7. 6
7. 6
© Springer-Verlag Berlin Heidelberg 2005
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.
 e toxic e ects 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].  ere are three isomeric forms of cresol, vis., o-, m- and p-cresols; the toxicity of each
isomer is somewhat di erent [2].  e composition ratios of cresol isomers are di erent 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, a er being absorbed into human bodies, is metabolized into glucuronide- and/or
sulfate-conjugated forms and excreted into urine.  e half-life of unchanged cresol in blood is
as short as about 1.5 h [3]; this means that it becomes undetectable several hours a er 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-ethylphenol
a
(internal standard, IS, Aldrich and other manufacturers)

added to blank specimens and processed in the same way to construct a calibration curve.
 e 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 specimen
c
is mixed with 10 µL of IS solution.
ii. A 5-µL volume of 4 M sodium acetate bu er 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. A er cooling to room temperature, 100 µL acetonitrile is placed in the above mixture with
stirring.
iv.  e following procedure is achieved according to the iii–v steps of the above section.
iii. Analysis of the sulfate-conjugated forms
e
i. A 100-µL volume of a specimen
c
is mixed with 10 µL of the IS solution.
ii. A 5-µL volume of 2.5 M Tris-HCl bu er solution (pH 7.5) and 5 µL of sulfatase are added
to the above mixture and incubated at 37 °C for 2 h.
iii. A er cooling to room temperature, 100 µL acetonitrile is added to the above mixture with
stirring.
iv.  e 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 di erence in composition ratio of acetonitrile in the supernatant solu-
tion may a ect the peak area ratio of cresol to IS; it is preferable to  x the composition ratio of
acetonitrile before injection into HPLC.
> Figure 6.1 shows HPLC chromatograms for the authentic cresol isomers and related

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 specimen
f
is mixed with 0.9 mL distilled water and 10 µL IS solution,
and poured into the activated cartridge.
iii.  e test tube, which had contained the specimen, is rinsed with 1 mL distilled water; the
rinsed water is also poured into the cartridge.
iv.  e cartridge is washed with 1mL distilled water, and the water inside the cartridge is re-
moved by aspiration under reduced pressure.
v.  e target compound(s) and IS are eluted with 1 mL ethyl acetate.
vi.  e organic eluate is condensed
g
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).
 e 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.
585
Poisoning cases, and toxic and fatal concentrations
Cresol poisoning case due to its percutaneous absorption: a male child was playing on a slide

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.  e 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 a er treatments for 4 days; cresols were
measured for the serum, which had been sampled about 24 h a er ingestion, and were ex-
pressed as a total amount of phenols, but free phenol could not be detected.  e victim in Case
6 with blood cresol concentration at only 10 ng/mL was su ering from severe liver cirrhosis,
Time courses of plasma concentrations of cresol isomers and their conjugates in a cresol-
poisoned patient after percutaneous absorption.
⊡ Figure 6.3
587
Time courses of plasma concentrations of cresol isomers and their conjugates in a cresol-
poisoned patient after its oral intake.
⊡ Figure 6.4
⊡ Table 6.1
Cresol poisoning cases
Case
No.
Age Sex Amount
of intake
(mL)
Route Blood or plasma

14 48 M – percut. 58** – 1 + alive [21]
* : cresol concentration, ** : cresol + phenol concentration, –: data not available, percut.: percutaneous.
Poisoning cases, and toxic and fatal concentrations
588 Cresol
and was thus considered exceptional as a fatal case.  e 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  rst 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.  e 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.  e 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 e ect 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)  e 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

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)
Poisoning cases, and toxic and fatal concentrations