In the presence of light and air, autoxidation of limonene readily occurs to give a variety of oxygenated monocyclic terpenes. The oxidation products of d-limonene identified (Karlberg & Dooms-Goossens, 1997) include:
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(+)-cis-limonene-1,2-oxide (1,2-epoxy-p-mentha-8-ene);
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(+)-trans-limonene-1,2-oxide (1,2-epoxy-p-mentha-8-ene);
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l-carvone ((R)-(-)-6,8-p-mentha-diene-2-one);
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(-)-cis-carveol (cis-2-hydroxy-p-mentha-6,8-diene);
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(-)-trans-carveol (trans-2-hydroxy-p-mentha-6,8-diene);
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cis-limonene-2-hydroperoxide (cis-2-hydroperoxy-p-mentha-6,8-diene); and
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trans-limonene-2-hydroperoxide (trans-2-hydroperoxy-p-mentha-6,8-diene).
The primary oxidation products are hydroperoxides, which are unstable and readily degrade to other secondary oxidation products such as carvone, if further subjected to daylight and air (Nilsson et al., 1999). If oxidation process continues, polymers are created and the liquid will become viscous (Karlberg and Dooms-Goossens, 1997).
When limonene is heated to decomposition it emits carbon monoxide and carbon dioxide (CCOHS, 2001).
5.4Impurities and additives
Impurities in d-limonene are mainly other monoterpenes, such as:
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myrcene (7-methyl-3-methylene-1,6-octadiene),
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-pinene (2,6,6-trimethyl-bicyclo[3.1.1]-hept-2-ene),
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-pinene (6,6-dimethyl-2metylene-bi-cyclo[3.1.1]heptane),
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sabinene (2-metyl-5-(1-metyl-ethyl)-bicyclo[3.1.0]hexan-2-ol),
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3-carene ((1S-cis)-3,7,7-trimetyl-bicyclo[4.1.0]hept-2-ene).
Information from industry indicates that the dl-limonene content of commercial grade dipentene is 10% to 70%. The name dipentene in scientific literature refers to racemic limonene, but in industry may refer to any mixture of terpene hydrocarbons, usually p-menthadienes [Zinkel, 1989]. Some imported dipentene is obtained from the manufacture of synthetic pine oil. However, other manufacturing methods may also be used.
Some limonene grades are supplied with antioxidants such as butylated hydroxy anisole (BHA), butylated hydroxy toluene (BHT) and preservatives, for example, sodium erythorbate and sodium benzoate.
5.5Conversion factors
The conversion factors for limonene are:
1 ppm = 5.56 mg/m3
1 mg/m3 = 0.177 ppm
6.1Identification
The isomers of limonene are usually determined by gas chromatography using flame ionisation detection (GC-FID) or mass spectrometry (GC-MS).
As limonene is easily oxidised in air, it is also important to analyse the oxidation products. Hydroperoxides of d-limonene can be characterised by GC if the sample is injected on-column (IPCS, 1998). GC-MS with chemical ionisation in negative ion mode was shown to be a successful method for the identification and determination of the molecular weight of chemically unstable limonene hydroperoxides (Nilsson et al., 1996). A high performance liquid chromatography (HPLC) method for isolation of individual compounds in autoxidised d-limonene has been developed by using 2 different stationary phases in normal phases mode (Nilsson et al., 1996). The method can be used for the isolation of individual contact allergens in sensitisation experiment vehicles, such as petroleum and olive oil.
A sensitive GC-MS method using stable-isotopically labelled internal standards has been reported (Zhang et al., 1999), for the quantitation of a metabolite of d-limonene, perillyl alcohol (POH), and its metabolites, perillic acid (PA) and cis- and trans-dihydroperillic acid (DHPA). The products were separated on a capillary column and analysed by an ion-trap GC-MS using NH3 chemical ionisation. The quantitation limits for POH, PA, cis- and trans-DPHA were < 10 ng/mL using 1-2 ml plasma.
The determination of limonene in ambient air can be achieved by either passive or active means. Passive sampling involves adsorption onto activated charcoal with desorption by carbon disulfide followed by capillary GC. Active methods include adsorption onto Tenax (a porous polymer) using an air flow rate of 100 to 300 ml/min (up to 3 L of ozone free air can be sampled on 200 mg Tenax) (Janson & Kristensson, 1991) or multisorbent sampling tubes (Chan et al., 1990) and separated by capillary GC. Thermal desorption at 250 oC for 5 minutes is followed by GC-MS. The limit of detection was not stated.
National Institute of Occupational Safety and Health (NIOSH) method 1552 and Occupational Safety and Health Administration (OSHA, USA) method PV2036, for the determination of occupational airborne limonene entails the passage of 10 L of sample volume over activated charcoal at sampling rates of 10 to 200 mL/min for long-term sampling (Eller & Cassinelli, 1994; OSHA, 1994) and 250 mL/min for short-term sampling (Josefsson, 1993). Desorption is achieved with carbon disulfide followed by GC-FID. The detection limit is 0.4 g/sample (4 g/m3). The detection limit of the overall procedure (DLOP) is 1.3 g/sample (0.13 g/m3). The DLOP is defined as the concentration of analyte that gives a response that is significantly different from the background response (OSHA, 1994).
6.3Biological monitoring methods
The presence of d-limonene in biological samples including body fluids, blood and tissues can be detected by a head-space technique (Falk-Filipsson et al., 1993). The samples (blood, urine) are transferred to gas-tight head-space vials (22.4 ml) and capped immediately with Teflon-lined membranes. The head-space air of the samples is analysed automatically on a GC equipped with a head-space auto-sampler, a FID, and a polar column. The concentration of d-limonene in the samples is determined by comparison with individual standard curves prepared in the same concentration range by adding d-limonene to the samples taken before the exposure. The limit of detection in blood is 1.4 g/L.
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