In-Depth Qualitative Analysis of Lime Essential Oils Using the Off-Line Combination of Normal Phase High Performance Liquid Chromatography and Comprehensive Two-Dimensional Gas Chromatography-Quadrupole Mass Spectrometry

The present research is focused on the in-depth qualitative analysis of three types of lime essential oil (EO), viz., Key (A and B) and Persian, using the off-line combination of normal phase high performance liquid chromatography (NP-HPLC) and comprehensive two-dimensional gas chromatography–quadrupole mass spectrometry (GC × GC-QMS). The first analytical dimension (NP-HPLC) was exploited for the isolation of the hydrocarbon constituents from the oxygenated ones. Each fraction was then reduced in volume and analyzed using (cryogenic modulation) GC × GC-QMS. Peak assignment was carried out through the combined use of mass spectral database and linear retention index matching processes. The powerful four-dimensional technology enabled the separation and identification of a very high number (153) of lime essential oil volatile compounds.


Introduction
Two varieties of sour lime, namely Key or Mexican (Citrus aurantifolia Swingle) and Persian (Citrus latifolia Tanaka), find wide use in the flavor industry. Distilled Key lime oil is the most common product, with its aroma deriving from transformation processes (hydration, elimination, rearrangement reactions) which occur during the distillation process. Cold-pressed lime oil is characterized by a fragrant citrus aroma and is used in perfumery, as well as in the flavor industry. Different types of cold-pressing processes provide different types of lime oils: (I) a screw press is used to attain a juice-oil-pulp mixture, followed by centrifugation to isolate the essential oil. Such a procedure is used only for Key limes and yields the type A oil; (II) the peel is subjected to gentle grating, with the oil

LC Pre-Separation
LC pre-separations were performed on the lime essential oils using the Shimadzu 5D Ultra-e system (Kyoto, Japan) consisting of: (1) An LC system, equipped with a CBM-20A communication bus module, two LC-30AD dual-plunger parallel-flow pumps, a DGU-20A online degasser, an SPD-M20A photodiode array detector, a CTO-20A column oven, and an SIL-30AC autosampler. Data were acquired by the LC solution v.5.92 software (Shimadzu).
(2) An AOC-5000 auto injector equipped with a dedicated dual side-port syringe, employed as a transfer device (not used in the present investigation). LC fractions were collected by disconnecting the transfer line (linking the outlet of the LC detector to the syringe) from the syringe side.
Prior to GC × GC-QMS injection, the fractions were reduced to a volume of 100 µL (under a gentle stream of nitrogen).

GC × GC-QMS Analysis
All GC × GC-QMS applications were carried out on system consisting of a GC2010 gas chromatograph and a QP2010 Ultra quadrupole mass spectrometer (Shimadzu).
The primary column, an SLB-5 ms 30 m × 0.25 mm ID × 0.25 µm d f column (Merck Life Science), was connected to an uncoated capillary segment (1.5 m × 0.18 mm ID, used to create a double-loop), using an SGE SilTite mini-union (Trajan, Ringwood, Victoria, Australia). The uncoated capillary was then connected to a segment of Supelcowax-10 (100% polyethylene glycol) 1.0 m × 0.10 mm ID × 0.10 µm d f column (Merck Life Science), using another union (Trajan). Modulation was carried out every 5 s by using a loop-type modulator (under license from Zoex Corporation, Houston, TX, USA). The duration of the hot pulse (400 • C) was 400 ms.
GC oven temperature program: 50 • C to 250 • C at 3 • C min −1 . Carrier gas, helium, was supplied at an initial pressure of 173.5 kPa (constant linear velocity). Injection temperature: 250 • C.

Results
As performed in previous research [9], peak identification was carried out through the combined use of MS database spectral searching and LRI information (comparison between the MS database and experimental LRI values). Three levels of identification were defined: level I-a similarity match ≥90% and an experimental LRI value within a ± 5 LRI tolerance window, with respect to the database result; level II-either a similarity match ≥90%, or an experimental LRI value within a ± 5 LRI tolerance window, with respect to the database result (a compound identified in such a manner cannot be characterized by a similarity match <80%, or an experimental LRI value outside a ± 10 LRI tolerance range); level III-a similarity match >75% and an experimental LRI value within a ± 15 LRI tolerance window, with respect to the database result. It must be emphasized that pure standard compounds were not used in the present research to confirm peak identity. However, the combined use of LRI data and MS information is nowadays accepted for the identification of essential oil constituents [12]. Finally, the main scope of the research was to demonstrate the power of the off-line four-dimensional (4D) method for this type of food sample.
After the LC pre-separation step, the two fractions (hydrocarbons and oxygenates) were reduced in volume (to 100 µL) and then subjected to three GC × GC-QMS analyses; the hydrocarbon fraction was analyzed twice, for the monoterpene (M) and sesquiterpene (S) hydrocarbons. For the latter compounds, present in lower quantities compared to the M hydrocarbons, a higher sample volume and lower split ratio were used. Fifty hydrocarbons were identified, considering the three oils: 46, 47, and 47 hydrocarbons in the Key A, Key B, and Persian lime oils, respectively, as shown in Table 1. With regard to the oxygenated compounds, an overall number of 103 constituents were identified: 77, 82, and 48 compounds in the Key A, Key B, and Persian lime oils, respectively, as shown in Table 2. The GC × GC-QMS chromatogram of the oxygenated fraction of the Persian lime oil is shown in four expansions in Figure 1A-D. As can be seen, more than half of the detected peaks in Figure 1A-D were not assigned.
Considering both the hydrocarbons and oxygenates, a total number of 153 constituents were identified in the three oils: 123, 129, and 95 compounds in the Key A, Key B, and Persian lime oils, respectively, as shown in Tables 1 and 2.    Compound not yet identified in cold-extracted laboratory oils [2]. c Compound identified previously in industrially cold-extracted lime oils and cold-extracted laboratory oils, reported since 1980 [2]. d Compound identified in only one of the samples. e Compound identified previously only in cold-extracted laboratory oils [2].  Table 2 for peak identification).

Discussion
The off-line combination of HPLC and GC × GC-QMS, and its application to the detailed qualitative analysis of lime Essential oils (Eos), gave origin to compound-rich chromatograms, due to the possibility of concentrating the two pre-separated fractions (hydrocarbon and oxygenated compounds), and the two fundamental GC × GC characteristics, namely, the enhanced separation  Table 2 for peak identification).

Discussion
The off-line combination of HPLC and GC × GC-QMS, and its application to the detailed qualitative analysis of lime Essential oils (Eos), gave origin to compound-rich chromatograms, due to the possibility of concentrating the two pre-separated fractions (hydrocarbon and oxygenated compounds), and the two fundamental GC × GC characteristics, namely, the enhanced separation power and sensitivity. As mentioned previously, fifty hydrocarbons were identified with the distribution of M, S, and aliphatic hydrocarbons illustrated in the graph reported in Figure 2.  Table 2 for peak identification).

Discussion
The off-line combination of HPLC and GC × GC-QMS, and its application to the detailed qualitative analysis of lime Essential oils (Eos), gave origin to compound-rich chromatograms, due to the possibility of concentrating the two pre-separated fractions (hydrocarbon and oxygenated compounds), and the two fundamental GC × GC characteristics, namely, the enhanced separation power and sensitivity. As mentioned previously, fifty hydrocarbons were identified with the distribution of M, S, and aliphatic hydrocarbons illustrated in the graph reported in Figure 2.  As can be observed, also in Table 1, the hydrocarbon profiles in the three types of lime oils were very similar. Considering the Key A oil, a number of compounds corresponding to 36, 9, and 1 were identified at levels I, II, and III, respectively; with regard to the Key B oil, a number of compounds corresponding to 32, 14, and 1 were identified at levels I, II, and III, respectively; finally, in the Persian oil, 38 and 9 compounds were identified at levels I and II, respectively. It is noteworthy that the LRI values were calculated by considering the total retention time (sum of the first and second dimension retention times) of the most intense modulated peak of both the alkanes and the lime oil hydrocarbons. Furthermore, the MS database LRI values were derived from analyses performed on the same (low polarity) column, as that used in the first analytical dimension. The retention of both the alkanes and the lime oil hydrocarbons, on the short medium-polarity (100% polyethylene glycol) second dimension, was negligible; for such a reason, there was a general good agreement between experimental and database LRI values.
Six hydrocarbons (all aliphatic) reported in Table 1, to the best of the present authors' knowledge, have not been previously reported in the literature (an in-depth investigation was carried out) in a cold-extracted lime oil. Furthermore, γ-elemene (a sesquiterpene) was found for the first time in Persian oil, even though it has been reported in Key A and B oils [2,10]. Five hydrocarbons were found in both types of Key oils (undecane, tetradecane, pentadecane, hexadecane, heptadecane), while six (tetradec-1-ene, tetradecane, γ-elemene, pentadecane, hexadecane, heptadecane) were present in the Persian oil.
The chemical class distribution of the 103 oxygenated compounds identified in the lime oils is illustrated in the graph shown in Figure 3. out) in a cold-extracted lime oil. Furthermore, γ-elemene (a sesquiterpene) was found for the first time in Persian oil, even though it has been reported in Key A and B oils [2,10]. Five hydrocarbons were found in both types of Key oils (undecane, tetradecane, pentadecane, hexadecane, heptadecane), while six (tetradec-1-ene, tetradecane, γ-elemene, pentadecane, hexadecane, heptadecane) were present in the Persian oil.
The chemical class distribution of the 103 oxygenated compounds identified in the lime oils is illustrated in the graph shown in Figure 3. The number of identified compounds was higher in the Key oils compared to the Persian one. Considering the Key A oil, a number of compounds corresponding to 46, 54, and 23 were identified at levels I, II, and III, respectively; with regard to the Key B oil, a number of compounds corresponding to 36, 73, and 20 were identified at levels I, II, and III, respectively; finally, in the Persian oil, 41, 46, and 8 compounds were identified at levels I, II, and III, respectively. Compared to The number of identified compounds was higher in the Key oils compared to the Persian one. Considering the Key A oil, a number of compounds corresponding to 46, 54, and 23 were identified at levels I, II, and III, respectively; with regard to the Key B oil, a number of compounds corresponding to 36, 73, and 20 were identified at levels I, II, and III, respectively; finally, in the Persian oil, 41, 46, and 8 compounds were identified at levels I, II, and III, respectively. Compared to the hydrocarbons, and in percentage terms, many more compounds were identified at levels II and III. Such an occurrence was, in part, due to the strong interaction of specific oxygenated compounds (e.g., alcohols) on the second dimension column, causing an increased divergence between the experimental and database LRIs.
After an in-depth investigation in the literature, no information was found on 65 compounds present in Table 2 and related to cold-pressed lime oil. Additionally, no previous description was found for the presence of tridecanal in Key B oil, even though it was identified in all the three oils [2]. Finally, (E)-nerolidol (a sesquiterpene alcohol) and dodecyl acetate were found in all the three oils, even though they have not been previously related to Persian lime oil [2].
To conclude, the applied LC//GC × GC-QMS method has enabled the in-depth elucidation of the chemical profile of three types of cold-pressed lime essential oils. The proposed method allows the formation of highly informative and ordered elution patterns that can be exploited for the creation of a fingerprint database as a support for quality assurance.
To the best of the authors' knowledge, many volatiles are here related to such samples for the first time. It cannot obviously be excluded that, in cases, peak identification may not be correct (especially for level III identifications), and that compounds present in the literature related to cold-pressed lime oil have been missed. Even so, the 4D method herein proposed is a powerful analytical not only for citrus (and non-citrus) essential oil analysis, but also in other areas of food research. For example, the 4D technology has been used for the determination of mineral oil contamination in baby foods [13].