Essential Oil and Hydrolat Analysis by GC/MS

An essential oil is not an ingredient in the sense that a synthetic preservative is an ingredient. It is a mixture of dozens to several hundred volatile compounds, in proportions that vary with species, chemotype, growing region, harvest, distillation and storage. The safety assessor cannot assess “lavender oil”. The assessor has to assess linalool, linalyl acetate, camphor, 1,8-cineole and the rest, each at its actual concentration, each against its own toxicological limit, and each, where it is a regulated allergen, against the labelling threshold in Annex III.

Gas chromatography-mass spectrometry is the method that turns the oil into that list. Oxford Biosciences runs GC/MS on essential oils, hydrolats, absolutes and perfume compounds in its own analytical laboratory, and the report is written for the safety assessor, not merely for the file.

Regulatory basis

In the EU and UK, Annex I, Part A, Section 1 of Regulation (EC) No 1223/2009 requires the qualitative and quantitative composition of the product, and, for perfume and aromatic compositions, the name and code number of the composition and the identity of the supplier. Section 2 requires the purity of the substances and the identification of impurities and traces. For a natural complex substance, that means the constituent profile.

Annex III lists the fragrance substances that must be named in the ingredient list when their concentration exceeds 0.001 percent in leave-on products or 0.01 percent in rinse-off products. The original list of 26 allergens was introduced by Directive 2003/15/EC. In the EU, Commission Regulation (EU) 2023/1545 extended it to more than 80 substances, including many that are principal constituents of common essential oils: alpha-pinene and beta-pinene, camphor, carvone, terpineol, menthol, vanillin, methyl salicylate, beta-caryophyllene and others. Products placed on the EU market from 31 July 2026 must comply with the extended list; products placed on the market before that date may be made available until 31 July 2028. The UK has not, at the date of this page, mirrored the 2023 extension, so a product sold in both markets has to be labelled to the EU list, which satisfies both.

Annex II and Annex III also name individual essential oil constituents that are prohibited or restricted regardless of allergen status, and the CPSR must demonstrate that the finished product respects each limit. The oil’s percentage in the product is only half of that calculation; the constituent’s percentage in the oil is the other half, and GC/MS is the only way to know it.

The same analysis serves the IFRA Standards, which restrict fragrance materials by product category on a basis that requires the constituent profile of every natural in the compound, and the ingredient safety substantiation expected under MoCRA in the United States.

Constituents that determine whether an oil can be used at all

Some constituents set the maximum concentration of an oil in a product more tightly than any allergen. The report quantifies each of the following where present:

Constituent	Found in	Regulatory position (EU and UK, Regulation 1223/2009)
Methyl eugenol	Basil, tarragon, bay, nutmeg, rose, some tea tree chemotypes	Annex III entry 102: 0.01 percent in fine fragrance, 0.004 percent in eau de toilette, 0.002 percent in fragrance cream, 0.0002 percent in other leave-on and oral products, 0.001 percent in rinse-off
Estragole (methyl chavicol)	Basil, tarragon, fennel, anise	Not listed in Annex III but classified and assessed as genotoxic by the SCCS; restricted through the safety assessment on a margin of exposure basis
Safrole	Sassafras, nutmeg, cinnamon leaf, camphor oil	Annex II entry 360: prohibited except as a natural constituent of essences, and then not exceeding 100 ppm in the finished product, or 50 ppm in oral hygiene products with no use in products for children
Furocoumarins (bergapten, xanthotoxin, psoralen and others)	Cold-pressed bergamot, lime, lemon, bitter orange, grapefruit, angelica root	Annex III entry 358: total furocoumarins not exceeding 1 mg/kg in sun protection and bronzing products; phototoxicity assessed in the CPSR for all leave-on products
Pulegone	Pennyroyal, peppermint, buchu	Hepatotoxic; assessed on exposure and margin of safety; concentration in peppermint oil reported for every batch
Thujone (alpha and beta)	Sage, wormwood, thuja, tansy	Neurotoxic; assessed on exposure and margin of safety
Menthofuran	Peppermint	Hepatotoxic metabolite precursor; reported with pulegone
Ascaridole	Boldo, some tea tree	Reported where present; oils containing it are generally excluded from cosmetic use
Alpha-santalol, alpha-bisabolol and other sensitising sesquiterpenes	Sandalwood, chamomile	Assessed as skin sensitisers at the concentration found

A batch of basil oil at 0.5 percent in a body cream can be within the methyl eugenol limit or fifty times over it, depending on the chemotype. Without the GC/MS profile of the batch actually used, the CPSR is a guess.

How the analysis is done

1. Sample preparation

The oil is diluted in a suitable solvent, typically hexane or ethanol, to a concentration that keeps the major constituents within the linear range of the detector while allowing minor constituents at 0.01 percent to be detected. Absolutes and resinoids, which contain non-volatile waxes, are diluted further and filtered. Hydrolats, which are aqueous with a dissolved volatile fraction usually below 0.1 percent, are extracted by liquid-liquid extraction or by headspace solid-phase microextraction before injection, and the result is reported both as a percentage of the volatile fraction and as mg/L of the hydrolat.

Where quantification of specific allergens is required against a labelling threshold, an internal standard is added at a known concentration before dilution so that the allergen concentration can be reported in absolute terms rather than as a relative peak area.

2. Chromatography

One microlitre of the diluted sample is injected in split mode into a capillary gas chromatograph. Separation is performed on a low-polarity 5 percent phenyl methylpolysiloxane column, 30 or 60 metres in length, 0.25 mm internal diameter and 0.25 micrometre film thickness, with helium as carrier gas at constant flow. The oven is programmed from 60 °C, held for a few minutes to resolve the monoterpene hydrocarbons, then ramped at 3 °C per minute to 240 °C or higher to elute the sesquiterpenes, oxygenated sesquiterpenes and diterpenes. A run takes about an hour.

Where constituents co-elute on the non-polar column, and several important pairs do, including some allergens, a second run is performed on a polar polyethylene glycol column, which separates on a different basis and resolves the pairs. Two-column confirmation is the norm for regulated allergens.

3. Mass spectrometry and identification

The column eluent passes into a mass spectrometer operating in electron ionisation mode at 70 eV, which fragments each molecule into a reproducible pattern of ions scanned across a mass range of about 35 to 500 atomic mass units. Each peak in the chromatogram is identified by two independent criteria applied together:

• Mass spectral match against reference libraries, principally the NIST library and the Adams library of essential oil constituents, with a match quality threshold below which an identification is reported as tentative.
• Linear retention index, calculated from the retention time of the peak relative to a homologous series of n-alkanes (C8 to C30) run under identical conditions, using the van den Dool and Kratz equation, and compared with published values for the same column phase.

A mass spectral match alone is not an identification. Many monoterpenes and sesquiterpenes are isomers with near-identical spectra and are distinguished only by retention index. Reports that list constituents on the basis of library match alone are the reason two laboratories can give different names to the same peak.

4. Quantification

For the full constituent profile, each identified compound is reported as a percentage of the total integrated peak area, the convention used in the ISO monographs for individual oils and in the literature. Where absolute quantification of a regulated allergen or restricted constituent is required, the compound is quantified against a calibration curve prepared from a certified reference standard, using the internal standard to correct for injection and matrix variation, and reported as a percentage by mass of the oil. The method for the allergens follows the IFRA analytical method for the determination of fragrance allergens by GC/MS.

The limit of quantification for the allergens is set well below the 0.001 percent leave-on labelling threshold at the intended use level of the oil, so that a negative result means the allergen is genuinely below the threshold rather than merely below the reach of the method.

5. Comparison with the ISO specification

Most commercially important oils have an ISO standard specifying the botanical source, the physicochemical constants and a chromatographic profile with minimum and maximum percentages for the characteristic constituents: ISO 3515 for lavender, ISO 4730 for tea tree, ISO 856 for peppermint, ISO 855 for lemon, ISO 3761 for rosewood, ISO 4720 for the nomenclature of the oils generally, and so on. The report compares the measured profile with the specification, identifies any constituent outside its range, and states whether the deviation is consistent with natural variation, a different chemotype, degradation or adulteration.

Adulteration and degradation

Essential oils are adulterated for commercial reasons more often than brands expect. The GC/MS profile detects the common patterns: dilution with a fixed oil or a solvent such as diethyl phthalate or benzyl benzoate, which appear as foreign peaks; addition of synthetic linalool and linalyl acetate to lavender, revealed by the absence of the minor constituents that accompany them in the natural oil and, where necessary, by chiral analysis of the enantiomer ratio; extension of expensive oils with cheaper oils of a related species; and reconstitution of an oil from isolates. Degradation is also visible: the oxidation of limonene to limonene oxide and carveol in aged citrus oils, and of linalool to its hydroperoxides, produces sensitisers that were not present in the fresh oil, and an old batch of an otherwise compliant oil can carry a sensitisation risk the certificate of analysis does not show.

Blends and perfume compounds

A blend is analysed as a single sample and reported as a single constituent list, which is what the CPSR needs, since the allergen labelling threshold applies to the finished product regardless of which oil contributed the allergen. Where a client supplies the blend recipe, the report reconciles the measured profile against the expected contribution of each oil and identifies any oil that is not behaving as its specification predicts. Perfume compounds from fragrance houses are analysed in the same way when the supplier’s allergen declaration is incomplete, out of date against the 2023 list, or needs verification.

What the report contains

• Sample identification, botanical source, supplier, batch and date of receipt
• Method summary: columns, temperature programme, detector conditions and identification criteria
• Full constituent table: retention time, linear retention index, identification, match quality and percentage
• Allergen table: each Annex III allergen present, its percentage in the oil, and its resulting concentration at the oil’s intended use level in the product, against the 0.001 and 0.01 percent thresholds
• Restricted and prohibited constituents: each with its percentage and the resulting concentration in the product against the Annex II or III limit
• Comparison with the applicable ISO specification
• Adulteration and degradation assessment
• The total ion chromatogram

What to send

5 mL of essential oil, absolute or perfume compound, or 50 mL of hydrolat, in an amber glass vial with a PTFE-lined cap, from the batch that will be used in the product. The supplier’s certificate of analysis, safety data sheet and any existing allergen declaration are useful for comparison but are not a substitute for the analysis. State the intended percentage of the oil in each product and whether the product is leave-on or rinse-off, so that the allergen contribution can be calculated in the report.

How it fits with the CPSR

The constituent profile is the basis of the toxicological assessment of every product containing the oil: the margin of safety for each constituent of concern, the allergen labelling decision, the phototoxicity assessment for citrus and other furocoumarin-bearing oils, and the check against every Annex II and III limit. Where we prepare the CPSR, the GC/MS report and the assessment are written by the same team from the same data. Where a client relies on a supplier’s certificate rather than an analysis of the batch in use, the CPSR records that reliance and the assumption it rests on, and we advise when that assumption is not safe.