Commercial Helichrysum italicum Essential Oils and Hydrosols from Adriatic and Continental Croatia: Quality Assessment and Chemical Composition

Abstract

Immortelle (Helichrysum italicum (Roth) G. Don, family Asteraceae) essential oils (HiEOs) and hydrosols (HiHYs) are widely used in cosmetic, pharmaceutical, and agricultural formulations. However, their composition and quality vary depending on geographical origin and production practices, while standardized reference values—particularly for hydrosols—are still lacking. The aim of this study was to investigate and compare the physicochemical properties and chemical composition of commercial HiEOs and HiHYs from the Adriatic and continental regions of Croatia. Samples were analysed using standard pharmacopoeial methods and gas chromatography–mass spectrometry (GC–MS). Physicochemical analyses (relative density, acid value, refractive index, pH, turbidity, and essential oil content) showed that all samples were within generally accepted quality ranges, with no significant differences observed between regions using the Mann–Whitney U test. HiEOs were dominated by sesquiterpene hydrocarbons (53.15–55.60%), whereas HiHYs contained predominantly oxygenated monoterpenes (43.54–69.86%). The main compounds identified in both fractions were α-pinene, neryl acetate, γ-curcumene, and β-selinene, which formed a consistent chemical signature and served as practical biomarkers for the quality of H. italicum EO and hydrosol. Principal Component Analysis (PCA) distinguished sample groupings based on physicochemical properties and chemical composition, indicating regional variability without exceeding accepted quality limits. This study presents the first comparative dataset of Croatian commercial HiEOs and HiHYs, and defines practical parameter ranges to support standardized specifications, ensure consistent quality, and enhance the industrial applicability of immortelle-based products.

1. Introduction

Immortelle (Helichrysum italicum, (Roth) G. Don, Asteraceae) is an aromatic and medicinal plant adapted to sandy, stony, and dry areas of the Mediterranean region [1]. The name derives from the Greek words helios (sun) and chrysos (gold), referring to the golden-yellow colour of its flowers [2]. It is a perennial shrub, 30–70 cm in height, with woody stems, linear-lanceolate grey-green leaves, and clustered yellow flower heads, blooming from May to June (Figure 1) [3,4].

Helichrysum italicum has traditionally been used to treat respiratory, digestive, and dermatological disorders [1]. Its essential oil (EO), produced and stored in glandular trichomes and epidermal structures of the inflorescences and leaves, is the plant’s most valuable product [5]. The yield and composition of EO are influenced by geographical origin, soil type, altitude, climate conditions, sun exposure, vegetative stage, cultivation methods, harvesting time, and distillation methods [6,7,8,9,10,11].

Due to its broad biological activities, including anti-inflammatory, antioxidant, antimicrobial, antidiabetic, insecticidal, and antineoplastic effects [12,13,14,15,16,17,18,19,20,21,22,23], H. italicum essential oil (HiEO) is widely used in the pharmaceutical, cosmetic, nutraceutical, and fragrance industries [24,25]. It also shows potential for reducing skin irritation, protecting against skin degeneration, supporting wound healing, and delaying skin aging [15,26,27,28,29,30]. The essential oil of H. italicum possesses a complex chemical profile characterized by monoterpenes (e.g., α-pinene, limonene, nerol, neryl acetate), sesquiterpenes (e.g., α- and β-selinene, γ-curcumene), and various phenolic compounds, including flavonoids, acetophenones, phloroglucinols, tremetones, coumarins, and polyphenolic acids [25,31,32].

In contrast to the extensively studied HiEO, the hydrosol of H. italicum (HiHY) remains largely unexplored. Hydrosols, also known as hydrolates, are aromatic aqueous solutions obtained as by-products of steam distillation during EO extraction [33]. They predominantly contain hydrophilic oxygenated compounds, including monoterpene alcohols, aldehydes, ketones, and sesquiterpene alcohols [34,35,36]. In contrast, lipophilic hydrocarbon monoterpenes are present in lower concentrations [33]. Typically, hydrosols (HYs) contain less than 1 g/L (0.10%) of water-soluble EO constituents [33,35].

Owing to their organoleptic properties and biological activities (antifungal, antibacterial, antioxidant, anti-inflammatory) [37,38], hydrosols are used in cosmetics, perfumery, aromatherapy, food preservation, and organic agriculture [33,34,35,38,39]. Hydrosol (HiHY) has demonstrated antioxidant and antimicrobial activity, with reports suggesting potential synergistic effects with antibiotics [38]. The main compounds of immortelle HY are monoterpenes and sesquiterpenes [36,39].

Despite increasing commercial interest, there is a lack of standardized quality parameters for both HiEO and HiHY. Accurate knowledge of their physical and chemical properties is essential for quality control, safety assurance, and product standardization. The main objective of this study was to compare the physicochemical parameters of commercial H. italicum essential oils (HiEOs) and hydrosols (HiHYs) originating from two geographically and climatically distinct regions of Croatia: the Adriatic and the continental region. For quality assessment, samples collected from the Croatian market over one year were analysed for six physicochemical indicators: relative density, refractive index, acid value, pH, turbidity, and EO content in HY. In HiEO samples, three key indicators—relative density, refractive index, and acid value—were determined. The results of the physicochemical parameters were evaluated against available general official standards for EO and HY, as well as relevant literature, due to the absence of standardized reference values for HiEO and HiHY. Additionally, a comparative analysis of selected Adriatic and continental samples was conducted to assess regional variation.

2. Materials and Methods

2.1. Reagents and Materials

Standard buffer solutions (pH 4.00 and pH 7.00), ethanol (96%), phenolphthalein (1% ethanol solution), sodium chloride (NaCl), and potassium hydroxide (KOH, 0.1 M) were obtained from Kemika (Zagreb, Croatia). All reagents were of analytical grade. Ultrapure deionised water for sample preparation was produced using a Milli-Q purification system (Millipore Corp., Bedford, MA, USA). Sample masses were measured using a Mettler Toledo XP205 analytical balance (Mettler-Toledo GmbH, Giessen, Germany) with a readability of 0.01 mg.

2.2. Samples

A total of 24 H. italicum preparations (15 HiHY and 9 HiEO) were collected in 2024 from the Croatian market, including specialized retail outlets, regional trade fairs, and registered small agricultural holdings. All samples were stored at room temperature until analysis. By geographical origin, the HiHY set comprised 9 Adriatic and 6 continental samples, while the HiEO set included 5 Adriatic and 4 continental samples (

Table 1. Commercial samples of immortelle (H. italicum) hydrosols (HiHY) and essential oils (HiEO) from the Adriatic and continental regions of Croatia.

Sample Type	Region	Sample Codes
Hydrosol	Adriatic	HiHY-01–HiHY-09
Hydrosol	Continental	HiHY-10–HiHY-15
Essential oil	Adriatic	HiEO-01–HiEO-05
Essential oil	Continental	HiEO-06–HiEO-09

). Detailed information on all commercial samples is provided in the Supplementary Material (Table S1, Figure S1).

2.3. Physicochemical Parameters

2.3.1. Relative Density

Relative density is defined as the ratio of the mass of a given volume of a substance to the mass of an equal volume of distilled water at the same temperature [40]. Measurements were performed using a 5 mL glass pycnometer at 20 °C. The masses of the empty pycnometer, the pycnometer with distilled water, and the pycnometer with sample (HiEOs and HiHYs) were measured, and the density was calculated using Equation (1):

$$d_{20/20} = {\displaystyle \frac{d \left(\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\right)}{d \left(\mathrm{d}\mathrm{i}\mathrm{s}\mathrm{t}\mathrm{i}\mathrm{l}\mathrm{l}.{\mathrm{H}}_2\mathrm{O}\right)}}= {\displaystyle \frac{\left(p_2 - p\right)}{\left(p_1 - p\right)}}$$

(1)

where d is the relative density, p is the mass of the empty pycnometer, p₁ is the mass of the pycnometer filled with distilled water, and p₂ is the mass of the pycnometer filled with the sample, all expressed in grams.

2.3.2. Refractive Index

The refractive index is defined as the ratio of the sine of the angle of incidence to the sine of the angle of refraction for light of a specified wavelength passing from one medium into another at a constant temperature [40]. The refractive index (nᴰ₂₀) of each sample was measured at 20 °C using a Hanon A670 automatic refractometer (Hanon Advanced Technology Group Co., Ltd., Hong Kong, China) with a resolution of 0.00001 at λ = 589.3 nm (sodium D-line). Calibration was performed using distilled water (nᴰ₂₀ = 1.3329). The instrument determines the refractive index by measuring the critical angle of total internal reflection at the prism–sample interface and calculating nᴰ₂₀ based on the known refractive index of the prism. The refractive index was calculated according to Equation (2):

n^D₂₀ = n_a sin θ

(2)

where n_a is the prism refractive index and θ is the critical angle for total internal reflection, dependent on the refractive index of the sample.

2.3.3. Acid Value

The acid value (AV), defined as the amount of KOH (mg) required to neutralize the free acids in 1 g of sample, was determined by titration. A 1.00 mL aliquot of each HiHY or HiEO sample was titrated with 0.1 M KOH using phenolphthalein as an indicator. Titrations were carried out in a 1:1 (v/v) mixture of 96% ethanol and petroleum ether, previously neutralized with 0.1 M KOH [40]. The AV was calculated according to Equation (3):

$$V={\displaystyle \frac{5.610 V_{0.1 \mathrm{M} \mathrm{K}\mathrm{O}\mathrm{H}}}{m}}$$

(3)

where V_{0.1 M KOH} is the titration volume (mL), and m is the sample mass (g).

2.3.4. pH Value

The pH of HiHY samples was measured at 25 °C using a Mettler Toledo MP220 pH meter (Mettler-Toledo GmbH, Giessen, Germany) with a resolution of 0.01. The instrument was calibrated with pH 4.00 and pH 7.00 buffer solutions. The pH was calculated from the measured electromotive forces (EMFs) using Equation (4):

$$\mathrm{p}\mathrm{H}={\mathrm{p}\mathrm{H}}_{\mathrm{S}}-{\displaystyle \frac{E-E_{\mathrm{S}}}{k}}$$

(4)

where E and E_S are the EMFs of the sample and standard buffer, respectively, and k is the potential change per pH unit (V).

2.3.5. Turbidity

Turbidity measurements of HiHY samples were performed using a Hach 2100P turbidimeter (Hach Company, Loveland, CO, USA), which determines turbidity based on the intensity of light scattered at a 90° angle. Results were expressed in Nephelometric Turbidity Units (NTU). Calibration was carried out using standards of 3, 6, 18, and 30 NTU prepared from a 4000 NTU formazin stock solution.

2.3.6. Essential Oil Content

The EO content (%) in HiHY samples was determined by water distillation for 90 min at 100 °C using an Unger apparatus. Approximately 10 g of each sample was weighed into a 500 mL round-bottom flask. Distilled water (160 mL), sodium chloride (2 g; added to reduce the solubility of volatile constituents and improve phase separation), and glass beads (to ensure even boiling) were added. The EO was collected in a vertical receiver–separator column pre-filled with 1 mL of petroleum ether due to the low EO content (<1%). After distillation, the petroleum ether phase was evaporated using a rotary evaporator (38–40 °C), and the flask containing the dry residue was placed in a desiccator overnight to remove moisture [40]. The EO content was calculated using Equation (5):

$$\mathrm{E}\mathrm{O} \left(\%\right)={\displaystyle \frac{(m_1 - m)}{m_2}}\times 100$$

(5)

where m₁ is the mass of the flask with EO, m is the mass of the empty flask, and m₂ is the mass of the sample in grams.

2.4. Preparation Samples and Identification of Essential Oil and Hydrosol Components

Four essential oil (EO) and four hydrosol (HY) samples of H. italicum were analysed by gas chromatography-mass spectrometry (GC-MS). Samples for GC-MS were selected to represent the four principal Croatian subregions (northern and southern Adriatic; central and eastern continental) and were sourced from different producers to reflect market diversity. This subset was analysed to verify regional representativeness. For hydrosol analyses, 2 mL of HY from each sample was placed in a glass bottle and sealed with a stopper. This was placed in a water bath, and a solid phase micro-extraction (SPME) needle was injected through the septum of the bottle cap. The first part of the process took place at 40 °C for 20 min to allow the compounds to evaporate from the water and settle on the SPME fiber. The fiber was then injected into the gas chromatography (GC) inlet and left for 20 min to ensure that all volatile compounds were reabsorbed by the solid phase microextraction (SPME) fiber into the injection liner. For EO analyses, samples were diluted in pentane/diethyl ether (ratio 2:1), with 2 mg of EO in 1 mL of the organic solution. Then, 2 µL of the diluted EO was injected directly into the liner. Gas chromatographic analysis of both lipophilic and hydrophilic samples was then performed using a gas chromatograph (factory Model 3900 produced by Varian Inc. from Lake Forest, CA, USA) equipped with a flame ionization detector and a mass spectrometer (factory Model 2100T produced by Varian Inc. from Lake Forest, CA, USA). Each sample was analysed on two columns: a non-polar capillary column VF-5ms (30 m × 0.25 mm i.d., 0.25 µm coating thickness produced by Palo Alto, CA, USA) and a polar CP Wax 52 (30 m × 0.25 mm i.d., 0.25 µm coating thickness produced by Palo Alto, CA, USA). The conditions for the VF-5ms column were 60 °C (isothermal) for 3 min, then increased to 246 °C at a rate of 3 °C/min and held for 25 min (isothermal). The conditions for the CP Wax 52 column were 70 °C (isothermal) for 5 min, then increased to 240 °C at a rate of 3 °C/min and held for 25 min (isothermal). The chromatographic conditions were as follows: the flame ionization detector temperature was 300 °C, and the injector temperature was 250 °C. The carrier gas was helium with a flow rate of 1 mL/min. The injection volume was 2 µL, and the split ratio was 1:20. The MS conditions were: ionization voltage, 70 eV; ion source temperature, 200 °C; mass scan range, 40–350 mass units. Individual peaks in all samples were identified by comparing their n-alkane retention indices with those of authentic samples and published studies [41,42], as well as with our libraries from previous work, and other published reports [43,44].

2.5. Statistical Analysis

All measurements were performed in triplicate, and results are expressed as mean ± standard deviation (SD). Before analysis, data distribution was assessed using the Kolmogorov–Smirnov test. As the data distribution deviated from normality, the Mann–Whitney U test was used to analyse differences between physicochemical parameters of HiHYs and HiEOs commercial samples from the Adriatic and continental regions of Croatia. Differences were considered significant at p < 0.05. Principal Component Analysis (PCA) was performed for the physicochemical parameters in hydrosols (relative density, acid value, pH, refractive index, turbidity, and EO content) and in EO (relative density, refractive index, and acid value) of immortelle. PCA was also performed for the ten most abundant compounds (α-pinene, limonene, linalool, terpinen-4-ol, α-terpineol, neryl acetate, β-patchoulene, italicene, γ-curcumene, and β-selinene) in two Adriatic and two continental HiHYs, as well as for the ten most volatile compounds (α-pinene, γ-terpinene, neryl acetate, α-copaene, italicene, ar-curcumene, γ-curcumene, β-selinene, α-selinene, and β-curcumene) of the four HiEOs (two Adriatic and two continental). Before PCA, each variable was standardized. Data analysis was conducted using Statistica software version 7 (StatSoft Inc., Tulsa, OK, USA).

3. Results

3.1. Physicochemical Parameters of HiHYs and HiEOs

In this study, the relative density, acid value, pH, refractive index, turbidity, and essential oil (EO) content of immortelle hydrosols (HiHYs) from coastal and continental regions of Croatia were analysed to assess their purity and quality parameters. The pH, turbidity, and EO content were not applicable for EO samples, as these parameters can be measured only in aqueous systems. The results are presented in

Table 2. Physicochemical indicators of Adriatic H. italicum hydrosols (HiHY) and essential oils (HiEO), expressed as mean ± SD (n = 3).

Sample	Relative Density (d20/20)	Refractive Index (nᴰ20)	Acid Value (mg KOH/g)	pH (25 °C)	Turbidity (NTU)	EO Content in HY (%)
HiHY-01	0.999 ± 0.001	1.3330 ± 0.0002	0.0058 ± 0.0002	3.82 ± 0.03	1.65 ± 0.02	0.13 ± 0.02
HiHY-02	0.985 ± 0.001	1.3329 ± 0.0003	0.0059 ± 0.0002	3.59 ± 0.01	2.88 ± 0.02	0.11 ± 0.02
HiHY-03	1.049 ± 0.002	1.3329 ± 0.0002	0.0055 ± 0.0001	3.52 ± 0.02	1.49 ± 0.02	0.07 ± 0.01
HiHY-04	1.010 ± 0.002	1.3336 ± 0.0002	0.0071 ± 0.0001	5.17 ± 0.02	1.56 ± 0.01	0.08 ± 0.01
HiHY-05	1.019 ± 0.001	1.3329 ± 0.0002	0.0058 ± 0.0002	4.12 ± 0.02	2.93 ± 0.03	0.12 ± 0.03
HiHY-06	0.957 ± 0.002	1.3329 ± 0.0001	0.0061 ± 0.0002	3.98 ± 0.01	3.77 ± 0.02	0.09 ± 0.02
HiHY-07	0.970 ± 0.003	1.3333 ± 0.001	0.0060 ± 0.0002	3.54 ± 0.03	3.12 ± 0.01	0.08 ± 0.02
HiHY-08	0.995 ± 0.001	1.3327 ± 0.002	0.0070 ± 0.0001	3.50 ± 0.02	1.35 ± 0.04	0.07 ± 0.03
HiHY-09	1.005 ± 0.001	1.3338 ± 0.0002	0.0050 ± 0.0001	4.13 ± 0.01	2.58 ± 0.01	0.11 ± 0.01
HiEO-01	0.900 ± 0.001	1.4810 ± 0.0001	0.0120 ± 0.0001	–	–	–
HiEO-02	0.890 ± 0.001	1.4793 ± 0.0001	0.0161 ± 0.0002	–	–	–
HiEO-03	0.875 ± 0.002	1.4649 ± 0.0002	0.0150 ± 0.0001	–	–	–
HiEO-04	0.900 ± 0.001	1.4766 ± 0.0003	0.0234 ± 0.0002	–	–	–
HiEO-05	0.880 ± 0.001	1.4732 ± 0.0001	0.0140 ± 0.0002	–	–	–

–, not applicable.

and

Table 3. Physicochemical parameters of continental H. italicum hydrosols (HiHY) and essential oils (HiEO) expressed as mean ± SD (n = 3).

Sample	Relative Density (d20/20)	Refractive Index (nᴰ20)	Acid Value (mg KOH/g)	pH (25 °C)	Turbidity (NTU)	EO Content in HY (%)
HiHY-10	0.983 ± 0.002	1.3329 ± 0.0001	0.0058 ± 0.0002	3.77 ± 0.03	5.20 ± 0.01	0.08 ± 0.01
HiHY-11	0.983 ± 0.001	1.3330 ± 0.0003	0.0056 ± 0.0002	4.66 ± 0.02	1.18 ± 0.01	0.08 ± 0.01
HiHY-12	1.075 ± 0.002	1.3328 ± 0.0002	0.0054 ± 0.0002	3.43 ± 0.03	1.69 ± 0.02	0.12 ± 0.02
HiHY-13	1.008 ± 0.003	1.3330 ± 0.0001	0.0057 ± 0.0002	4.68 ± 0.02	1.23 ± 0.01	0.03 ± 0.02
HiHY-14	0.982 ± 0.003	1.3329 ± 0.0001	0.0068 ± 0.0001	4.57 ± 0.01	1.86 ± 0.02	0.03 ± 0.01
HiHY-15	1.045 ± 0.002	1.3338 ± 0.0002	0.0050 ± 0.0002	5.80 ± 0.02	3.12 ± 0.01	0.02 ± 0.01
HiEO-06	0.904 ± 0.001	1.4771 ± 0.0002	0.0141 ± 0.0001	–	–	–
HiEO-07	0.890 ± 0.002	1.4790 ± 0.0001	0.0190 ± 0.0001	–	–	–
HiEO-08	0.890 ± 0.002	1.4802 ± 0.0001	0.0201 ± 0.0002	–	–	–
HiEO-09	0.897 ± 0.001	1.4796 ± 0.0002	0.0180 ± 0.0001	–	–	–

–, not applicable.

and are expressed as the mean ± standard deviation of all measurements.

PCAs were conducted for the physicochemical parameters in both hydrosol (Figure 2a) and lipophilic (Figure 2c) samples of immortelle. Principal component (PC) 1 and PC 2 explained 58.71% of the variance for HiHYs and distinguished two clusters. The first cluster comprises samples collected mainly in the Adriatic region (Ha1, Ha2, Ha3, Ha5, Ha6, Ha7, Ha8). The two continental samples included in this group are Hc1 and Hc3. These samples have higher EO content, higher turbidity, and acid value. The second cluster consists of one Adriatic (Ha4) and three continental (Hc2, Hc4, Hc5) samples. These samples have a higher pH value. Two samples (Ha9, Hc6) differ from the others and are characterized by higher relative density and refractive index. The PCA loading plots of the physicochemical parameters for PC 1 and PC 2 are shown in Figure 2b.

The PCA explained 89.97% of the variance in the lipophilic samples of immortelle and distinguished two clusters (Figure 2c). The first cluster comprises two Adriatic (Ea2, Ea4) and three continental (Ec2, Ec3, Ec4) samples, which are characterized by a higher acid value. The second cluster consists of one Adriatic (Ea1) and one continental (Ec1) sample, both with higher relative density and refractive index. The PCA loading plots for the physicochemical parameters for PC 1 and PC 2 are shown in Figure 2d.

PCA distinguished sample groupings based on physicochemical properties, indicating regional variability, although the Mann–Whitney U-test did not show a statistically significant difference between the Adriatic and continental regions of Croatia.

3.2. Identification of Volatile Components in Commercial Samples of Immortelle (H. italicum) Essential Oils and Hydrosols

The results for the composition of the lipophilic fraction (essential oil, HiEO) and the water fraction (hydrosol, HiHY) of all investigated immortelle samples are shown in

Table 4. Phytochemical composition (%), identification and major groups of chemical components of essential oil (EO) of H. italicum.

Component	RIa	RIb	EO Adriatic/HiEO-01	EO Adriatic/HiEO-02	EO Continental/HiEO-06	EO Continental/HiEO-07
Monoterpene hydrocarbons			18.63	8.64	12.84	20.37
α-Pinene *	938	1032	12.98 ± 0.01	2.12 ± 0.01	7.12 ± 0.01	14.46 ± 0.01
α-Fenchene	945	1061	0.56 ± 0.01	–	–	–
Camphene *	962	1059	0.36 ± 0.01	0.81 ± 0.01	0.06 ± 0.01	–
Sabinene	971	1128	–	0.26 ± 0.01	0.16 ± 0.01	–
β-Pinene	982	1113	0.87 ± 0.01	0.43 ± 0.01	1.85 ± 0.01	0.96 ± 0.01
Myrcene	992	1174	0.18 ± 0.01	0.72 ± 0.01	–	0.77 ± 0.01
α-Terpinene	1016	1251	0.23 ± 0.01	0.41 ± 0.01	0.89 ± 0.01	0.59 ± 0.01
p-Cymene	1021	1268	0.11 ± 0.01	0.71 ± 0.03	0.34 ± 0.01	0.17 ± 0.01
β-Phellandrene	1025	1213	0.51 ± 0.01	–	–	–
Limonene	1032	1204	1.01 ± 0.01	0.97 ± 0.01	0.97 ± 0.01	0.43 ± 0.01
γ-Terpinene	1057	1255	1.82 ± 0.01	2.21 ± 0.01	1.45 ± 0.01	2.99 ± 0.01
Oxygenated monoterpenes			21.09	26.65	21.22	15.26
1,8-Cineole	1026	1210	0.21 ± 0.01	1.81 ± 0.01	1.01 ± 0.01	0.34 ± 0.01
cis-Sabinene hydrate	1065	1561	0.52 ± 0.01	0.15 ± 0.01	–	–
Linalool *	1099	1548	1.67 ± 0.01	0.66 ± 0.01	3.86 ± 0.01	0.56 ± 0.01
cis-p-Mentha-2,8-dien-1-ol	1134	1666	–	1.14 ± 0.01	0.75 ± 0.01	1.02 ± 0.01
Borneol *	1176	1719	0.15 ± 0.01	1.69 ± 0.01	0.81 ± 0.01	0.87 ± 0.01
Terpinen-4-ol	1184	1611	0.63 ± 0.01	1.11 ± 0.01	1.04 ± 0.01	1.32 ± 0.01
α-Terpineol	1186	1646	0.32 ± 0.01	1.06 ± 0.01	1.65 ± 0.01	0.46 ± 0.01
Myrtenol	1197	1782	0.42 ± 0.01	–	0.11 ± 0.01	–
endo-Fenchyl acetate	1218	1480	0.12 ± 0.01	–	–	–
Nerol	1226	1786	0.51 ± 0.01	0.85 ± 0.01	0.51 ± 0.01	1.25 ± 0.01
Bornyl acetate	1285	1570	0.41 ± 0.01	0.33 ± 0.01	0.37 ± 0.01	0.22 ± 0.01
Neryl acetate	1358	1692	16.13 ± 0.01	17.85 ± 0.01	11.11 ± 0.01	9.22 ± 0.01
Sesquiterpene hydrocarbons			53.15	54.29	53.91	55.60
α-Copaene	1377	1484	1.22 ± 0.01	1.08 ± 0.01	7.82 ± 0.01	1.81 ± 0.01
β-Patchoulene	1380	1524	1.27 ± 0.01	1.22 ± 0.01	1.04 ± 0.01	0.45 ± 0.01
β-Bourbonene	1383	1508	0.69 ± 0.01	–	1.66 ± 0.01	–
Italicene	1400	1481	1.11 ± 0.12	0.81 ± 0.01	2.01 ± 0.01	8.31 ±0.01
α-Gurjunene	1407	1520	0.46 ± 0.01	–	0.46 ± 0.01	–
cis-α-Bergamotene	1411	1549	–	0.22 ± 0.01	–	2.33 ± 0.01
Caryophyllene	1424	1585	0.98 ± 0.01	0.82 ± 0.01	1.68 ± 0.01	0.84 ± 0.01
β-Farnesene	1454	1639	–	–	0.16 ± 0.01	–
α-Humulene	1456	1654	0.44 ± 0.01	–	–	–
ar-Curcumene	1472	1743	7.62 ± 0.01	4.33 ± 0.01	10.05 ± 0.01	1.08 ± 0.01
γ-Curcumene	1480	1692	27.28 ± 0.01	23.71 ± 0.01	1.21 ± 0.01	7.18 ± 0.01
β-Selinene	1488	1727	2.72 ± 0.01	11.31 ± 0.01	16.71 ± 0.01	23.71 ± 0.01
δ-Selinene	1491	1755	2.51 ± 0.01	2.11 ± 0.01	1.02 ± 0.01	–
Viridiflorene	1496	1697	0.08 ± 0.01	0.13 ± 0.01	–	0.55 ± 0.01
β-Bisabolene	1494	1729	0.22 ± 0.01	0.56 ± 0.01	0.76 ± 0.01	–
α-Selinene	1498	1740	3.42 ± 0.01	3.42 ± 0.01	1.78 ± 0.01	5.66 ± 0.01
Bicyclogermacrene	1500	1718	0.14 ± 0.01	0.26 ± 0.01	–	0.13 ± 0.01
β-Curcumene	1514	1737	2.34 ± 0.01	4.31 ± 0.01	6.41 ± 0.01	3.01 ± 0.01
δ-Cadinene	1517	1745	0.65 ± 0.01	–	1.14 ± 0.01	0.54 ± 0.01
Oxygenated sesquiterpenes			1.67	3.90	1.94	1.97
Caryophyllene oxide *	1581	1955	1.03 ± 0.01	1.56 ± 0.01	0.88 ± 0.01	0.23 ± 0.01
γ-Eudesmol	1632	2135	0.07 ± 0.01	–	–	0.35 ± 0.01
α-Cadinol	1655	2208	0.13 ± 0.01	1.43 ± 0.01	0.25 ± 0.01	0.27 ± 0.01
Selin-11-en-4α-ol	1658	2245	–	0.23 ± 0.01	–	0.45 ± 0.01
α-Bisabolol	1688	2116	–	–	0.81 ± 0.12	0.67 ± 0.01
α-Bisabolol oxide	1748	2511	0.44 ± 0.01	0.68 ± 0.01	–	–
Hydrocarbons			0.21	0.25	3.30	–
Eicosane *	2000	2000	–	0.25 ± 0.01	0.25 ± 0.01	–
Heneicosane *	2100	2100	–	–	–	–
Docosane *	2200	2200	0.13 ± 0.01	–	–	–
Tricosane *	2300	2300	–	–	–	–
Tetracosane *	2400	2400	0.08 ± 0.01	–	2.14 ± 0.01	–
Pentacosane *	2500	2500	–	–	0.91 ± 0.01	–
Total identified (%)			94.75	93.73	93.21	93.2

Retention indices were determined relative to a series of n-alkanes (C8–C40) on the capillary column: RI^a, VF-5MS column, RI^b, CP Wax 52 column; RI, indicates comparison of RIs with those listed in a homemade library, reported in the literature [41], and/or of authentic samples; comparison of mass spectra with those listed in the mass spectral libraries NIST02 and Wiley 7; *, coinjection with reference compounds; –, component not identified.

and

Table 5. Phytochemical composition (%), identification and major groups of chemical components of hydrosol (HY) of H. italicum.

Component	RIa	RIb	HY Adriatic/ HiHY-01	HY Adriatic/ HiHY-02	HY Continental/ HiHY-10	HY Continental/ HiHY-11
Monoterpene hydrocarbons			20.6	5.1	8.33	7.26
α-Pinene *	938	1032	6.56 ± 0.01	0.86 ± 0.01	1.02 ± 0.01	0.72 ± 0.01
α-Fenchene	945	1061	–	0.32 ± 0.01	0.16 ± 0.01	–
Camphene *	962	1059	0.66 ± 0.01	–	0.06 ± 0.01	–
Sabinene	971	1128	–	–	0.16 ± 0.01	0.38 ± 0.01
β-Pinene	982	1113	4.53 ± 0.01	0.55 ± 0.01	1.85 ± 0.01	0.52 ± 0.01
Myrcene	992	1174	0.21 ± 0.01	–	–	–
α-Terpinene	1016	1251	0.24 ± 0.01	1.21 ± 0.01	1.21 ± 0.01	0.88 ± 0.01
p-Cymene	1021	1268	0.61 ± 0.01	–	0.34 ± 0.01	0.44 ± 0.01
β-Phellandrene	1025	1213	0.55 ± 0. 1	0.36 ± 0.01	–	–
Limonene	1032	1204	4.82 ± 0.01	0.92 ± 0.01	2.97 ± 0.01	3.89 ± 0.01
γ-Terpinene	1057	1255	2.42 ± 0.01	0.88 ± 0.01	0.56 ± 0.01	0.43 ± 0.01
Oxygenated monoterpenes			43.54	54.79	47.35	69.86
cis-Sabinene hydrate	1065	1561	0.32 ± 0.01	–	–	–
Linalool *	1099	1548	13.77 ± 0.01	11.21 ± 0.01	1.71 ± 0.01	4.02 ± 0.01
cis-p-Mentha-2,8-dien-1-ol	1134	1666	–	0.64 ± 0.01	1.32 ± 0.01	0.64 ± 0.01
Borneol *	1176	1719	0.92 ± 0.01	–	0.81 ± 0.01	–
Terpinen-4-ol	1184	1611	7.24 ± 0.01	1.34 ± 0.01	1.04 ± 0.01	1.76 ± 0.01
α-Terpineol	1186	1646	1.43 ± 0.01	3.13 ± 0.01	1.65 ± 0.01	8.97 ± 0.01
Myrtenol	1197	1782	0.21 ± 0.01	–	0.11 ± 0.01	–
endo-Fenchyl acetate	1218	1480	0.17 ± 0.01	–	–	–
Nerol	1226	1786	0.37 ± 0.01	0.67 ± 0.01	1.23 ± 0.01	0.27 ± 0.01
Bornyl acetate	1285	1570	1.45 ± 0.01	4.97 ± 0.01	0.37 ± 0.01	2.32 ± 0.01
Neryl acetate	1358	1692	17.66 ± 0.01	32.83 ± 0.01	39.11 ± 0.01	51.88 ± 0.01
Sesquiterpene hydrocarbons			28.58	34.55	38.68	15.47
α-Copaene	1377	1484	0.24 ± 0.01	–	–	0.33 ± 0.01
β-Patchoulene	1380	1524	5.84 ± 0.01	0.64 ± 0.01	6.66 ± 0.01	2.92 ± 0.01
β-Bourbonene	1383	1508	0.53 ± 0.1	–	–	–
Italicene	1400	1481	2.31 ± 0.01	4.68 ± 0.01	4.21 ± 0.01	1.12 ± 0.01
α-Gurjunene	1407	1520	0.04 ± 0.01	–	0.89 ± 0.01	–
Caryophyllene	1424	1585	1.01 ± 0.01	0.68 ± 0.01	1.68 ± 0.01	0.39 ± 0.01
β-Farnesene	1454	1639	–	–	0.16 ± 0.01	–
α-Humulene	1456	1654	0.46 ± 0.01	–	–	–
ar-Curcumene	1472	1743	5.62 ± 0.01	4.97 ± 0.01	0.31 ± 0.01	–
γ-Curcumene	1480	1692	6.91 ± 0.01	11.66 ± 0.01	12.23 ± 0.01	5.04 ± 0.01
β-Selinene	1488	1727	2.68 ± 0.01	4.32 ± 0.01	6.69 ± 0.01	2.35 ± 0.01
δ-Selinene	1491	1755	0.65 ± 0.01	5.67 ± 0.01	1.02 ± 0.01	1.89 ± 0.01
β-Bisabolene	1494	1729	0.22 ± 0.01	–	–	0.34 ± 0.01
Viridiflorene	1496	1697	–	–	–	0.14 ± 0.01
α-Selinene	1498	1740	1.44 ± 0.01	–	1.78 ± 0.01	–
Bicyclogermacrene	1500	1718	0.14 ± 0.01	0.44 ± 0.01	–	0.61 ± 0.01
β-Curcumene	1514	1737	0.45 ± 0.01	0.62 ± 0.01	1.91 ± 0.01	0.34 ± 0.01
δ-Cadinene	1517	1745	0.04 ± 0.01	0.87 ± 0.01	1.14 ± 0.01	–
Oxygenated sesquiterpenes			2.87	2.36	1.94	2.12
Caryophyllene oxide *	1581	1955	1.42 ± 0.01	1.37 ± 0.01	0.88 ± 0.01	0.38 ± 0.01
γ-Eudesmol	1632	2135	0.67 ± 0.01	0.31 ± 0.01	–	0.54 ± 0.01
α-Cadinol	1655	2208	0.15 ± 0.01	–	0.25 ± 0.01	0.43 ± 0.01
Selin-11-en-4α-ol	1658	2245	–	–	–	0.77 ± 0.01
α-Bisabolol	1688	2116	–	0.68 ± 0.01	0.81 ± 0.01	–
α-Bisabolol oxide	1748	2511	0.63 ± 0.01	–	–	–
Total identified (%)			95.59	96.8	96.3	94.71

Retention indices (RI) determined relative to a series of n-alkanes (C8–C40) on the capillary column: RI^a, VF-5MS column, RI^b, CP Wax 52 column; RI, comparison of RIs with those listed in a homemade library, reported in the literature [41], and/or authentic samples; comparison of mass spectra with those listed in the mass spectral libraries NIST02 and Wiley 7; *, coinjection with reference compounds; –, component not identified.

. The phytochemical composition of HiEOs is categorised into five classes: monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpene hydrocarbons, oxygenated sesquiterpenes, and hydrocarbons (Table 4). Hydrocarbons were not identified in the hydrosol samples, so the compounds are divided into four classes (Table 5). The compounds α-pinene, ar-curcumene, neryl acetate, γ-curcumene and β-selinene are the most abundant in all HiEOs (Table 4). These are also the main compounds in the studied HiHY samples (Table 5). Other significant differences between the HiEO and HiHY fractions of immortelle are that α-pinene was present at a lower percentage in the HiHY samples than in the HiEO samples, while neryl acetate was more abundant in the HiHY samples (Table 4 and Table 5). Linalool was identified at a higher percentage in the hydrosol fractions (Table 5) compared to the corresponding oil fractions, except in the HiEO-06 sample, where a higher percentage of linalool was found in the oil fraction (Table 4). Further comparison of the compositions of all HiEO and HiHY samples shows that α-copaene is present in all oils but only in two hydrosol samples (HiHY-01 and HiHY-11), with values less than 1% (Table 5).

PCA was conducted on the most abundant compounds of the HiHYs (Figure 3a). PC 1 and PC 2 explained 81.79% of the variance and separated a cluster consisting of one Adriatic (Ha4) and one continental (Hc1) hydrosol sample. These samples are characterized by a higher content of italicene, γ-curcumene, and β-selinene. The samples Ha1 (characterized by a higher content of α-pinene, terpinen-4-ol, linalool, and β-patchoulene) and Hc6 (characterized by a higher content of α-terpineol and neryl acetate) differ significantly from the other samples. The PCA loading plots of the compounds for PC 1 and PC 2 are shown in Figure 3b.

PCA was also conducted on the most abundant compounds in the HiEO samples (Figure 3c). PC 1 and PC 2 explained 88.34% of the variance in the HiEO samples and distinguished a cluster consisting of samples Ea1 and Ea4 from the Adriatic region (Figure 3c). This cluster is characterized by a higher content of γ-curcumene and neryl acetate. Two samples from the continental region (Ec1, Ec6) differ from the other samples. Sample Ec1 is characterized by a higher content of α-copaene, β-curcumene, and ar-curcumene, while sample Ec6 contains a higher content of italicene, β–selinene, and α-pinene. The PCA loading plots of the compounds for PC 1 and PC 2 are shown in Figure 3d.

4. Discussion

4.1. Quality Parameters

Given the increasing use of immortelle EOs and HYs in medical, cosmetic, food, and agricultural applications, there is a growing need to establish standardized analytical protocols and define reference values for their key physicochemical parameters, which are regulated by the International Organization for Standardization (ISO) [45] and pharmacopoeial guidelines. According to pharmacopoeial standards, the quality of EOs is typically assessed based on physical properties (relative density, refractive index, optical rotation, freezing and boiling points, and solubility) and chemical parameters (acid value, peroxide value, and saponification number) [40,46]. Analysis of these properties in HYs and EOs can provide valuable insight into product purity, stability, and potential contamination. As pH and turbidity apply only to aqueous systems, these measurements were not included in the EO analysis. In the absence of specific reference values for HiEOs and HiHYs, the experimental results obtained in this study were compared with values reported for related specimens in the literature [37,47].

Relative density (d_20/20) is closely related to the chemical composition of the EOs. Essential oils rich in terpenes typically have densities below 0.900, while those containing aromatic compounds may exceed 1.000 [48]. The measured densities of HiEOs in this study ranged from 0.875 to 0.900 (Adriatic region) and 0.890 to 0.904 (continental region). The European Pharmacopoeia reports comparable relative density values for lavender (Lavandula angustifolia L.) essential oil (0.878–0.892) and rosemary (Salvia rosmarinus Spenn.; syn. Rosmarinus officinalis L.) essential oil (0.895–0.920) [40]. As expected, the relative densities of HiHYs were higher, ranging from 0.957–1.049 (Adriatic) and 0.982–1.075 (continental), reflecting their predominantly aqueous composition and the low concentrations of dissolved volatile compounds (<0.1% w/v). These values are comparable to those reported for bay laurel (Laurus nobilis L.) hydrosols (0.998) [49].

The refractive index (n^D₂₀) is a simple and reliable indicator of the purity and quality of fatty and essential oils. The refractive index values for Adriatic (1.4649–1.4810) and continental (1.4771–1.4802) HiEO samples are comparable to those of Lamiaceae EO, such as lavender (1.455–1.466) and rosemary (1.464–1.473) [40]. Given the predominantly aqueous composition of hydrosols, their refractive index is expected to be close to that of water (1.3330). The refractive index values measured for Adriatic and continental HiHYs (1.3327–1.3338 and 1.3328–1.3338, respectively) were consistent with those reported for other authentic hydrosols [47]. Similar values have been documented for bay laurel hydrosol (1.333–1.334) and rosemary hydrosol (1.334–1.336) [49,50].

The acid value (AV) serves as an indicator of oil degradation. Elevated values are often associated with prolonged or improper storage, as aldehyde oxidation and ester hydrolysis lead to the formation of free fatty acids [51]. According to the European Pharmacopoeia, the acid value of EOs, determined from 5.00 g of lavender and rosemary essential oils, should not exceed 1.0 [40]. In our study, the AV for the Adriatic and continental HiEOs ranged from 0.0120–0.0234 and 0.0141–0.0201 mg KOH/g, respectively, which is well within the acceptable pharmacopoeial values. As no pharmacopoeial standards exist for determining the acid value of hydrosols, the validated pharmacopoeial procedure used for essential oils was applied. As expected, lower acid values of 0.006 mg KOH/g were observed for both Adriatic (0.0050–0.0071 mg KOH/g) and continental (0.0050–0.0068 mg KOH/g) HiHYs samples, which is consistent with the acid values of bay laurel hydrosol (0.0088–0.0096 mg KOH/g) [47]. Although some compounds (e.g., neryl acetate) may undergo hydrolysis and increase the acid value, the very low AVs measured in both HiEOs and HiHYs indicate minimal ester hydrolysis, which does not influence the observed AVs.

The pH value serves as a quality indicator, reflecting the acidity of the sample and indicating potential degradation processes [40]. In HYs, lower pH improves antibacterial properties and storage stability. The pH of HY samples ranges from 4.5 to 5.5, depending on botanical origin and chemical composition. Literature data for HiHY report pH values between 3.5 and 3.8 [52]. The pH values determined in this study for Adriatic (3.50–5.17) and continental (3.43–5.80) HiHY samples are slightly higher than those previously reported for bay laurel hydrosol (3.62–4.09), but still fall within the general pH range observed for HYs [33].

Although the pH of the analysed HiHY samples varied, all values were within the general range reported for hydrosols. Given its importance for microbial stability and product quality, a practical pH range of 4.5–5.5 may be appropriate for commercial H. italicum hydrosols and may serve as a reference for future quality specifications.

Turbidity is an indicator of water and HYs quality, reflecting physical purity and possible contamination [53]. Although no specific turbidity standards exist for HiHYs, the World Health Organization (WHO) guidelines for drinking water suggest values below 5 NTU [54]. The measured turbidity values for Adriatic (1.35–3.77 NTU) and continental (1.18–5.20 NTU) HiHY samples fall within these limits, indicating acceptable quality. The same range was observed for bay laurel hydrosols (0.74–3.71 NTU) [47].

The EO content in HiHY is influenced by geographical origin, soil, altitude, and climate [7,8,9]. Reported EO yields in Croatian H. italicum samples range from 0.02% to 0.32% [9,10,24]. As hydrosols contain mainly hydrophilic volatiles and traces of EO, lower values are expected, as confirmed in this study, with ranges from 0.07–0.13% for Adriatic and 0.02–0.12% for continental HiHYs. Despite their low concentration, EO components in HYs contribute to their overall biological activity [55,56].

According to the available quality requirements, all HiEOs and HiHYs complied with prescribed standards, and no significant differences were observed in the measured physicochemical parameters between Adriatic and continental HiEOs or HiHYs. Given the established ranges of physicochemical parameters for commercial HiEOs and HiHYs, which are consistent with pharmacopoeial requirements and literature values for related species, these ranges may serve as a basis for defining standardized reference values and improving the overall quality control of immortelle-based products on the market.

4.2. Phytochemical Analysis

The phytochemical composition of free volatile compounds in commercial H. italicum samples from Croatia was investigated to provide a preliminary, regionally balanced comparison between the Adriatic and continental regions. Eight representative samples were selected for this purpose: four EOs and four HYs originating from different locations within the Adriatic and continental regions (Table 4 and Table 5). All samples were analysed by GC-MS. Table 4 lists the compounds identified in the essential oils of the studied immortelle samples, categorized into five classes. Table 5 lists the compounds identified in the hydrosol composition of the investigated immortelle samples, divided into four classes. To aid interpretation, the phytochemical fingerprints are presented alongside the physicochemical parameters in Section 4.1. The predominance of sesquiterpene hydrocarbons in HiEOs and oxygenated monoterpenes in HiHYs corresponds with the observed relative densities, refractive indices, and low acid values typical of authentic, properly distilled H. italicum products.

Hydrocarbons were not identified in the hydrosols, as expected due to their low water solubility. The highest proportion of hydrocarbons in essential oil was found in the continental (HiEO-06) sample (3.3%), while in the other two Adriatic EO samples, the values were low (below 1%). No hydrocarbons were identified in the continental EO (HiEO-07) sample (Table 4). This variation reflects natural chemotypic differences and distillation-related factors that influence the distribution of volatile classes in H. italicum [57,58].

The classes of monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpene hydrocarbons, and oxygenated sesquiterpenes are common to all analysed EO and HY samples.

The class of sesquiterpene hydrocarbons predominates in the composition of all EO samples (53.15–55.60%) (Table 4). This class is also dominant in the composition of immortelle EO in the study by Andjić et al. [59]. In the four hydrosol samples, the predominant class is oxygenated monoterpenes, ranging from 43.54% in the Adriatic (HiHY-01) sample to 69.86% in the continental (HiHY-11) sample (Table 5). Monoterpenes are also prevalent in the hydrosol composition of one-year-old and two-year-old cultivated immortelle species at the Institute of Hop Growing and Brewing of Slovenia [36]. This compositional polarity (sesquiterpenes in EOs versus oxygenated monoterpenes in HYs) further explains the measured physicochemical parameters, such as the higher refractive index of EOs compared to HYs and the uniformly low AV, which support product authenticity, appropriate distillation conditions, low chemical degradation, and overall product purity.

The monoterpene hydrocarbon components: α-pinene, β-pinene, α-terpinene, limonene, and γ-terpinene are the most abundant constituents in the EO and HY composition of the studied immortelle samples. α-pinene was identified in significantly higher amounts in oil samples than in hydrosols (Table 4 and Table 5). Comparing our data with those reported for immortelle oil and hydrosol extracts from Bosnia and Herzegovina, the proportion of α-pinene in the oil extracts is three times higher than in the hydrosol [60]. Across all eight samples, the most frequently identified compound is neryl acetate, which was found in the highest amount in the continental (HiHY-11) sample, at 51.88% (Table 5).

Ninčević et al. [12] reported that neryl acetate was identified in Tuscan immortelle oil at 10–22%, values similar to our results. In general, these authors, referring to previous research, stated that immortelle oils from Croatia have a composition similar to oils from Italy [12]. The analysis of Corsican immortelle oils revealed that neryl acetate was the dominant compound, with its percentage varying depending on the vegetation period, ranging from 15.8% in plants at the early shoot phase to 42.5% at full bloom. Neryl acetate is an acetate ester formed by the condensation of the hydroxyl group of nerol with the carboxyl group of acetic acid. Although neryl acetate is a stable compound, the oil must be stored properly to prevent its decomposition and the formation of acetic acid [61,62].

Key markers (neryl acetate, α-pinene, γ-curcumene, β-selinene) fall within the ranges reported for high-quality H. italicum products and are consistent with recognized Mediterranean chemotypes, thus linking the chemical composition to established EO and HY quality criteria. Neryl acetate, a characteristic authenticity marker for H. italicum, also corresponds with the low AVs observed, indicating minimal ester hydrolysis and appropriate storage conditions (Section 4.1). Sesquiterpene hydrocarbons are the most significant class, accounting for over half of the total identified compounds in all four investigated HiEOs (Table 4). Sesquiterpene hydrocarbons identified in all essential oil and hydrosol samples are β-patchoulene, italicene, ar-curcumene, γ-curcumene, β-selinene, and β-curcumene (Table 4 and Table 5). Comparing seasonal variations in immortelle essential oil cultivars from Serbia, the authors concluded that the γ-curcumin + ar-curcumin chemotype is typically the dominant chemotype in the Western Balkan region, suggesting that climatic conditions and precipitation influence curcumin synthesis [63]. The authors of this article reached the same conclusions as other researchers, such as Mancini et al. [64], who investigated volatile compounds from immortelle, and noted that the phytochemical composition varies depending on factors such as geography, the plant’s vegetation period, and the preparation of plant material for research.

Although regional differences were detected, all samples remained within recognized chemotypic profiles and did not deviate from the physicochemical ranges defined in Section 4.1. This coherence between chemical fingerprinting and physicochemical parameters supports product authenticity and uniform quality across regions.

5. Conclusions

In this study, all the evaluated physicochemical parameters of Helichrysum italicum essential oils (HiEOs) and hydrosols (HiHYs) were within the expected quality ranges, with no indication that geographical origin adversely affected product purity or stability.

Comparative GC–MS analysis of free volatile compounds in commercial HiEOs and HiHYs from the Adriatic and continental regions of Croatia showed consistent chemical profiles, while also highlighting clear compositional differences between HiEOs and HiHYs. Helichrysum italicum essential oils were dominated by sesquiterpene hydrocarbons, whereas HiHYs contained high proportions of oxygenated monoterpenes. Despite these differences, several key constituents (such as neryl acetate, α-pinene, γ-curcumene, and β-selinene) were present in all samples. These compounds define a consistent chemical signature of high-quality H. italicum products and serve as practical biomarkers for EO and hydrosol quality assessment. Their alignment with the established physicochemical ranges (such as characteristic refractive indices and low acid values) indicates authenticity, proper distillation, and minimal degradation, thus providing reliable thresholds for routine quality control.

Although PCA revealed clustering related to chemical composition, these differences reflected natural regional variation in secondary metabolite profiles and remained within typical ranges for H. italicum. Consequently, the observed compositional patterns did not affect the overall quality of the products.

These findings confirm the high quality of Croatian immortelle products. Furthermore, the physicochemical parameter ranges determined in this study provide the first solid basis for defining standardized reference values tailored specifically to H. italicum essential oils and hydrosols, thereby supporting more consistent quality control and strengthening the market value of immortelle-based products in the cosmetic, food, and agronomic industries.