In this chapter, a method for headspace GC/MS analysis of naphthalene is presented utiliz-ing its volatile property.. For spiked tests, the above each stock solution is diluted 10-fold w
Trang 1© Springer-Verlag Berlin Heidelberg 2005
Introduction
Naphthalene ( naphthalin) is being widely used as a moth repellant (insecticide), and being sold
at supermarkets and general stores Since one package of the compound usually contains as much as 1.8–5 g of naphthalene, the accidental ingestion of such a product by a small infant
may cause fatality However, recently, p-dichlorobenzene products, to be dealt with in the next
chapter, have become more popular as a moth repellant
Naphthalene exists in the form of colorless crystals at room temperature, but easily sub-limes into its gas with an aromatic odor exerting the repellant eff ect for insects
In this chapter, a method for headspace GC/MS analysis of naphthalene is presented utiliz-ing its volatile property
Reagent and their preparation
i Reagents
Naphthalene can be purchased from Sigma (St Louis, MO, USA); stable-isotopic naphthalene-d8
(internal standard, IS) from Aldrich (Milwaukee, WI, USA) Other common chemicals used are
of the highest purity commercially available
ii Preparation
Naphthalene and naphthalene-d8 (IS) solutions: 2-mg aliquot each is dissolved in 2 mL metha-nol to prepare stock solution separately A 1-µL aliquot of each stock solution is injected into GC/MS to measure each mass spectrum
For spiked tests, the above each stock solution is diluted 10-fold with methanol; a diff erent volume of the naphthalene solution (1–10 µL containing 0.1–1 µg) and 10 µL (containing
1 µg) of naphthalene-d8 (IS) solution are placed in each glass vial with a Tefl on-septum cap containing 0.2 mL of blank whole blood
GC/MS conditions
GC column b: an Rtx-1 fused silica medium-bore capillary column (30 m × 0.32 mm i d., fi lm thickness 0.25 µm, Restek, Bellefonte, PA, USA)
GC conditions; instrument: a GC-17A gas chromatographc (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 fl ow rate: 3 mL/min; injection mode: splitless for 1 min aft er injection, followed by the split mode
Trang 2MS conditions; instrument: a Shimadzu QP-5050 quadrupole mass spectrometerc (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 specimend, 10 µL (containing 1 µg) of IS solution and 0.8 mL distilled water are placed in a 7-mL volume glass vial with a Tefl on-septum screw cap, capped and mixed gently
ii Th 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 syringee is also heated on the same block Th e needlef size for the syringe
is 23 G Aft er heating, a 1-mL volume of the headspace vapor is withdrawn into the heated syringe, and carefully injected into GC/MS not to infl uence the vacuum degree of the MS instrument
iii Detection is made using ions at m/z 128 for naphthalene and at m/z 136 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, 10 µL IS solution and a diff erent amount of naphtha-lene are added Th e mixture is treated according to the above procedure Th e calibration curve consists of peak area ratio of naphthalene to IS on the vertical axis and naphthalene concentration on the horizontal axis Th e peak area ratio obtained from a blood specimen
is applied to the calibration curve to obtain a naphthalene concentrationg
Assessment of the method
> Figure 6.1 shows mass spectra of naphthalene and IS By this method, most reliable
identi-fi cation of naphthalene can be achieved Th e molecular ions constituted the base peaks; the
peaks at m/z 128 and 136 are not interfered with each other > Figure 6.2 shows SIM
chro-matograms for naphthalene and IS, which had been extracted by the headspace method Th e detection limit of naphthalene in the SIM mode was about 100 ng/mL in whole blood
Th e effi ciency (recovery) of naphthalene was tested; about 25–30 % of total naphthalene, which had been added to blank whole blood, were distributed in the headspace gas in the 7-mL volume vial aft er 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 However, it is not the case in the present method; each peak
appears in a sharp shape (> Fig 6.2) Th e author adopted a relatively low temperature (50 °C)
of the column (oven) upon injection of the headspace vapor, and confi rmed that an entire amount of naphthalene contained in the headspace gas injected was completely trapped inside the column at 50 °C
It should be kept in mind that naphthalene is contained in gasoline, kerosene and heavy oil [1] and also in tobacco smoke [2]
Trang 3Mass spectra of naphthalene and naphthalene-d8 (IS).
⊡ Figure 6.1
SIM chromatograms for naphthalene and IS headspace-extracted from whole blood The
amounts of IS and naphthalene spiked to 0.2 mL blood were 1 µg and 50 ng, respectively.
⊡ Figure 6.2
Trang 4Poisoning case and fatal concentrations
A 6-month-old male [3] opened two packages of naphthalene (commercial name: Neopas®) and seemed to ingest a part of the total amount (18 g) of the compound, because an appreciable amount
of its crystals were found attached around his mouth and to the face and scalp hair At 3 h later, he was cheerful and showed no symptoms upon arrival at a hospital His skin was immediately washed, and various treatments, such as gastrolavage, administration of activated charcoal and a purgative (magnesium citrate) and enforced diuresis, were given to him On the 5th day of admis-sion, exanthemas, followed by blisters and erosions, appeared in his back; they were improved by the oral administration of Predonine® (prednisolone) together with the topical deterging On day
6, hemolysis appeared together with the decrease of hemoglobin value from 12.7 to 10.6 g/dL During careful monitoring of his conditions, no abnormalities were observed for his neurological, hepatic and renal functions On day 14, he was discharged with complete recovery In this case, the real amount of naphthalene ingested by him seemed much smaller than that estimated
When a large amount of naphthalene is taken orally, vomiting takes place in every case; there appears abdominal spasm associated with diarrhea When naphthalene is absorbed into the body, central nervous system symptoms, such as headache, vertigo, unconsciousness, con-vulsion and coma appear Naphthalene is metabolized into α- and β- naphthols and naphtho-quinone, which cause severe hemolysis, followed by hemoglobinemia, methemoglobinemia and then renal dysfunction aft er several days [4, 5]
Oral fatal doses estimated are 1–2 g for infants and 5–15 g for adults with great variations among individuals [3]
Th e number of reports describing the concentrations of naphthalene in blood and tissues
in its poisoning cases is very limited Ijiri et al [6] reported a case, in which a neurotic mother had given her 2-month-old male baby milk mixed with 6 g naphthalene; he had died 30 min–
1 h later Th e concentrations of naphthalene in blood, the liver and kidney were 0.55, 0.12 and 0.03 µg/g or mL, respectively
Notes
a) Stable-isotopic naphthalene is commercially available from Aldrich and Sigma (product
No 17,604-4) Its price is not expensive In MS techniques, the use of a stable-isotopic compound is most desirable for quantitation; this is called “ isotope dilution technique” b) Any type of non-polar dimethylsilicone capillary columns can be used, irrespective of their manufacturers An Rtx-Volatiles fused silica capillary column (30 m × 0.32 mm, fi lm thick-ness 1.5 µm) was also tested, but the Rtx-1 column gave better results
c) Any type of GC/MS instruments including sector, quadrupole and ion-traps types can be used
d) Since whole blood can be analyzed by this method, urine seems also analyzable with the same procedure
e) Either a usual 5-mL volume glass syringe or a gas-tight syringe can be used
f) Th e tip of the usual injection-syringe needle is being cut obliquely and sharply Th is can be used in this method However, the author is using a special type of 23 G needles, which has
a tip being cut conically; this needle is advantageous, because such a tip prevents the needle from being clogged by septum debris
Trang 5g) It is possible to make quantitation analysis without IS using an external calibration curve Also in place of the stable-isotopic compound, 1-methylnaphthalene or 2-methylnaphtha-lene can be used as IS
References
1) Bertsch W, Holzer G (1995) Analysis of accelerants in fire debris by gas chromatography/mass spectrometry In: Yinon J (ed) Forensic Applications of Mass Spectrometry CRC Press, Boca Raton, pp 129–169
2) Schmeltz I, Tosk J, Hoffmann D (1976) Formation and determination of naphthalenes in cigarette smoke Anal Chem 48 645–650
3) Japan Poisoning Information Center (ed) (2000) Poisoning Accidents and their Countermeasures with Special Reference to Actual Cases, revised ed Jiho Inc., Tokyo, pp 65–68 (in Japanese)
4) Nishi K (ed) (1999) Emergency Manual for Poisoning Iyaku Journal, Osaka, pp 244–245 (in Japanese)
5) Ukai T (ed) (1999) Manual of Treatments in Acute Poisoning, 3rd edn Jiho Inc., Tokyo, pp 156–157 (in Japanese)
6) Ijiri I, Shimosato K, Ohmae M et al (1987) A case report of death from naphthalene poisoning Jpn J Legal Med 41:52–55