FARMACIA, 2016, Vol. 64, 6 ORIGINAL ARTICLE QUALITATIVE ASSAY OF ESSENTIAL OILS OF LAVENDER AND PEPPERMINT IN COMMERCIAL PRODUCTS THROUGH SPECTRAL AND CHROMATOGRAPHIC METHODS

SILVIA IMRE1, SIGRID EŞIANU1, AMALIA MIKLOS1*, IOANA TIUCA2, IONELA DICHER1, AMELIA TERO-VESCAN1, DANIELA-LUCIA MUNTEAN1, RADU OPREAN2

1University of Medicine and Pharmacy Tîrgu Mureş, Faculty of Pharmacy, 38 Gheorghe Marinescu Street, 540139, Târgu Mureş, Romania 2“Iuliu Haţieganu” University of Medicine and Pharmacy, Faculty of Pharmacy, 4 Pasteur Street, 400349, Cluj-Napoca, Romania

*corresponding author: [email protected] Manuscript received: February 2016 Abstract Essential oils are largely employed for their therapeutic properties, being marketed extensively in pharmaceutical and cosmetic industry. The aim of our study was to assess the purity and quality of peppermint and lavender volatile oils, available on the market, from various commercial producers. The methods used for testing the quality of these oils were refractometry, polarimetry, thin layer and gas chromatography, which are official techniques in Pharmacopoeia monographies of essential oils, and IR spectrometry, a non-official one. Following the analytical results, not all samples proved to be of excellent quality and high purity, some of them being contaminated with other substances or even diluted. Gas chromatography coupled with mass spectrometry and IR spectrometry methods had proved effective in assessing without doubt the qualitative difference between samples, the other techniques remaining a fast alternative, although there are limitations in terms of specificity and sensitivity.

Rezumat Uleiurile esențiale sunt folosite în mare măsură pentru proprietățile lor terapeutice, fiind comercializate intens în industria farmaceutică și cosmetică. Scopul studiului nostru a fost de a evalua puritatea și calitatea uleiurilor volatile de mentă și lavandă existente pe piață, de la diferiți producători comerciali. Tehnicile de laborator aplicate pentru controlul calității uleiurilor volatile au fost cele prevăzute de farmacopee, refractometrie, polarimetrie, cromatografie în strat subţire, cromatografie de gaze, și o metodă neoficială în monografiile uleiurilor volatile, spectrometria IR. Nu toate probele s-au dovedit a prezenta o calitate excelentă, unele dintre ele fiind contaminate cu alte substanțe sau chiar diluate. Tehnicile de cromatografie de gaze cuplată cu spectrometrie de masă şi spectrometrie IR au permis diferențierea calitativă certă dintre probe, celelalte tehnici rămânând o alternativă rapidă, cu rezerva limitărilor acestora în ceea ce privește specificitatea și sensibilitatea.

Keywords: volatile oils, peppermint, lavender, qualitative analysis

Introduction (up to 4%) and carvone (up to 1%) are present in smaller proportions [16]. Nowadays, the use of essential oils as alternative Lavender oil, also, is widely used for its medicinal therapies has gained the worldwide concern, owing actions including antispastic, anti-inflammatory, to their various biological activities. Considerable sedative, antimicrobial and general tonic action. attention has been devoted to peppermint oil, which Externally, it can be used for wounds and superficial is widely used for its important properties including burns. It contains as major compounds linalool antimicrobial, anti-inflammatory, antispasmodic, (20 - 45%), linalyl acetate (25 - 46%), mono- cytoprotective, hepatoprotective, with strong anti- terpenes, alcohols and esters [1, 10, 13]. oxidant actions. It is also used for its cooling effect, to Even if essential oils are marketed extensively in enhance the dermal penetration of pharmaceuticals pharmaceutical and cosmetic industry, not all and is one of the most important flavouring products available to use are properly controlled in additives in the world [5, 14]. terms of quality of their composition. The analytical The major components of peppermint oil are methods applied for the analysis of essential oils menthol (30 - 55%) and menthone (14 - 32%). are numerous and include methods exploiting Monoterpenes including limonene (1 - 5%), cineole volatility (gas chromatographic methods) or their (3.5 - 14%), menthofuran (1 - 9%), isomenthone optical activity (polarimetric method), along with (1.5 - 10%), menthyl acetate (2.8 - 10%), pulegone 857 FARMACIA, 2016, Vol. 64, 6 the IR spectral methods, refractometry and thin absorbance spectra were recorded in 4000 - 700 cm-1 layer chromatography [6, 7, 17]. spectral region. Gas chromatography coupled with mass spectrometry GC-MS analysis (GC-MS) is the elected technique for analysing the Equipment: Agilent 7890A GC System with Agilent main constituents of essential oils. The scientific 5975C Inert XL EI/CI MSD. Chromatographic literature regarding GC-MS analysis of essential oils conditions: injector temperature 250°C, column reported a large number of components qualitatively temperature 60°C, isotherm for 2 min, 3°C/min, to and quantitatively analysed [2-4, 8-9, 11, 15]. 240°C, isotherm for 5 min. Column: HP - 5 ms 30 m x Other techniques, inexpensive, remain a fast 0.25 mm x 0.25 µm. Injection 1 µL/split less. Detector alternative, although there are limitations in terms parameters: temperature 230°C, scan m/z = 50 - 800, of specificity and sensitivity. solvent delay 4 min, (automatic) reference voltage, This study was designed to assess the purity and gas flow He 1 mL/min. The samples were diluted quality of peppermint and lavender volatile oils 1:1000 in hexane before the analysis. The identification available in herbal stores, through standardized or was made by mass spectra comparison. modified methods provided in pharmacopoeia, and to test the ability of IR spectrometry, a powerful Results and Discussion identification method, to compare the same type of volatile oils from different producers. TLC analysis Peppermint essential oil. Identification of menthol: Materials and Methods by comparing the retention factor (Rf = 0.25) of standard solution spot with the retention factors of Five peppermint oil samples, M1-M5, and four samples, it was observed that all samples contained lavender oil samples, L1-L4, from different menthol, M1 and M4 containing the highest amount. producers were analysed through standardized or Identification of menthyl acetate: all samples contained modified methods provided by pharmacopoeia: this compound, M4 and M5 having the highest refractive index, optical rotation, thin layer amount. The comparison of the chromatographic chromatography, GC-MS and through a non- profiles for the five samples (Figure 1) showed that official method, ATR-FTIR spectrometry. The gas M1 and M2 were virtually identical in terms of chromatography coupled with mass spectrometry number of spots, colour and size. M3 and M4 was used as reference method. samples presented a spot just below the Rf = 0.5 Thin-layer chromatographic analysis (TLC) and for M5 a yellowish orange, not intense, spot Reference substance: linalool, linalyl acetate, has appeared just below the blue-violet spot located menthol, menthyl acetate. Reagents: ethyl acetate at Rf 0.79 which proves that this sample contains a (Lachner), toluene (Lachner), ethanol (Chimopar), compound that is not found in other studied oils. anisaldehyde (Sigma-Aldrich). Sample preparation: a volume of 20 µL commercial essential oil was diluted with l mL of toluene. Chromatographic method: volumes of 5 µL samples were applied and eluted with toluene: ethyl acetate 95:5 as mobile phase on 20 x 10 cm chromatographic plate of Silicagel 60, 0.25 mm (Macherey Nagel, Duren, Germany); the plates were then sprayed with anisaldehyde solution and kept at 100 - 105°C in oven, for 5 - 10 minutes. Physico-chemical constants determination Figure 1. Polarimetric measurements were carried out on a Thin-layer chromatograms of peppermint essential oils Polamat A (Karl Zeiss Jena) polarimeter with 1 dm (the samples from left to right: menthol, M1, M2, M3, tube. Refractometry measurements were carried out menthy lacetate, M4, M5, menthol, menthyl acetate) on Abbé refractometer and the temperature was maintained at 20 C. ° Lavender essential oil. Identification of linalool: all IR spectrometry analysis samples contain linalool (R = 0.30) and linalyl IR spectrometry analysis were made with FTIR f acetate (R = 0.59), L3 with lower concentrations Nicolet 380 spectrometer (Thermo Electron Corp.), f than other L samples. Figure 2 shows clearly that equipped with ATR Smart (Multi – bounce HATR) sample L3 differs qualitatively from the other with Zn Se 45° plate. Omnic software version 8.3 samples: the common identified spots have low (Thermo Scientific) was used for instrumental intensity, which means diluted product, and the spectral recording and data acquisition. All FT-IR compound with Rfx = 0.87 is missing.

858 FARMACIA, 2016, Vol. 64, 6 Physico-chemical parameters The results were analysed in terms of Pharmacopoeia provisions for peppermint and lavender oil monography, respectively. Lavender samples. According to Pharmacopoeia provisions [18-19], L1, L2 and L4 samples were within the limits for refractive index and optical rotation. Table I shows that L3 lavender oil has a very high refractive index and an angle of rotation nearly 0, which proves again dilution or contamination. Figure 2. Peppermint oil samples. Regarding the refractive Thin-layer chromatograms of lavender essential oil index and the optical rotation, all peppermint oil samples (the samples from left to right: linalool, samples fall within Pharmacopoeia limits (Table II). linalyl acetate, L1, L3, L3, L4, linalool, linalyl acetate)

Table I Refractive index and optical rotation for lavender oil samples

Sample code Eu. Ph. limits L1 L2 L3 L4 20 nD 1.455 ÷ 1.466 1.4660 1.4610 1.4750 1.4600 !" ° ° α! -12.5° ÷ -6.0° -11 -11° -0.2° -6.8

Table II Refractive index and optical rotation for peppermint oil samples

Sample code Eu. Ph. limits M1 M2 M3 M4 M5 20 nD 1.457 ÷ 1.467 1.4615 1.4615 1.4620 1.4615 1.4595 !" ° α! -10° ÷ -30° -24.8° -24.8° -16.8° -23° -22.2

IR spectrometry analysis (Figure 3). Making the comparison with the reference Peppermint oil samples. The spectrum of each spectra from the spectrometer library, menthol and commercial product was obtained and the spectral sample M1, for example, has a similarity score of fingerprint was found virtually identical in all cases 81.70 and menthone, 66.10.

Figure 3. IR spectra of peppermint essential oil samples (from top to bottom M5, M1, M2, M3, M4 samples)

Lavender oil samples. The samples spectra were presented a supplementary band around 1600 cm-1, identical for all samples, except L3 sample, which in contrast to the other samples with a single band 859 FARMACIA, 2016, Vol. 64, 6 in this spectral region (Figure 4). For L3 sample the reference spectra, standardized Lavender oil and fingerprint region showed significant differences. linalyl acetate spectra, the similarity score was Making the comparison of samples spectra with the consistent for L1, L2 and L4 samples.

Figure 4. IR spectra of lavender essential oil samples (from top to bottom L4, L1, L2, L3 samples)

GC-MS analysis comparison with L1, L2 and L4: 19 identified The GC-MS confirmed the preliminary tests results. compounds were common, including linalool and The M samples had identical chromatographic linalyl acetate with low concentrations, and 14 patterns. The L3 sample chromatogram (Figure 5) compounds were different. and mass spectrometry profile were different in

Figure 5. GC-MS chromatograms of lavender essential oils

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