GC/MS chemical analysis of lavandin (Lavandula x intermedia) hydrolat: successive extraction fractions

Hydrolats are valuable co-products of the essential oil distillation process, whose volatile compounds can be quantified by various methods. In this paper, we have tried to estimate the liquid-liquid extraction cycle number threshold for volatile compounds quantification of lavandin (Lavandula x intermedia) hydrolat. For this purpose, ten consecutive hydrolat extractions with n-hexane were analyzed GC/MS with hexadecane (C16) as an internal standard and compared with the lavandin essential oil. The chemical composition of the lavandin hydrolat showed similarity with its essential oil to the great extent, while volatile compounds dissolved in hydrolat exclusively belonged to the class of oxygenated monoterpenes. The total amount of extracted compounds has been estimated to 2174.2 mg/L, where the most dominant compounds in lavandin hydrolat were cisand trans-furanoid linalool oxide (676.3 and 634.1 mg/L, respectively), followed by much smaller amounts of linalool, camphor, and 1,8-cineole (167.6, 157.0, and 148.2 mg/L, respectively). Cumulative recoveries of total compounds yield after the third, fifth, and eighth extractions were 88 %, 96 %, and 99 %, respectively. Combined fraction analysis confirmed that in the first 5 cycles more than 95 % of the total yield (from 10 cycles) of extracted volatile compounds can be collected. Based on the results of this study, for volatile compounds quantification in lavandin hydrolat, 5 cycles of n-hexane liquid-liquid extraction can be recommended.


INTRODUCTION
Hydrolats are valuable co-products obtained from aromatic and other plants by steam distillation. During this process, some components of the essential oil are dissolved in water in a certain ratio. Beside hydrolat, there are many different names for this co-product among which hydrosol, aromatic water, floral water, essential water, and herbal distillates are the most common. Hydrolats have many industrial applications such as cosmetics, perfumery, pesticides, aromatherapy, pharmaceutical, medical, and religious (Rajeswara Rao, 2013). Since some essential oils have a relatively high proportion of water-soluble compounds, a significant amount of the essential oil could retain in the water during the distillation process. Therefore, hydrolats of excellent quality are obtained when cohobation is an integral part of the distilling process (Price and Price, 2004). Essential oil components are lipophilic and have different solubility in water at room temperature, mostly below 2 % (Chen et al., 2014). However, due to different water-solubility of the components, the chemical analysis of the hydrolate will show a different profile compared to the essential oil from which it originates (Catty, 2001). The quality of the hydrolate may also depend on the collection time so that the early collected contain more low boiling point and the later collected more terpenoids with higher boiling points. (Rajeswara Rao, 2013). Some research indicates that for the therapeutic value of hydrolates, separation of fractions is more desirable than the entire distillation water collected at the end of the distillation process (Rose, 1999).
Considering that it is generally accepted that less than 2 % of essential oil is dissolved in hydrolats and that there is a lack of methods for quantification of individual components in the literature, the aim of this paper was to test the method of hydrolat compounds quantification using an internal standard on the example of lavandin distillation. In order to evaluate the yield of total and individual extracted volatile compounds dissolved in the hydrolate, individual and combined n-hexane liquid-liquid extractions were analyzed.

Standards and reagents
Sodium sulfate anhydrous (Na 2 SO 4 ) and hexadecane were purchased from Sigma Chemicals Co. (USA), while n-hexane and distilled water were purchased from Zorka Pharma, Šabac (Serbia). All chemicals used in the experimental procedure were of analytical grade purity.

Plant material
Flowers of lavandin (Lavandula × intermedia) were purchased from the Production sector of the Institute for Medicinal Plant Research "Dr. Josif Pančić", Belgrade.

Essential oil and hydrolat extraction procedures
Isolation of essential oil and hydrolat was performed using the hydro-distillation method using a Clevenger type apparatus according to the procedure I of the Ph. Jug. IV (1984). Lavandin flowers (40 g) were placed in flat-bottom flask (1 L) and filled with tap water until mass of 493 g. After setting the Clevenger apparatus on the heating body, an additional 7 mL of water was introduced into the system through a pipe above the burette, making a total mass of water of approximately 460 g, and together with the sample 500 g. Hydrolat was collected from the burette after the distillation process. The essential oil was collected and dried over anhydrous sodium sulfate. The essential oil yield, expressed as a percentage, was calculated on a moisture-free basis.

Sample preparation
Oil samples (20 µL) were dissolved in n-hexane (2 mL) and stored at 4°C until further analysis. Hydrolat was extracted with n-hexane. For optimization of the number of extraction cycles liquid-liquid extraction (LLE) of volatile compounds dissolved in water was performed ten times (5 mL of hydrosol with 10×1 mL n-hexane). Hexadecane (C16) was used as an internal standard for quantification of volatile compounds dissolved in hydrolat. For this purpose, an internal standard stock solution has been made (5.5 mg in 1 mL n-hexane). In each extraction party, 100 µL of internal standard has been added so that the final concentration of internal standard was 500 mg/L. All samples were stored in the freezer until further analysis. In order to validate the yield ratio of the extracted compounds from the lavandin hydrolat, the first five (1-5) and the last five (6-10) LLE fractions were combined into separate groups. Extractions were performed on newly prepared hydrolat from the same plant material and by the same procedure as for the analysis of individual fractions. In 1 mL of combined hexane fractions, 100 µL of the internal standard has been added so that the final concentration of internal standard was 500 mg/L. Samples were stored in the freezer until further analysis. Chemical analysis of combined fractions was performed under the same GC/MS conditions, as for analysis of individual fractions.

Chemical analysis
The chemical composition of the essential oil and hydrolat extraction parties was analyzed using GC/MS technique. GC/MS analyses were performed on a Shimadzu GCMS-QP2010 ultra mass spectrometer fitted with a flame ionic detector and coupled with a GC2010 gas chromatograph. The InertCap5 capillary column (60.0 m×0.25 mm×0.25 µm) was used for separation. Helium (He), at a split ratio of 1:5 and a linear velocity of 35.2 cm/s was used as a carrier gas. Initially, the oven temperature was 60°C, which was held for 4 min, then increased to 280°C at a rate of 4°C/min, and held for 10 min. The injector and detector temperatures were 250°C and 300°C, respectively. The ion source temperature was 200°C . The identification of the constituents was performed by comparing their mass spectra and retention indices (RIs) with those obtained from authentic samples and/or listed in the NIST/Wiley mass-spectra libraries, using different types of search (PBM/NIST/AMDIS) and available literature data (Adams, 2007).

Hydrolat chemical composition
The chemical composition of the lavandin hydrolat is generally similar to the main components of the essential oil (Table 2). In our experiment, no sesquiterpene component was detected in the aqueous solution. The volatile compounds dissolved in water exclusively belonged to the class of oxygenated monoterpenes, which is mostly similar to previously published analyzes (Baydar and Kineci, 2009;Politi et al., 2020;Smigielski et al., 2013). The most abundant compounds in lavandin hydrolat were cisand trans-furanoid linalool oxide (676.3 and 634.1 mg/L, respectively), followed by much smaller amounts of linalool, camphor, and 1,8-cineole (167.6, 157.0, and 148.2 mg/L, respectively). The next compounds regarding quantity were cis-pyranoid linalool oxide, which co-eluted together with borneol at the same retention time, hotrienol, α-terpineol, and trans-pyranoid linalool oxide (89.7, 79.7, 67.0, and 63.4 mg/L, respectively). Two furanoid linalool oxide isomers, which amount in essential oil fluctuated about 4 % each, were expressed in hydrolat in the highest amounts among all water-soluble compounds. Tannous et al. (2004) reported that oxygenated compounds are more water-soluble. So, further oxidation of the monoterpene alcohols, cisand trans-linalool to the corresponding oxides, probably leads to their better solubility in water, which is why they are the compounds with the highest share. The higher amount of cisand trans-linalool oxide in lavandin hydrolat than in essential oil was reported byŚmigielski et al. (2013). Baydar and Kineci (2009) reported the much higher content of linalool oxide in hydrolat than in essential oil, also. Furthermore, two pyranoid linalool oxides, which were not observed in the essential oil, were detected in hydrolat in significant amounts. Also, in the essential oil, co-elution of cis-pyranoid linalool oxide with borneol was not detected. This indicates that the total amounts of these pyranoid linalool oxide isomers from the plant material are dissolved in the hydrolat. Our results of lavandin hydrolat chemical composition are not in accordance with the majority of previously published reports, where linalool was the most abundant compound (42-56 %), followed by cam- phor (13-24 %) and 1,8-cineole (8-24 %), while linalool oxide isomers were detected in smaller amounts (3-6 %) (Baydar and Kineci, 2009;Politi et al., 2020;Yohalem and Passey, 2011). Some other sources reported components such as 1,8-cineole and camphor as the most dominant in lavandin hydrolat (53 and 67 %, respectively) (Garzoli et al., 2020;Jeon et al., 2013). Reports dealing with the chemical composition of true lavender (L. angustifolia) hydrolats also indicate a high content of linalool (26-52 %) (Kaloustian et al., 2008;Prusinowska et al., 2016;Śmigielski et al., 2013). However, none of the previously published reports list linalool oxides as the dominant compounds in the hydrolat, while pyranoid types of linalool oxides were not even mentioned. These differences can arise as a consequence of different starting plant material, as well as different distillation techniques. Kunicka-Styczyńska et al.
The first extraction with n-hexane had the highest yield of all dissolved compounds, while each subsequent extraction had a decreasing amount. The recovery of the compounds from the hydrolat did not proceed uniformly during the extractions. The number of extracted compounds decreased from the first to the fourth extraction, after which traces of five compounds were retained until the last extraction. Those compounds were 1,8-cineole, cisand trans-furanoid linalool oxides, and cisand trans-pyranoid linalool oxides. It may be important to note here that borneol did not co-eluted with cis-pyranoid linalool oxide after the third extraction. Figure 1 shows the n-hexane recovery of the eight most dominant compounds from lavandin hydrolat. Hereby, the recovery ratio of total compounds yield after the third, fifth, and eighth extractions is presented here (88 %, 96 %, and 99 %, respectively). Śmigielski et al. (2013) also reported higher volatile compounds recovery with an increased number of extraction cycles. They also reported that, extraction of 97 % of the volatile compounds from the hydrolat was achieved with five LLE cycles.
In Figure 2, chromatograms of essential oil and chosen hydrolat extraction cycles (1 st , 5 th , and 10 th ) have been shown. This presentation provides insight into the similarity of the chemical composition of essential oil and hydrolat, as well as the gradual depletion of compounds from aqueous solution in higher extraction cycles. The main weakness of this method is that even after ten consecutive n-hexane extractions of volatile compounds from the hydrolat, not all components are depleted to level below the detection on the gas chromatogram. Therefore, other non-polar solvents should be explored in further research. Furthermore, the choice of aliphatic internal standard could be further considered since hexadecane (C16) in our GC method elutes at a time very close to eluation time of trans-caryophyllene (RT~40 min, Figure 2A). This did not interfere with the hydrolat analysis, as only oxigenated monoterpene compounds dissolved in the hydrolat and no traces of sesquiterpenes were detected, but it would be desirable to design an internal standard elution at the time where other components do not elute nearby.

Combined fractions
In order to verify the obtained results of quantification of volatile compounds in individual extraction cycles, we quantified the extractions combined into two groups of 1-5 cycles and 6-10 cycles ( Table 2). The chemical composition of the combined extractions differed slightly from that of the individual fractions study. The most important difference is that the entire amount of 1,8-cineole was extracted in the first five cycles, while the lavander lactone was abundant in the second five cycles. This discrepancy may be due to sampling, as a new distillation was performed for grouped fractions yield validation purposes. However, the total yield of extracted volatile compounds from the hydrolat almost coincides with the total yield of components from the ten cycles of the dynamic study (2174.2 mg/L and 2251.4 mg/L). Judging by the cumulative recovery, it can be concluded that in the first 5 cycles more than 95 % of the total yield (from 10 cycles) of extracted volatile compounds has been collected.

CONCLUSION
The chemical composition of the lavandin hydrolat showed high similarity with its essential oil. However, analysis revealed that the volatile compounds dissolved in water exclusively belonged to the class of oxygenated monoterpenes. In ten consecutive liquid-liquid extractions of lavandin hydrolat total amount of 2174.2 mg/L extracted compounds has been quantified. The most dominant compounds in lavandin hydrolat were cisand trans-furanoid linalool oxide (676.3 and 634.1 mg/L, respectively), followed by much smaller amounts of linalool, camphor, and 1,8-cineole (167.6, 157.0, and 148.2 mg/L, respectively). The number of extracted compounds decreased from the first to the fourth extraction, after which traces 1,8-cineole, cisand trans-furanoid linalool oxides, and cisand trans-pyranoid linalool oxides were retained until the last extraction. Cumulative recoveries of total compounds yield after the third, fifth, and eighth extractions were 88 %, 96 %, and 99 %, respectively. Method verification confirmed that in the first 5 cycles more than 95 % of the total yield (from 10 cycles) of extracted volatile compounds can be collected. Therefore, based on the results of this study, for the purpose of volatile compounds quantification in lavandin hydrolat 5 cycles of n-hexane liquid-liquid extraction can be recommended.