187–191 (in Japanese) 13) Ogata N, Matsushiba N, Shibata T (1995) Pharmacokinetics of wood creosote, glucuronic acid and sulfate con- jugation of phenolic compounds. Pharmacology 51:195–204 14) Fuke C, Ito A, Tamaki N et al. (1997) The analysis of cresols in biological materials from a case of cresol poisoning by high-performance liquid chromatography. In: Takatori T (ed) Proceedings of the 14th Meeting of the Inter- national Association of Forensic Sciences, Vol. 2. Shunderson Communications, Ottawa, pp 258–261 15) Masselter SM, Zemann AJ, Bobleter O (1993) Separation of cresols using coelectroosmotic capillary electropho- resis. Electrophoresis 14:36–39 16) Green MA (1975) A household remedy misused fatal cresol poisoning following cutaneous absorption, a case report. Med Sci Law 15:65–66 17) Arthurs GJ, Wise CC, Coles GA (1977) Poisoning by cresol. Anaesthesia 32:642–643 18) Kashimura S, Kageura M, Hara K et al. (1987) A case of severe liver cirrhosis, in which the victim died after in- gesting an insecticide – death of disease or poisoning? Res Pract Forensic Med 30:171–175 (in Japanese with an English abstract) 19) Thomas BB (1969) Peritoneal dialysis and lysol poisoning. Br Med J 3:720 20) Ohashi N, Kiyono H (1988) Cresol and phenol. Jpn J Acute Med 12:1342–1346 (in Japanese) 21) Tabata T, Yoshioka T (1996) Percutaneous intoxication of cresol with or without phenol: report of two cases. Jpn J Toxicol 9:101–105 (in Japanese with an English abstract)

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