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Open AccessArticle

Quantitative Analysis of Fragrance and Odorants Released from Fresh and Decaying Strawberries

Atmospheric Environment Laboratory, Department of Environment & Energy, Sejong University, Seoul 143-747, Korea
Palo Duro Research Center, West Texas A&M University, Canyon, TX 79016, USA
Author to whom correspondence should be addressed.
Sensors 2013, 13(6), 7939-7978;
Received: 18 April 2013 / Revised: 7 June 2013 / Accepted: 13 June 2013 / Published: 20 June 2013


The classes and concentrations of volatile organic compounds (VOC) released from fresh and decaying strawberries were investigated and compared. In this study, a total of 147 strawberry volatiles were quantified before and after nine days of storage to explore differences in the aroma profile between fresh strawberries (storage days (SRD) of 0, 1, and 3) and those that had started to decay (SRD = 6 and 9). In terms of concentration, seven compounds dominated the aroma profile of fresh strawberries (relative composition (RC) up to 97.4% by mass, sum concentration): (1) ethyl acetate = 518 mg∙m−3, (2) methyl acetate = 239 mg∙m−3, (3) ethyl butyrate = 13.5 mg∙m−3, (4) methyl butyrate = 11.1 mg∙m−3, (5) acetaldehyde = 24.9 mg∙m−3, (6) acetic acid = 15.2 mg∙m−3, and (7) acetone = 13.9 mg∙m−3. In contrast, two alcohols dominated the aroma profile of decayed samples (RC up to 98.6%): (1) ethyl alcohol = 94.2 mg∙m−3 and (2) isobutyl alcohol = 289 mg∙m−3. Alternatively; if the aroma profiles are re-evaluated by summing odor activity values (ΣOAV); four ester compounds ((1) ethyl butyrate (6,160); (2) ethyl hexanoate (3,608); (3) ethyl isovalerate (1,592); and (4) ethyl 2-methylbutyrate (942)) were identified as the key constituents of fresh strawberry aroma (SRD-0). As the strawberries began to decay; isobutyl alcohol recorded the maximum OAV of 114 (relative proportion (RP) (SRD = 6) = 58.3%). However, as the decay process continued, the total OAV dropped further by 3 to 4 orders of magnitude—decreasing to 196 on SRD = 6 to 7.37 on SRD = 9. The overall results of this study confirm dramatic changes in the aroma profile of strawberries over time, especially with the onset of decay.
Keywords: fresh and decaying strawberry; strawberry fragrances; mass concentration; threshold; odor activity value (OAV) fresh and decaying strawberry; strawberry fragrances; mass concentration; threshold; odor activity value (OAV)

1. Introduction

Strawberries are one of the most widely consumed fruits with a good flavor and high nutritional value [1,2]. The aroma and odor-quality of strawberries depend on the type and concentration of volatile hydrocarbons (HC) in the aroma profile [35]. In fact, it is estimated that more than 360 volatile organic compounds (VOC) are emitted from strawberries [6,7]. However, only a small number of these contribute significantly to the strawberry fragrance and impact its perceived quality [8]. The intensity of strawberry fragrance has also been found to vary with the degree of freshness of the fruit. If the characteristics of VOC emitted from strawberries are evaluated thoroughly over time, this information can be used to improve our understanding of the natural strawberry fragrance and allow growers and retailers to optimize their harvesting, packing, storage and retail display procedures.

In order to assess the VOCs released from strawberry, researchers have used several different analytical approaches. Gas chromatographs (GCs) equipped with either flame ionization (FID) or mass spectrometer (MS) detectors have been the most common choices [911]. Recently, strawberry fragrances were also evaluated by combining olfactometry and GC techniques—i.e., harnessing state-of-the-art analytical technology alongside the particular selectivity of the human nose [1,2,12].

In this study, the concentrations and chemical types of strawberry volatiles were analyzed to characterize the fragrance (aroma profile: freshness staging) and offensive odorants (due to decay). All volatile compounds released from strawberry samples were collected at five different intervals (up to 9 days of storage period) at 25 °C. For the quantification of volatile components, liquid-phase standard was prepared containing a total of 19 odorous compounds for external calibration (Table 1S) The numbering of all supplementary (S) Tables and Figures are made with an S symbol following the number and placed in the Appendix section at the end. These calibration results were then used to develop predictive equations based on effective carbon number (ECN) [13]. These equations were then used for an extensive list of ‘compounds lacking authentic standards/surrogates (CLASS)’ due to the absence of standard material (i.e., authentic compounds) or to the synthesis complexities or costs involved in standard preparation [14]. The use of the predictive equations based on response factor vs. effective carbon number (ECN) linear correlation allowed robust, statistical estimation of all CLASS. The results of this approximation method allowed us to characterize the emission pattern of most fragrance and odorous components released from strawberry samples in a quantitative manner. In this research, we undertook measurements of strawberry aromas and odorants to provide detailed descriptions on their emission patterns in relation to storage duration. The results of this study will thus help us understand the characteristics of the flavor changes in strawberries that occur during storage.

2. Materials and Methods

In this research, a total of 19 VOCs that had relatively strong odor intensities with a wide range of volatility and polarity were selected for external calibration (Table 1S). The calibration results obtained using this standard mixture was used to derive predictive equations based on ‘effective carbon number (ECN)’ theory [13]. These ECN-based predictive equations were then used to calculate the concentrations of ‘CLASS’ due to the absence of standard material (i.e., authentic compounds) or to the complexity involved in standard preparation [14].

Liquid-phase working standards (L-WS) of 19 VOCs in methanol were prepared to include: (1) five aldehydes: acetaldehyde (AA), propionaldehyde (PA), butyraldehyde (BA), isovaleraldehyde (IA), and n-valeraldehyde (VA), (2) six aromatics hydrocarbons: benzene (B), toluene (T), styrene (S), p-xylene (p-X), m-xylene (m-X), and o-xylene (o-X), (3) two ketones: methyl ethyl ketone (MEK) and methyl isobutyl ketone (MIBK), (4) one alcohol: isobutyl alcohol (i-BuAl), (5) one ester: n-butyl acetate (BuAc), and (6) four volatile fatty acids: propionic acid (PPA), butyric acid (BTA), isovaleric acid (IVA), and n-valeric acid (VLA) (Table 1S). The detailed procedures to make the L-WS are described in Table 2S.

The concentrations of CLASS were derived from the predictive equations based on linear regression equations between RF values of target standard compounds (Table 3S) and their effective carbon numbers (ECNs). The ECN was determined by counting the number of the atoms (C, H, and O) and moieties in functional groups (e.g., ether, carbonyl, and methyl groups) in terms of ‘carbon number equivalent (CNE)’ in light of their approximate relative contribution to the sensitivity (RF) in the MS system: ECN = I × (CNE of C) + J × (CNE of H) + K × (CNE of O) + (CNE of > C = O) + M × (CNE of -O-) + N × (CNE of -CH3) (Figure S1): (1) C = 1, (2) H = -0.035, (3) O = 0, (4) >C = O = -0.95, (5) -O- = 0.55, and (6) -CH3 = 0.15). As 10 out of 147 volatiles detected from the strawberry samples matched with 19 VOCs contained in the L-WS, they were quantified directly using the calibration data of the L-WS. However, we did not prepare standards for the remaining 137 volatiles for many different reasons. For simple quantitation of those strawberry aroma components, we treated them as CLASS to quantify their concentrations based on the ECN approach (Table 4S).

2.1. Approaches for the Collection of Volatile Components and Instrumental Setup

2.1.1. The Collection of Strawberry Volatiles

The sorbent tube sampling method was used for the collection of the VOCs released from the cut strawberry sample. In the case of ammonia and RSC, the bag sampling method (polyester aluminum-PEA bag) was used as discussed in the next subsection. The sorbent tube was prepared as a three-bed sorbent by packing with 100 mg of Tenax TA, Carbopack B, and Carboxen 1000 (Supelco, Bellefonte, PA, USA) in a SS tube holder (tube size: length: 9 cm, OD: 6 mm, and ID: 5 mm; Camsco, Houston, TX, USA). The strawberries for this study were grown in Jin Ju city, Gyeong Sang Nam Do Province, Korea. The strawberries (1 kg in a Styrofoam tray) were purchased from a local market within one day after harvesting. An approximately 50 gram of strawberry sample was sliced and placed inside a 750 mL capacity impinger with the gas inlet and outlet positioned as the side arm and nozzle cap, respectively (ID: 3 mm and length: 30 mm). The strawberry samples were cut into four pieces (3 cm ×3 cm × 5.5 cm) (The weight of sample was adjusted to ∼50 g) (Figure S2). The inlet and outlet of the impinger were connected to a 10 L PEA bag filled with back-up gas (ultra-pure nitrogen > 99.999%) and the inlet of the sorbent tube (ST), respectively. A Teflon tube was used to connect the impinger and the PEA bag at one end and the ST at the other. The outlet of the ST was connected to the mini vacuum pump interfaced with mass flow controller (MFC) (Shibata ΣMP-30, Saitama, Japan) using silicon tubing. The VOCs emitted from the strawberry were flushed onto the ST by pumping the nitrogen gas through the impinger containing the strawberry samples at a flow rate of 50 mL·min −1 for 1 min. The impinger was maintained at 25 °C using a temperature-controlling water bath (Figure 1). VOCs emitted from the strawberry sample were collected five times during the entire storage period of 9 days set for this study at 0, 1, 3, 6 and 9 days (Table 5S). For each selected day, the collection of samples was made as replicate for each target group (VOC, RSC, ammonia, and olfactory analysis). To initiate each sampling at a given day, a pre-purge was conducted by supplying ultra-pure nitrogen into the impinger at a flow rate of 50 mL·min −1 for 20 min. Throughout the storage period, the strawberry sample in the impinger was maintained in an aerobic state as the inlet and outlet of the impinger were left open to the air in the laboratory.

2.1.2. Instrumental Setup for VOC Analysis

All the analyses in this study were carried out using a GC-2100 (Shimadzu, Kyoto, Japan) equipped with a QP2010 MS (Shimadzu) and a UNITY thermal desorber (TD: Markes International, Ltd, Llantrisant, UK). The TD focusing cold trap was packed with Tenax TA and Carbopack B in a 1:1 volume ratio (inner diameter = 2 mm and total sorbent bed length: 50 mm). The VOCs were separated on a CP Wax column (diameter = 0.25 mm, length = 60 m, and film thickness = 0.25 μm) using a 50 min GC analytical cycle. The separated VOCs were detected by MS system and identified through library searching (NIST mass spectral library, NIST, Gaithersburg, MD, USA). The detailed conditions are also presented in Table 5S.

2.2. Calibration of the Liquid Working Standards of VOCs

Five-point calibration curves were prepared by analyzing sorbent tubes loaded with 1 μL of L-WS at these different concentration levels: (1) 1.30, (2) 6.52, (3) 13.0, (4) 26.1, and (5) 65.2 ng·μL−1) (Table 2S). Details of the approach used to introduce the liquid standards to the sorbent tubes in the vapor phase have been described in detail elsewhere [15]. In short, a micro-syringe was used to introduce the liquid standard into the sampling end of the ST in a 50 mL·min−1 flow of nitrogen for 10 minutes. Each loaded sorbent tube was then analyzed by the TD-GC-MS system described below.

The sensitivity of the instrumental system remained fairly constant leading to stable response factors throughout the 9-day study period (Table 3S). All coefficients of variation (CV (%): SD/mean × 100) for the RF values were fairly stable (CV = 1.46 ± 1.29% (<4%)) allowing mean RF values to be applied to all the data. The correlation coefficients (R2) of nearly all VOCs were above 0.99 (mean = 0.9954 ± 0.0075%), although there was a slight anomaly in the case of AA (0.9619 (SRD = 0) and 0.9698 (SRD-9)). In addition, to assess reproducibility, the L-WS with a mean of 26.1 ng·μL−1 was analyzed repeatedly prior to analysis of each batch of samples (SRD -0, 1, 3, 6, and 9). If the RF values of the 19 VOCs determined using this consecutive series of analyses, the RSE values generally fell below 4% (mean RSE = 1.39 ± 0.82%).

2.3. The Analysis of Ammonia and Reduced Sulfur Species

Although strawberries are noted for their attractive fragrance, the fruit can release unpleasant odors if stored for too long. Important offensive odorants such as ammonia and reduced sulfur compounds (RSC) [16,17] were therefore also quantified in this study. As expected, these compounds were found to be difficult to detect in the fresh fruit but they became increasingly abundant as the decay progressed. The TD-GC-MS setup optimized for the VOC analysis in this study is not optimal choice for ammonia or RSCs, thus these compounds were determined using alternative analytical approaches.

For the collection of samples to analyze for ammonia and RSC, the bag sampling method (PEA bag) was employed. The inlet and outlet of the impinger filled with the strawberry samples were connected to a gas cylinder filled with ultra-pure air (>99.999%) and a 10 L empty PEA bag, respectively. Teflon tubing was used to connect the impinger and the gas cylinder at one end and the empty PEA bag at the other. Air for the cylinder was plowed through the impinger containing the strawberry sample and into the 10 L empty PEA bag at a flow rate of 100 mL·min−1 for 100 min afterapre-purge was conducted by supplying ultra-pure air into the impinger at a flow rate of 100 mL·min−1 for 10 min. Ammonia was analyzed using absorption photometry (Genysys 10 series, Thermo Scientific, Waltham, MA, USA) based on the indophenol method, a well-known approach for amino compounds [18,19]. The reduced sulfur compounds were analyzed using an on-line thermal desorption system (UNITY-Air Server, Markes International, Ltd.) coupled with a GC (CP-3800, Varian, Palo Alto, CA, USA) and pulsed flame photometric detector (PFPD: Varian). The RSCs in the PEA bag were transferred to the TD system using pump and collected into the focusing trap (cold trap) in TD system. The RSCs loaded on the focusing trap were then thermally desorbed and transferred to the GC column for separation and detection. Finally, the dilution-to-threshold (D/T) ratios of the strawberry samples were also determined using an air dilution sensory (ADS) test, as a direct means to assess odor intensity [20].

3. Results and Discussion

3.1. Major Volatile Components Emitted from Strawberry Samples

The concentration and occurrence frequency of these species is classified in terms of functional groups in Table 1. The types and concentration levels of 147 VOCs detected from all strawberry samples are also summarized in Table 6S. If they are arranged by the chemical grouping and occurrence frequency, they can be classified as follows: (1) ester = 61, (2) alcohol = 21, (3) aldehyde = 11, (4) ketone = 9, (5) fatty acid = 5 and (6) miscellaneous (etc.) = 40.

Esters (n = 47), with total concentration of 52,648 μg· m−3 (76.8% by mass), represented more than half of the 81 VOCs detected at the start of the study (SRD = 0). Similarly, approximately 130 different types of esters have been reported from strawberry fragrances [21], where they were found to represent 25 to 90% of strawberry volatiles [22-24]. At SRD-1, esters (n = 38) still recorded the highest concentration (42,713 μg· m−3). By day 3, while the total concentration of esters had continued to increase (sum concentration = 705,447 μg· m−3) their numbers had fallen down to 31. However, once decay had started, their concentration dropped dramatically down to 533 (SRD = 6) and 45.7 μg·m−3 (SRD = 9). In contrast, alcohols exhibited a reversed trend. Although alcohols were much less abundant in fresh strawberries (sum concentration of SRD-0, 1, and 3 = 1,582 μg· m−3), they tended to peak noticeably at 380 mg·m−3 on SRD-6. Thus, the best indicators of the fresh and decayed stages of strawberries are identified as esters and alcohols, respectively. If the other classes of chemicals are considered, aldehydes were detected in all samples and recorded the highest concentration (19,054 μg· m−3) on day 3. Ketones and fatty acids were also relatively abundant in fresh strawberries, although they faded away during decay (Figure 2).

In order to evaluate the indicative fragrance of strawberry, the relative composition (RC) of the strawberry volatiles was assessed initially by normalizing the concentration of an individual compound against the total concentration of all species at each sampling day (Table 2). If any compound with more than 0.05% of RC (total mass) on one or more sampling day was selected, 53 were observed. The compound contribution pattern of these 53 VOCs was then analyzed both in terms of concentration and odor intensity. The sum concentration for these major VOCs (RC > 0.05%) generally exceeded 99.9% of the total mass of VOCs from each individual measurement (99.4% (SRD-0) to 99.99% (SRD-6).

Ethyl acetate [mass concentration = 17,240 μg·m−3 (25.1%)] and methyl acetate 11,945 μg·m−3 (17.4%) were the highest from the SRD-0 sample along with ethyl butyrate (7,290 μg·m−3), methyl butyrate (4,977 μg·m−3), ethyl hexanoate (4,270 μg·m−3), and methyl hexnoate (2,492 μg· m−3). These six esters thus showed the highest RC (70.3%) at SRD-0. Other than esters, acetone and acetic acid had relatively high concentrations of 5,960 and 6,177 μg· m−3 at SRD-0.

If the results of all fresh stages (SRD-0, 1, and 3) are combined together, esters maintained the maximum abundance (n = 23) with the sum of 80,015 μg· m−3 (93.4%). However, patterns changed dramatically during decay, esters dropped down to 20 μg· m−3 (n = 22) in SRD-6 after excluding ethyl acetate (SRD-6 = 467 μg·m−3). Acetaldehyde also underwent 15-fold reduction to 1,313 μg· m−3 in SRD-6 compared to its maximum at (SRD-3). In contrast, two alcohols rose significantly to 287,758 μg·m−3 [75.3% (isobutyl alcohol)] and 91,537 μg·m−3 [23.9% (ethyl alcohol)] at SRD-6. In the case of SRD-9, the concentrations of those alcohols decreased to 967 (15.9%) and 2,665 μg·m−3 (43.8%), respectively. Moreover the sum quantity (μg· m−3) of strawberry volatiles detected recorded the lowest value of 6,055 at SRD-9 [(RC [SRD-9/Σ SRD-all] × 100) = 0.49%] compared with all other periods (58,281 (SRD-2) to 730,144 μg· m−3 (SRD-3)).

Although our analysis focused mainly on volatile organics by GC-MS, we also analyzed some offensive odorants like reduced sulfur compounds (RSC) and NH3 (Table 7S). It can be seen that three RSCs and ammonia were detected from the strawberry sample. Especially, methane thiol and dimethyl disulfide were seen fairly consistently and recorded fairly high concentrations of 267 μg· m−3 and 196 μg m−3 in SRD-1, respectively. In contrast, ammonia was detected apparently only under the decaying conditions (concentration (μg· m−3) = 169 (SRD-6) and 445 (SRD-9)) relative to the fresh period below 81.3 (SRD-0, 1, and 3).

3.2. The Variety of VOC Threshold Values for Strawberry Volatiles and Their Relationship with Molecular Weights

The odor threshold of a compound is defined as the lowest concentration that can be detected by human olfaction [25]. The lower the odor threshold, the stronger the odorant will be. However, many authors have investigated the threshold values of various volatiles and results for individual compounds can be very variable. In this study, a literature survey was conducted for the odor strengths (thresholds) of the strawberry volatiles. Although we measured a total of 147 VOCs during this study period, we were only able to obtain threshold values for up to of 79 species (Table 6S). The results of this survey are also summarized in Table 8S.

As reported previously, the odor strengths of VOCs tend to exhibit strong relationships with their physicochemical properties, e.g., the number of carbons and molecular weight [26]. Hence, a number of combinations between such variables (e.g., log thresholds vs. molecular weights) were tested to seek for such linear relationship. For this comparative analysis, fatty acids and some miscellaneous groups were however excluded due to the lack of threshold data. As shown in Table 9S, an inverse correlation was seen consistently between the log (thresholds) and molecular weights of VOCs without a single exception. However, the magnitude of slope values differed greatly between the VOC groups, while the strongest correlation with molecular weights was seen from the maximum (out of all available) threshold values.

As shown in Figure 3, the strongest correlations were seen from a pair of log-maximum threshold values and molecular weight among all matching combinations (<1> for all data (n = 62): (1) R2 (maximum) = 0.4260, (2) R2 (minimum) = 0.2171, and (3) R2 (geometric mean) = 0.3384, and <2> For optimal fit (n = 54): (1) R2 (maximum) = 0.5743, (2) R2 (minimum) = 0.2897, and (3) R2 (geometric mean) = 0.4473).

3.3. The Evaluation of the Odor Strengths with Changes in Freshness Status

In previous sections, the changes of VOC quantities and their threshold values were evaluated from fresh to decayed stages of strawberry. To learn more about strawberry fragrance, our results were examined further with respect to type and strength of strawberry odors. The selection of reasonable threshold value is important to help understand the contribution of a compound at its given concentration level to the overall perception of odor. It is however difficult to assign a single meaningful figure because the threshold of a given compound is often available as multiple reported values.

In this study, the odor strengths of strawberry were calculated in terms of odor activity values (OAV) by dividing the concentrations of the VOCs with the corresponding threshold in the same concentration unit: OAV = concentration (ppbv)/threshold value (ppbv) [27]. For the 53 selected major VOCs, multiple threshold values have been reported for many (21 (one value), 21 (two values), 10 (three values), and 1 (four values)). In case of two or more reported value thresholds, the one with maximum value was used to calculate the OAVs in light of consistency as seen in correlation analysis.

Table 3 presents the specific description of odor types for each of the major VOCs with their OAV (n = 53). Information of the odor types was obtained by surveying the GC-olfactometry analysis of VOC [refer to a list of references (n = 24) in Table 3]. The ΣOAV values of the strawberries tended to decrease abruptly with storage time from their maximum at SRD-0 (OAV from SRD-0 to SRD-9 were 12,972, 6,992, 2,524, 196 and 7.37, respectively). The OAV values at SRD-0 decreased in order of ethyl butyrate (6,160), ethyl hexanoate (3,608), ethyl isovalerate (1,592), and ethyl 2-methylbutyrate (942).

To assess the relative contribution of a given compound in terms of OAV, its relative proportion (RP) was also calculated by dividing OAV (a given compound) with ΣOAV (all) (Table 3). This RP term for OAV is distinguished from the RC term used for relative mass concentration. If OAVs of these four esters are summed, their RP represents 94.8% of total OAV at SRD-0. It thus suggests that the fragrance of fresh strawberries is governed predominantly by these four esters. The scent of these esters is characterized as fruity, apple, and sweet (Table 3). Although their OAVs decreased from SRD-0 to SRD-1, they still recorded the highest OAVs among all the VOCs evaluated at SRD-1 with ΣRP = 91.1%. The fragrance pattern of SRD-0 and -1 is not likely to have changed because the key volatiles (the four esters) remained constant. In case of SRD-3, esters had high OAV along with significantly large ΣRP (97.2%), although their ΣOAV decreased considerably to 2,453 in SRD-3 relative to the earlier period (SRD-0 (12,917) and SRD-1 (6,942)). As a result, we were able to confirm that the esters should dominate the quality of fresh strawberry fragrance (SRD-0, 1, and 3).

To evaluate the occurrence patterns of fresh strawberry volatiles, our results were compared to a number of previous studies. Du et al. [1] analyzed the volatiles emitted from fresh strawberries using solid-phase micro-extraction (SPME)-GC-MS analysis. A total of 54 volatiles from two cultivar samples (Strawberry Festival and Florida Radiance) were selected as the main target compounds (with their respective standards). It is interesting to note that 52 target volatiles were found in both samples, while 23 of them were seen consistently in all fresh stage samples in this study. They further calculated the OAVs of detected volatiles using thresholds of 44 compounds. Accordingly, OAVs were seen to be dominated by two esters (ethyl butyrate and methyl butyrate), 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF), and linalool in two samples [(1) Strawberry Festival = 461 (ethyl butyrate: RP = 28.7%), 358 (methyl butyrate: 22.3%), 424 (DMHF: 26.4%), and 102 (linalool: 6.3%) and (2) Florida Radiance = 553 (ethyl butyrate: 33.7%) and 261 (methyl butyrate: 15.9%), 359 (DMHF: 21.9%), and 162 (linalool: 9.9%)]. In this study, ethyl butyrate exhibited relatively high OAV in fresh stages with its RP ((1) 47.5% (SRD-0), 65.4% (SRD-1), and 27.2% (SRD-3)). Likewise, methyl butyrate also showed high OAV with the RP values of 1.30% (SRD-0), 2.79% (SRD-1), and 0.50% (SRD-3). In contrast, the RP values of DMHF and linalool were relatively insignificant, although they were selected as the major VOCs in this study.

Nuzzi et al. [12] analyzed strawberry fragrances of six different cultivars such as ApoScaligera ((1) Darselect, (2) Eva, and (3) VR4) and Cesena area in Italy ((4) Alba, (5) Dora, and (6) CS4). The volatiles from these samples were collected by a charcoal adsorption tube and extracted using dichloromethane solution. Lastly, the GC-MS analysis of these extracts yielded a total of 37 volatiles (ester = 28, alcohol = 2, sulfide = 2, lactone = 2, and 3 others (2-pentanone, limonene, and linalool)) in their fresh stage. If the OAV is computed for each compound, nine of them showed the highest contributions with ΣRP = 96.7% (consisting of methyl 2-methylbutyrate (3.46%) to dimethyl trisulfide (23.2%)). Eight out of the nine volatiles with the high RP measured in studies of Nuzzi et al. [12] were also detected from our fresh strawberry samples (SRD-0 ,1, and 3). Especially, ethyl butyrate, ethyl 2-methylbutyrate, ethyl hexnoate, and ethyl isovalerate had the highest contribution to OAV in fresh periods. As such, the results of previous studies confirmed the significant role of esters in the fresh strawberries [1,12].

During decay, ΣOAVs of all detected esters (except ethyl 2-methylbutyrate with OAV = 46.9) were below 10, while accounting for 12.9% by ΣRP (ester (n = 12)). In contrast, isobutyl alcohol exhibited the highest OAV of 114 in SRD-6 (RP = 58.3%). As the odor of isobutyl alcohol is characterized as plastic and bad, is is distinguishable from pleasant fresh scents. As a result, odor intensity decreased with the progress of strawberry decay with the emergence of some offensive odors (e.g., ΣOAV of 7.37 in SRD-9). In SRD-9, only two compounds (acetaldehyde and isovaleraldehyde) showed OAVs above 1. For the reader's reference, volatiles that showed up at least once in terms of either OAV (above 100) or RP (above 5%) during the whole study period are illustrated in Figure 4.

If the OAVs of the RSCs and ammonia detected in strawberry aroma profiles are examined, their values from decayed strawberry samples (SRD-6 and 9) generally had very low OAV (below 1). Only methanethiol had high OAVs above 10 in SRD-0 and SRD-1 (OAV = 14.9 (SRD-0) and 64.6 (SRD-1)] compared with other RSCs or ammonia. However, if these results are compared with ΣOAV (all) of all hydrocarbons in fresh strawberry samples (SRD-0 and 1), the RP value of methane thiol were as low as 0.13% (SRD-0) and 1.04% (SRD-1). As a result, it is reasonable to infer that the volatile hydrocarbons should represent the odor/fragrance characteristics of strawberry most effectively.

3.4. Comparison between Odor Activity Value (OAV) and Dilution-to-Threshold Ratio

In this section, the TD-GC-MS odor profiles were compared between fresh and decaying strawberries. The results were then evaluated to assess the relationship between the classes of volatile components and their odor intensity. To estimate the key volatiles which dominate the strawberry scent, the odor strengths (OAV) were examined against the relative (mass) composition. Evaluation of our data indicated that the use of ΣOAV was useful to assess the actual occurrence of fragrance/odor from strawberry samples. As another means to explore the odor intensities of strawberry samples, we estimated the dilution-to-threshold (D/T) ratio derived experimentally based on air dilution sensory (ADS) test [20]. The D/T ratio is commonly used as a tool to measure the level of dilution by which the odor threshold is recognized [20]. As the D/T ratio of strawberry samples was measured concurrently with the analysis of their chemical composition, the ΣOAV values at each SRD interval can also be evaluated in relation to the D/T ratio.

The results of correlation analyses between storage day and log odor intensities (log ΣOAV and log D/T ratio) are plotted in Figure S3(A). In compliance with general expectation, the log D/T ratio decreased with the progress of decay to show the slope values of -0.2185 (with R2 = 0.8646 and p-value = 2.21E-2) (log D/T ratio). A similar trend is also observable from ΣOAV. As such, the results of this correlation analysis between the log ΣOAV and log D/T ratio show a strong correlation with R2 = 0.9338 (p-value = 7.33E-3). As a result, the computation of ΣOAV values can be used as sensitively as that of the D/T ratios to assess the fragrance occurrence pattern. In other words, the ΣOAV should be considered a good criterion to assess the fragrance or odor intensity of strawberry samples.

4. Conclusions

The volatiles emitted from strawberry are important components to accurately assess its fragrance. Although the fragrance of the strawberry can influence its flavor and taste, its smell types can vary greatly with aging conditions. If the volatiles from strawberry were analyzed at the fresh stage, the results should be useful enough to understand the natural flavor of strawberries. In this study, the mass concentrations and odor strength (odor activity values: OAV) from strawberries were analyzed by the sorbent tube method at storage times of 0, 1, 3, 6 and 9 days at 25 °C.

The results of our analysis indicated that the mass concentration (μg·m−3) of all strawberry volatiles varied greatly over time: 68,569 (SRD-0), 58,503 (SRD-1), 730,593 (SRD-3), 382,245 (SRD-6), and 6,086 (SRD-9). The concentrations of strawberry volatiles released at fresh period before (SRD-3) were absolutely dominated in this order, ethyl acetate, methyl acetate, ethyl butyrate, methyl butyrate, acetaldehyde, acetic acid, and acetone (ΣRC of 97.4%). However, as the strawberry samples began to decay, its RC was dominated by two alcohols (isobutyl alcohol and ethyl alcohol) with significant reductions in their summed concentration.

In this study, we were able to quantify a total of 147 strawberry volatiles (with 79 corresponding odor threshold values from literature survey). In order to assess the fragrance/odorant characteristics of strawberries, the OAV values were calculated for a total of 53 major volatiles that comprised more than 0.05% in mass concentration of all strawberry volatiles. The OAV values decreased abruptly with storage time (ΣOAV: SRD-0 (12,972) to SRD-9 (7.37)). If the ΣOAV values of a given strawberry volatiles are computed for the whole fresh period, their magnitude was dominated by four esters with fruity and sweet scents [(1) ethyl butyrate (11,422), (2) ethyl hexanoate (4,395), (3) ethyl isovalerate (2,751), and (4) ethyl 2-methylbutyrate (2,290)] [ΣRP (four esters) of 92.8%]. However, relative dominance of esters as the key strawberry fragrance changed abruptly with the onset of decay. Hence, as the transition proceeds, relationships between key parameters tended to vary widely. In case of SRD-6, the ΣOAV of the four esters decreased to 60.9 with ΣRC of below 1%, while isobutyl alcohol exhibited the highest OAV of 114 with RC of 75.3%. The ΣOAV was reduced further to reach the minimum value of 7.37 in SRD-9.

In this study, the strawberry volatiles were analyzed from fresh stage to 9 day storage at 25 °C, and the concentration of the strawberry volatiles were evaluated in relation to their occurrence patterns and olfaction data derived as D/T ratio. The overall results of our study suggest that strawberry volatiles are useful indicators to characterize the flavor changes of strawberry at the latter stages of its storage period.


This work was supported by a grant from the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (MEST) (No. 2009-0093848).

Conflict of Interest

The authors declare no conflict of interest.


Table 1S. List of 19 VOCs selected as model compounds for the predictive estimation of concentration values for “compounds lacking authentic standards/surrogates (CLASS)” that are emitted from strawberry.
Table 1S. List of 19 VOCs selected as model compounds for the predictive estimation of concentration values for “compounds lacking authentic standards/surrogates (CLASS)” that are emitted from strawberry.
OrderGroupCompoundsShort nameMW (g·mol−1)Density (g·cm−3)Boiling point (°C)FormulaCAS number


12KetoneMethyl ethyl ketoneMEK72.110.80579.64C4H8O78-93-3
13Methyl isobutyl ketoneMIBK100.20.802117–118C6H12O108-10-1

14AlcoholIsobutyl alcoholi-BuAl74.120.801108C4H10O78-83-1

15Estern-Butyl acetateBuAc116.20.881126C6H12O2123-86-4

16Fatty acidPropionic acidPPA74.10.99141C3H6O279-09-04
17n-Butyric acidBTA88.10.958163.5C4H8O2107-92-6
18i-Valeric acidIVA1020.925175–177C5H10O2503-74-2
19n-Valeric acidVLA1020.938186–187C5H10O2109-52-4
Table 2S. Preparation of liquid phase VOC standard for the analysis by the TD-GC-MS system.
Table 2S. Preparation of liquid phase VOC standard for the analysis by the TD-GC-MS system.
A. Preparation of liquid phase standard for 19 VOCs

Primary grade chemicalConcentration (%)

PSaVolume (μL)13,700900300300300300300300300300300300300300300300300300300300
Concentration (ng·μL−1)34,97211,61111,95411,59611,78613,10412,92513,45412,84512,84512,80411,95411,97011,89513,14914,70214,22613,73613,929

1st L-WSbvolume (μL)19,800200 (of PS)

Concentration ( ng·μL−1)350116120116118131129135128128128120120119131147142137139
B. Preparation of final working standard (F-WS) for 5 point calibration: absolute mass (ng) of VOC loaded on tube sampler

OrderMixing volume (μL)Concentration c (ng μL−1)


aPS: Dilution of pure chemical (primary grade chemical) to make 20 mL solutionb1st L-WS: Dilution of PS to make 20 mL solutioncAnalysis volume: 1 μL

Table 3S. Comparison of calibration results determined at the start and end of experiments: Response factor (RF), coefficient of determination (R2), and relative standard errors (RSE).
Table 3S. Comparison of calibration results determined at the start and end of experiments: Response factor (RF), coefficient of determination (R2), and relative standard errors (RSE).
OrderGroupCompoundRFR2RSEa (%)

Exp_day 0Exp_day 9MeanCVbExp_day 0Exp_day 9Mean







aFive replicate analyses of 26.1 ng (mean mass) of analytes per 1 μL injection of F-WS (4th calibration point)bCV (coefficient of variation) = SD/mean * 100

Table 4S. Comparison of RF values between actual experiment and the effective carbon number (ECN) approach.
Table 4S. Comparison of RF values between actual experiment and the effective carbon number (ECN) approach.
OrderGroupCompoundsShort nameNumber of atom and functional groupaECNcRF valuesPDe

CIbHJbOKb(>C=O)Lb(-O-)Mb(-CH3)NbActucal expECN approachd


12KetoneMethyl ethyl ketoneMEK4811023.0748,77345,9645.76
13Methyl isobutyl ketoneMIBK61211035.08117,515107,4608.56

14AlcoholIsobutyl alcoholi-BuAl41010124.5093,22389,7153.76

15Estern-Butyl acetateBuAc61221125.48115,453119,6983.68

16Fatty acidPropionic acidPPA3621112.5426,26929,74813.2
17n-Butyric acidBTA4621113.5469,54660,34413.2
18i-Valeric acidIVA51021124.5597,01591,2455.95
19n-Valeric acidVLA51021114.4079,28386,6569.30

Mean:6.52 ± 4.99

aCarbon number equivalent for each atom and functional group (CNE): (1) C = 1, (2) H = −0.035, (3) O = 0, (4) >C=0 = −0.95, (5) -O- = 0.55, and (6) − CH3 = 0.15bI, J, K, L, M and N = number of C, H, O, >=O, -O-, CH3 (atoms or functional groups) in each VOC, respectivelycECN = I + J*(CNE of -H) + L*(CNE of >C=O) + M*(CNE of -O-) + N*(CNE of -CH3)dThe predictive equation (by ECN approach) for estimation of VOC concentration was determined using 18 liquid working standards except for AA: (1) RF = 30,595, (2) intercept = -47,963, and (3) R2 = 0.9901ePercent different (PD, %) = ABS{[RF (actual exp)-RF (ECN approach)] / RF (actual exp) * 100}

Table 5S. Operational conditions of TD-GC-TOF MS system for the analysis of fresh and decaying strawberry.
Table 5S. Operational conditions of TD-GC-TOF MS system for the analysis of fresh and decaying strawberry.
[A] Sampling information of strawberry volatiles

a. Information of strawberry for sampling

OrderSample codeStorage time (day)Storage temp. (°C )


Initial weight = 50.09 g
b. Sampling approach

Sampler:3 bed sorbent tube
Purge gas:Nitrogen (>99.999%)
Purge gas flow:50 mL·min−1
Pump model:MP-Σ30 (Sibata, Japan)
Heater model:TC200P (Korea)

[B] Instrumental setups for VOC analysis

a. GC (Shimadzu GC-2010, Japan) and MS (Shimadzu GCMS-QP2010, Japan) Column: CP Wax (diameter: 0.25 mm, length: 60 m, and film thickness: 0.25 μm )

Oven settingDetector setting

Initial temp:35 °C (10 min)Ionization mode:EI (70 eV)
Ramp rate:6 °C·min −1Ion source temp.:200 °C
Max oven temp:215 °C (10 min)Interface temp.:200 °C
Total time:50 minTIC scan range:35∼260 m/z
Carrier gas:He (99.999%)
Carrier gas flow:1 mL·min −1

b. Thermal desorber (Unity, Markes, UK)

Cold trap sorbent:Tenax TA + Carbopack B (volume ratio=1:1) (diameter: 2 mm and sorbent bed length = 5 mm)
Split ratio:1:5Adsorption temp.:-10 °C
Split flow:5 mL·min−1Desorption temp.:320 °C
Trap hold time:20 minFlow path temp:150 °C

c. Sorbent (Sampling) Tube

Sorbent material:Tenax TA + Carbopack B + Carboxen 1000 (mass (mg)=100 : 100: 100 )
Desorption flow:50 mL·min−1
Desorption time:5 minDesorption temp.:300 °C
Table 6S. A list of individual VOC determined by the TD-GC-MS system from all strawberry samples throughout the study period.
Table 6S. A list of individual VOC determined by the TD-GC-MS system from all strawberry samples throughout the study period.
OrderCompoundsMWFormulaConcentrationOdor thresholdAveraged Similarity * (%)

ppb(μg m−3)

(g mole−1)SRD-0SRD-1SRD-3SRD-6SRD-9SRD-0SRD-1SRD-3SRD-6SRD-9(ppbv)(μg m3)Reference
A. Ester (61)
1Methyl acetate74C3H6O23,9493,87571,0771.570.7511,94511,720214,9874.762.281,700-6,1705,142-b, c98.4
2Ethyl acetate88C4 H8 O24,7934,277134,969130.011.8017,24015,384485,47646742.5610-3291,183-9,460a, b, c, e98.8
3Methyl propionate88C4H8O240.851.
4S-Methyl thioacetate90C3 H6 O S94.21300.0310.0310.031346475.80.1130.1130.1131.65.9a97.0
5Methyl trans-crotonate100C5 H8 O22.773.168.510.0050.00511.312.934.80.0210.02192.0
6Isopropyl acetate102C5H10O252.816.727.50.0050.00522069.5114.50.0210.021290-2,4001,209-a, b, c98.3
7Methyl isobutyrate102C5H10O20.0050.0050.0050.430.0050.0210.0210.0211.770.0211.910,006 7.9b91.0
8Ethyl propionate102C5 H10 O252.70.00553.50.0050.005219.80.0222230.0220.022729b98.5
9Propyl acetate102C5 H10 O20.0050.0051850.720.0050.0220.0227693.020.022568-9602,367-4,002a, b, c97.5
10Methyl butyrate102C5 H10 O211941,38488.80.470.0054,9775,7723701.950.0222.8-7.112-30a, b98.0
11Ethyl crotonate114C6 H10 O27.134.4926.20.0030.00333.2220.911220.0160.01695.7
12Methyl tiglate114C6H10O20.0030.0031.260.0030.0030.0160.0165.880.0160.01695.0
13Ethyl isobutyrate116C6 H12 O23.801.298.570.860.00318.06.1140.64.070.0160.220.10b95.0
14Methyl 2-methylbutanoate116C6H12O221.821.713.21.080.00310410362.75.140.0160.10.5a97.3
15Isobutyl acetate116C6H12O29.117.0088.91.860.00343.233.24218.800.0168-47938-2,271b, c98.0
16Methyl isovalerate116C6 H12 O254.856.97.780.0030.00326027036.90.0160.0161.1-2.25.2-10a, b97.7
17Ethyl butyrate116C6 H12 O21,5371,1421711.500.0037,2905,4158127.100.0170.04-0.20.19-1.2a, b97.5
18Butyl acetate116C6 H12 O234.236.526.40.050.0031621731250.240.01710.7-19550.9-925a, b, c93.0
19Methyl valerate116C6 H12 O23.911.740.0030.0030.00318.58.260.0170.0170.0172.210b95.5
20S-Methyl thiobutyrate118C5H10OS0.0064.470.0060.0060.0060.02721.570.0270.0270.02792.0
21Methyl 2-vinylbutanoate128C7 H12 O20.770.0030.0030.0030.0034.030.0130.0130.0130.01396.0
224-Penten-1-yl acetate128C7 H12 O22.650.803.740.0030.00313.84.2019.60.0140.01491.3
23Ethyl tiglate128C7H12O20.0021.4116.60.0020.0020.0137.4086.70.0130.01394.5
24cis-2-Penten-1-yl acetate128C7 H12 O24.960.0030.0030.0030.00325.950.0130.0130.0130.01391.0
25Prenyl acetate128C7H12O20.482.830.0020.0020.0022.5214.810.0130.0130.01396.0
26Methyl (2E)-2-hexenoate128C7H12O21.660.890.660.0030.0038.664.683.440.0130.013
27Isopropyl butyrate130C7H14O230.725.90.0020.0020.002163.0137.80.0130.0130.0136.233b93.5
28Ethyl 2-methylbutyrate130C7H14O263.023.866.33.130.00233512635216.60.0130.070.36a97.0
29Ethyl isovalerate130C7 H14 O214267.236.10.700.0027533571923.730.0130.013-0.090.069-0.47a, b96.3
30Isoamyl acetate130C7 H14 O281.369.11651.210.002432367.158786.430.0133.820e98.0
31Ethyl valerate130C7 H14 O27.311.730.0030.0030.00338.839.220.0130.0130.0130.11-0.330.58-1.78a, b96.5
32Methyl 4-methylvalerate130C7H14O22.580.960.0020.0020.00213.725.090.0130.0130.01394.5
33Methyl hexanoate130C7 H14 O246918511.360.0030.0032,49298260.340.0130.01315.682.8a97.0
343-Methyl-2-butyl acetate130C7 H14 O20.0020.0020.480.0020.0020.0130.0132.550.0130.01393.0
35Hex-5-enoic acid, ethyl ester142C8H14O21.310.0020.0020.0020.0027.620.0110.0110.0110.01192.0
36cis-3-Hexenyl acetate142C8 H14 O22.430.0020.0020.0020.00214.10.0110.0110.0110.0113.319a95.0
37(4E)-4-Hexenyl acetate142C8H14O25.412.680.0020.0020.00231.415.50.0110.0110.01191.0
38trans-2-Hexenyl acetate142C8 H14 O238.61.780.0020.0020.00222410.40.0110.0110.01142.8249a92.0
39Ethyl 2-hexenoate142C8H14O23.291.240.0020.0020.00219.17.210.0110.0110.01195.5
40Methyl amyl acetate144C8 H16 O20.0020.0020.370.0020.0020.0110.0112.160.0110.01193.0
41Isobutyl butyrate144C8H16O20.0021.130.910.0020.0020.0116.655.350.0110.0111.69.4b93.5
42Propyl isovalerate144C8H16O20.390.480.750.0020.0022.292.854.400.0110.0110.0560.330b91.0
43Butyl butylate144C8 H16 O20.781.370.0020.0020.0024.608.080.0110.0110.0114.8-22.128-130a, b96.5
44Ethyl hexanoate144C8H16O272513720.90.030.0024,2708081230.160.0110.21.2a97.0
45Hexyl acetate144C8H16O211025.54.700.0020.00264715027.70.0110.011212a97.7
46Valeric acid, thio-, S-ethyl ester146C7 H14 O S0.890.0030.0030.0030.0035.290.0150.0150.0150.01595.0
47Ethyl 3-hydroxy-3-methylbutanoate146C7 H14 O30.0020.0020.0020.040.0020.0120.0120.0120.210.01297.0
48Ethyl benzoate150C9H10O20.390.0020.0020.020.0022.370.0090.0090.150.00990.0
49Isobutyl isovalerate158C9H18O20.0010.0010.330.0010.0010.0090.0092.150.0090.0095.234b94.0
50Neopentyl butyrate158C9H18O20.0010.590.0010.0010.0010.0093.780.0090.0090.00993.0
51Isoamyl butyrate158C9 H18 O21.581.530.0010.0010.00110.29.870.0090.0090.00994.0
52Heptyl acetate158C9H18O20.290.0010.0010.0010.0011.850.0100.0100.0100.01091.0
53Methyl octanoate158C9 H18 O25.840.880.0010.0010.00137.75.7100.0100.0100.01036.7237a95.5
54Octyl acetate172C10 H20 O20.550.0010.0010.0010.0013.860.0080.0080.0080.00895.0
55Hexyl butyrate172C10H20O20.520.0010.0010.0010.0013.650.0080.0080.0080.00892.0
56Ethyl octanoate172C10H20O24.550.0010.0010.0010.00132.00.0080.0080.0080.00894.0
57Octyl acetate172C10H20O21.250.0010.0010.0010.0018.800.0080.0080.0080.00891.0
58Cyclopentanecarboxylic acid, decyl ester254C16H30O20.00050.00050.00050.00050.05550.00490.00490.00490.0050.5892.0
592-(Dodecyloxy)ethyl acetate272C16H32O30.00040.00040.110.00040.00040.00470.00471.210.0050.004792.0
60Linalylanthranilate N-(tert-butoxycarbonyl)-glycyl-273C17 H23 N O20.440.00040.00040.00040.00044.950.00440.00440.0040.004493.0

Σconcentration (ester)52,64842,713705,44753345.7
B. Alcohol (21)
62Ethyl alcohol46C2H6O0.0370.03730948,6851,4170.0700.07058091,5372,665520-28,800978-54,150b, c95 .6
63Isopropyl Alcohol60C3H8O8.764.5845.392.827821.511.2111228680.7310,200-25,015-b, c95.6
64n-Propyl alcohol60C3 H8 O0.0140.01430.4046.636.10.0350.03574.54114.488.5194-2,400231-5,886b, c94.3
65sec-Butyl alcohol74C4 H10 O0.0070.0070.0070.560.550.0220.0220.0221.701.65220-1,700665-5,142b, c93.5
66Isobutyl alcohol74C4 H10 O0.0070.00731.8495,136319.80.0210.02196.30287,75896711-83233-2,517b, c97.7
67n-Butyl alcohol74C4H10O0.0082.408.780.860.370.0237.2626.62.611.12490-26,0001,482-b, c96.3
681-Penten-3-ol86C5 H10 O0.0050.0050.610.0050.0050.0170.0172.130.0170.01794.0
69Isopropenylethyl alcohol86C5H10O0.0050.0050.0052.414.470.0170.0170.0178.4815.72,5008,788e96.0
703-Methyl-1-butanol88C5 H12 O1.091.2850.726.943.43.914.61182.2696.91562-456-161b, c93.2
712-Methyl-1-butanol88C5 H12 O0.0050.0050.0059.4819.60.0170.0170.01734.1270.5041147e98.0
72n-Pentanol88C5 H12 O0.0050.0050.0050.990.860.0170.0170.0173.573.10100-468360-1,683b, c96.0
731-Chloro-2-propanol94C3 H7 CL O20.0380.30.0090.0090.00977.03090.0340.0340.0341,0003,842f93.0
743-Methylpentanol102C6 H14 O0.0030.0030.0030.050.340.0130.0130.0130.221.4193.0
75n-Hexanol102C6 H14 O0.520.0031., c, e92.8
762-Heptanol116C7 H16 O0.0020.0020.0020.420.170.0110.0110.0112.010.8193.5
77Phenethyl alcohol122C8 H10 O0.0020.0020.0020.150.130.0100.0100.0100.730.6592.0
791-Octen-3-ol128C8 H16 O0.0020.0020.0020.020.120.0100.0100.0100.090.622.2611.8a91.0
802-Ethylhexanol130C8 H18 O0.420.0020.0020.
81Linalool154C10 H18 O5.145.080.0010.0010.00132.432.00.0070.0070.0070.191.18a93.0

C. Aldehyde (11)
83Acetaldehyde44C2 H4 O1,6181,64610,5847305402,9092,95919,0351,3139711.5-1862.7-335b, c99 .2
84Allyl aldehyde (Acrolein)56C3 H4 O0.0820.0820.0825.086.900.1890.1890.18911.6215.83.6-1748.2-398b, c98.0
85Methylacryl aldehyde70C4 H6 O0.0160.0160.0161.803.330.0450.0450.0455.149.528.524b
86Isobutyraldehyde72C4H8O0.0150.0150.0150.243.800.0430.0430.0430.7111.170.35-40.71.03-120b, c
87Butyraldehyde72C4H8O0.0150.0150.0150.350.0150.0450.0450.0451.040.0450.67-8.911.97-26.2b, c96.0
89Isovaleraldehyde86C5 H10 O0.0090.0090.0091.032.450.0300.0300.0303.608.620.1-2.240.4-7.87b, c96.0
90n-Caproaldehyde100C6H12O1.380.0050.770.0050.0055.660.0203.140.0200.0200.28-13.81.14-56.4a, b, c91.0
91Benzaldehyde106C7 H6 O0.0030.0033.710.500.220.0150.01516.12.160.9741.7181c94.3
92n-Nonylaldehyde142C9 H18 O0.550.0020.0020.0020.0023.170.0110.0110.0110.0110.34-2.241.97-13.0b, c91.0
932,4-dihydroxy-6-(2′-oxoheptyl)benzaldehyde250C14 H18 O40.0010.0010.0010.0010.360.0060.0060.0060.0063.6496.0

D. Ketone (9)
94Acetone58C3 H6 O2,5143,1312247.3528.05,9607,42253217.4366.54,580-42,00010,858-b, c, e97.8
95Methyl vinyl ketone70C4 H6 O0.0160.0161.250.0160.0160.0450.0453.570.0450.045183524d91.0
96Methyl ethyl ketone72C4 H8 O0.0140.0140.0141.151.350.0410.0410.0413.393.96270-7,760795-22,837b, c, e93.0
97Methyl n-propyl ketone86C5H10O54.047.10.0080.0080.0081901660.0270.0270.02728-1,55098-5,449b, c93.5
98Dimethyl diketone86C4H6O213.70.03013.93.210.03048.100.10448.8911.30.1044.3715.4c96.3
99Methyl Isobutyl Ketone100C6H12O4.627.501.110.0040.00418.8730.74.530.0170.017121-537495-2,195b, c, e95.0
100Methyl amyl ketone114C7 H14 O5.646.520.0030.510.6126.2830.40.0152.362.8535.6-141166-657a, b, c97.8
101Spiro[3.6]deca-5,7-diene-1-one148C10 H12 O0.0010.0010.150.0010.0010.0090.0090.910.0090.00994.0
102Phenyl methyl ketone120C8 H8 O1.560.0020.0020.100.137.670.0120.0120.500.623631,780c91.3

E. Fatty acid (5)
103Acetic acid60C2H4O22,5191,9181,761210866,1774,7034,3195142126-14515-356b, c, e93 .0
1042-Propynoic acid70C3 H2 O269.60.0230.0230.0230.0231990.0670.0670.0670.06794269i93.0
105Methacrylic acid86C4H6O23.920.0090.0090.0090.00913.80.0330.0330.0330.03393.0
1062-Methylbutanoic acid102C5 H10 O20.0050.0050.0050.310.0050.0220.0220.0221.300.0228-18633-775a, c, e93.0
107Undec-10-ynoic acid182C11H18O20.0010.0010.0010.0010.040.0080.0080.0080.0080.2892.0

F.etc. (40)
108Isoprene68C5 H83.986.114.080.0070.7711.0617.011.340.0202.1348-455133-1,265b, e95.3
109Ethylidenecyclopropane68C5 H80.0070.0070.0070.060.0070.0200.0200.0200.170.02093.0
110cis-Piperylene68C5 H81.541.490.0070.170.0074.294.140.0200.460.02089.3
112Divinylene oxide68C4H4O0.0080.0080.0080.0080.600.0230.0230.0230.0231.6697.0
113Propylethylene70C5 H100.0070.0070.740.0070.0070.0200.0202.120.0200.02090.0
114n-Pentane72C5 H1212.10.0070.0070.292.2035.70.0190.0190.866.481,400-31,6004,120-92,997b, c96.3
116Ethyl ether74C4 H10 O0.0070.0070.700.411.450.0220.0222.121.234.38330998h93.7
1171,3-Hexadiene82C6 H1025.01.770.0050.0050.00583.95.940.0150.0150.0152,0006,703g97.0
118Furan, 2-methyl-82C5 H6 O0.0050.0050.0050.0050.600.0160.0160.0160.0162.0024,67882,713d93.0
120n-Hexane86C6H141.,500-21,9005,273-76,983b, c94.3
1212,2-Dimethylbutane86C6 H143.490.0040.0040.0040.00412.270.0140.0140.0140.01490.0
122Toluene92C7H80.0030.0030.0030.0032.480.0120.0120.0120.0129.32160-1,550602-5,829b, c, e97.0
123Phenol94C6H6O0.0040.0040.0040.220.0040.0140.0140.0140.840.0145.60-11021.5-423b, c91.0
126Styrene104C8 H810.0511.9919.413.031.0942.751.082.512.94.6434.4-35146-149b, e93.2
127Ethylbenzene106C8H101.650.0020.0020.0020.107.160.0100.0100.0100.442.88-17012.5-737b, c94.5
128p-Xylene106C8 H100.320.0020.0020.0020.021.390.0110.0110.0110.1058-490251-2,123b, c93.0
1293-Cyclohexenyl cyanide107C7 H9 N0.0030.0030.0030.0030.130.0130.0130.0130.0130.5592.0
131n-Octane114C8 H180.0020.0020.0020.0020.120.0110.0110.0110.0110.571,700-5,7507,921-26,793b, c91.0
1342-Methyl-6-methylene-2,7-octadiene136C10 H160.0010.420.0010.0010.0010.0082.340.0080.0080.00892.0
135l-Limonene136C10 H160.250.520.0010.0010.0011.362.880.0080.0080.00890.0
136(E)-.beta.-Ocimene136C10 H160.240.0010.0010.0010.0011.330.0080.0080.0080.00890.0
1382,2,3,3-Tetramethylhexane142C10 H220.0011.650.0010.0010.0010.0089.570.0080.0080.00894.0
142n-Butanoic anhydride158C8H14O30.0020.530.0020.0020.0020.0133.440.0130.0130.01393.0
145l-Caryophyllene204C15 H240.0010.0010.0010.150.110.0050.0050.0051.280.8892.0
146.alpha.-Muurolene204C15 H240.0010.0010.0010.010.010.0050.0050.0050.110.1191.0
1472,3-Epoxy-.beta.-ionone208C13 H20 O20.0011.280.0010.0010.0010.00610.90.0060.0060.00698.0


Below detection limitReferences: a. Van Gemert [50], b. Nagata [26], c. Devos et al. [51], d. Ruth [52], e. Woodfield and Hall [53], f. Chemwatch [54], g. Evans et al. [55], h. U.S. Department of Labor [56], and i. Amoore and Hautala [57].*Mean similarity of mass spectra between actual experiment and library (NIST)

Table 7S. Concentrations of reduced sulfur compounds (RSC) and ammonia measured separately by GC-PFPD and absorption photometry.
Table 7S. Concentrations of reduced sulfur compounds (RSC) and ammonia measured separately by GC-PFPD and absorption photometry.
OrderCompoundsSRD-0SRD-1SRD-3SRD-6SRD-9Thresholdsb (μg m−3)Reference
[A] Concentration (μg m−3)

1Hydrogen sulfide0.02a0. et al. [58]
2Sulfur dioxide0.,635Nagata [26]
3Methanethiol61.42670.790.020.494.13Iowa State University [59]
4Dimethyl sulfide0.023.830.910.020.027.60Nagata [26]
5Dimethyl disulfide18.919635.80.570.7947.3Schiffman et al. [58]
6Ammonia81.381.381.31694451,042Nagata [26]
[B] Odor activity value (OAV: concentration/ threshold)

1Hydrogen sulfide
2Sulfur dioxide
4Dimethyl sulfide0.500.12
5Dimethyl disulfide0.404.140.760.010.02

aValues below detection limit are underlined (calculated as method detection limit)

Table 8S. The basic statistics on threshold valuesa (n = 79) of VOC.
Table 8S. The basic statistics on threshold valuesa (n = 79) of VOC.
[A] Number of contrasting sources for the VOC with odor thresholds

No. of sources for odor thresholds0 (no data)1234Total
No. of VOC733531121152

[B] The basic statistics of threshold values of VOCs (n = 79)

Statistical parameterMeanSDCV bMinimumMaximumMedianSum

Geo mean3,0779,5523100.08869,896123243,085

aWe determined the 79 threshold values out of the 147 VOCs detected from strawberry samples from previous studies(references from Table 5S)bCV (coefficient of variation) = SD/mean × 100

Table 9S. Log normal relationship between different types of odor threshold (ppbv) and molecular weight to assess the odor strength patterns of strawberry volatiles.
Table 9S. Log normal relationship between different types of odor threshold (ppbv) and molecular weight to assess the odor strength patterns of strawberry volatiles.
GroupsA. EsterB. AlcoholC. AldehydeD. KetoneSum of all groups b

All dataOptimal fit aAll dataAll dataOptimal fitAll dataOptimal fitAll dataOptimal fit
Number of data(n = 31)(n = 28)(n = 14)(n = 9)(n = 6)(n = 8)(n = 6)(n = 62)(n = 54)
[A] Slope c

Geo mean−0.0263−0.0367−0.0432−0.0098−0.0142−0.0197−0.0268−0.0286−0.0329

[B] R2

Geo mean0.17430.31880.71640.19340.87300.18850.65470.33840.4473

[C] p-value

Geo mean1.94E-021.75E-031.35E-042.36E-016.33E-032.82E-015.12E-027.10E-073.25E-08

aAfter excluding outlying data pointsbSum of the four major VOC groups (Ester, Alcohol, Aldehyde, and Ketone)cResults of linear regression analysis between odor threshold and molecular weights: for this comparison, results are compared between minimum, maximum, and geometric mean of threshold values available from previous studies

Figure 1S. The linear regression analysis between the response factors (RF) and effective carbon number (ECN) for each atom and function group (e.g., C, H, O, >C=O, -O-, and -CH3).
Figure 1S. The linear regression analysis between the response factors (RF) and effective carbon number (ECN) for each atom and function group (e.g., C, H, O, >C=O, -O-, and -CH3).
Sensors 13 07939f5 1024
Figure 2S. Photographs showing strawberry samples in impinge.
Figure 2S. Photographs showing strawberry samples in impinge.
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Figure 3S. Relationship between Σodor activity values (OAV) and dilution-to-threshold (D/T) ratio.
Figure 3S. Relationship between Σodor activity values (OAV) and dilution-to-threshold (D/T) ratio.
Sensors 13 07939f7 1024


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Figure 1. Illustration of the impinger system for the collection of VOCs emitted from strawberry samples. Labels: (1) 10 L polyester aluminum bag filled with ultra-pure nitrogen; (2) Silicon tubing; (3) Impinger bottle; (4) Aluminum container; (5) Water heated to 25 °C; (6) Heater; (7) Sensor; (8) Temperature regulator; (9) Strawberry slices; (10) Sorbent tube; (11) Mini vacuum pump; and (12) Teflon tubing.
Figure 1. Illustration of the impinger system for the collection of VOCs emitted from strawberry samples. Labels: (1) 10 L polyester aluminum bag filled with ultra-pure nitrogen; (2) Silicon tubing; (3) Impinger bottle; (4) Aluminum container; (5) Water heated to 25 °C; (6) Heater; (7) Sensor; (8) Temperature regulator; (9) Strawberry slices; (10) Sorbent tube; (11) Mini vacuum pump; and (12) Teflon tubing.
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Figure 2. Comparison of frequency) pattern and log concentration of strawberry volatiles (compounds sorted by functional group).
Figure 2. Comparison of frequency) pattern and log concentration of strawberry volatiles (compounds sorted by functional group).
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Figure 3. Plots of correlation between molecular weight and log (odor thresholds (ppbv)) for alldata (n = 62) and optimal fit (n = 54) of the four major VOC groups (Ester, Alcohol, Aldehyde, and Ketone) emitted from strawberry samples.
Figure 3. Plots of correlation between molecular weight and log (odor thresholds (ppbv)) for alldata (n = 62) and optimal fit (n = 54) of the four major VOC groups (Ester, Alcohol, Aldehyde, and Ketone) emitted from strawberry samples.
Sensors 13 07939f3a 1024Sensors 13 07939f3b 1024
Figure4. Comparison between (odor activity value) OAV and relative proportion (RP) of the major VOCs which had OAV and RP of above 100 and 5%, respectively at storagetime of 0, 1, 3, 6, and 9 days.
Figure4. Comparison between (odor activity value) OAV and relative proportion (RP) of the major VOCs which had OAV and RP of above 100 and 5%, respectively at storagetime of 0, 1, 3, 6, and 9 days.
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Table 1. Occurrence frequency and summed concentration of VOC groups emitted from strawberry samples during the whole study period.
Table 1. Occurrence frequency and summed concentration of VOC groups emitted from strawberry samples during the whole study period.
Sample CodeFunctional GroupTotal

A. EsterB. AlcoholC. AldehydeD. KetoneE. Fatty AcidF.etc.
[A] Frequency (Number of chemical compounds for each functional group)
SRD-138 (34) a5 (4)1 (1)4 (4)1 (1)12 (7)61(51)
SRD-331 (24)8 (3)3 (1)5 (2)1 (1)7 (5)55 (36)
SRD-618 (14)17 (7)8 (2)5 (2)2 (1)15 (3)65 (29)
SRD-94 (3)16 (16)8 (7)4 (4)2 (1)21 (8)55 (39)


[B] Summed concentration (ug m−3)


aNo. of compounds detected consistently from the previous sample is given in the parenthesis: 33 out of 37 compounds in SRD-1 were for example, also seen in SRD-0.

Table 2. Concentration (in both ppb and μg· m−3) and the corresponding relative (mass) composition (RC) of the major strawberry volatiles (mass concentration abundance a ≥ 0.05 %: n = 53).
Table 2. Concentration (in both ppb and μg· m−3) and the corresponding relative (mass) composition (RC) of the major strawberry volatiles (mass concentration abundance a ≥ 0.05 %: n = 53).
OrderCompoundsConcentrationRelative composition a (RC: %)


A. Ester
1Methyl acetate3,9493,87571,0771.570.7511,94511,720214,9874.762.2817.420.029.41.25E-030.04
2Ethyl acetate4,7934,277134,96913011.817,24015,384485,47646742.525.126.366.40.120.70
3Methyl propionate40.851.
4S-Methyl thioacetate94.2129.33.07E-023.07E-023.07E-023464761.13E-011.13E-011.13E-010.510.81
5Isopropyl acetate52.816.727.54.95E-034.95E-0322069.51142.06E-022.06E-020.320.120.02
6Ethyl propionate52.75.19E-0353.55.19E-035.19E-032202.17E-022232.17E-022.17E-020.320.03
7Propyl acetate5.19E-035.19E-031850.725.19E-032.17E-022.17E-027693.022.17E-020.117.90E-04
8Methyl butyrate1,1941,38488.80.475.19E-034,9775,7723701.952.17E-
9Methyl 2-methylbutanoate21.821.713.21.083.35E-0310310362.75.141.59E-
10Isobutyl acetate9.117.0088.91.863.35E-0343.233.24218.801.59E-
11Methyl isovalerate54.856.97.783.35E-033.35E-0326027036.91.59E-021.59E-020.380.460.01
12Ethyl butyrate1,5371,1421711.503.48E-037,2905,4158127.101.65E-0210.
13Butyl acetate34.236.526.40.053.49E-031621731250.241.65E-020.240.300.026.19E-05
14Isopropyl butyrate30.725.92.43E-032.43E-032.43E-031631381.29E-021.29E-021.29E-020.240.24
15Ethyl 2-methylbutyrate63.023.866.33.132.43E-0333512635216.61.29E-020.490.220.054.35E-03
16Ethyl isovalerate14267.236.10.702.43E-037533571923.731.29E-021.100.610.039.76E-04
17Isoamyl acetate81.369.11651.212.43E-034323678786.431.29E-020.630.630.121.68E-03
18Ethyl valerate7.311.732.51E-032.51E-032.51E-0338.89.221.33E-021.33E-021.33E-020.060.02
19Methyl hexanoate46918511.42.51E-032.51E-032,49298260.31.33E-021.33E-023.631.680.01
20trans-2-Hexenyl acetate38.61.781.90E-031.90E-031.90E-0322410.41.11E-021.11E-021.11E-020.330.02
21Ethyl hexanoate72513720.80.031.90E-034,2708081230.161.12E-026.231.380.024.28E-05
22Hexyl acetate11025.54.701.90E-031.90E-0364715027.71.12E-021.12E-020.940.263.79E-03
23Methyl octanoate5.840.881.49E-031.49E-031.49E-0337.75.719.64E-039.64E-039.64E-030.06

ΣConcentration or its RC (ester)52,34642,553705,11652644.776.372.796.50.140.74

B. Alcohol
1Ethyl alcohol3.72E-023.72E-0230948,6851,4177.00E-027.00E-0258091,5372,6650.0823.943.8
2Isopropyl Alcohol8.764.5845.392.827821.511.21112286810.
3n-Propyl alcohol1.42E-021.42E-0230.446.636.13.49E-023.49E-0274.511488.
4Isobutyl alcohol7.01E-037.01E-0331.895,1363202.12E-022.12E-0296.3287,7589670.0175.315.9
5Isopropenylethyl alcohol4.86E-034.86E-034.86E-032.414.471.71E-021.71E-021.71E-028.4815.72.22E-030.26

ΣConcentration or its RC (alcohol)1373561,049379,7814,6510.200.610.1499.476.4

C. Aldehyde
2Allyl aldehyde (Acrolein)8.24E-028.24E-028.24E-025.086.901.89E-011.89E-011.89E-0111.615.83.04E-030.26
3Methylacryl aldehyde1.59E-021.59E-021.59E-021.803.334.54E-024.54E-024.54E-025.149.521.34E-030.16

ΣConcentration or its RC (aldehyde)2,9092,95919,0351,3341,0164.245.062.610.3516.7

D. Ketone
2Methyl ethyl ketone1.38E-021.38E-021.38E-021.151.354.05E-024.05E-024.05E-023.393.968.86E-040.07
3Methyl n-propyl ketone54.047.17.55E-037.55E-037.55E-031901652.66E-022.66E-022.66E-020.280.28
4Methyl Isobutyl Ketone4.627.501.114.11E-034.11E-0318.930.74.531.68E-021.68E-
5Methyl amyl ketone5.646.523.23E-030.510.6126.330.41.50E-022.362.850.040.056.18E-040.05

ΣConcentration or its RC (ketone)6,1957,64953623.273.39.0413.10.076.06E-031.20

E. Fatty acid
1Acetic acid2,5191,9181,76121086.36,1774,7034,3195142129.018.040.591.35E-013.48
22-PROPYNOIC ACID69.62.35E-022.35E-022.35E-022.35E-02199.36.71E-026.71E-026.71E-026.71E-020.29

ΣConcentration or its RC (fatty acid)6,3764,7034,3195142129.308.040.591.35E-013.48

E. etc.
2Ethyl ether7.28E-037.28E-030.700.411.452.20E-022.20E-

ΣConcentration or its RC (etc)17560.588.230.458.

ΣConcentration or its RC (all)68,13958,281730,144382,2096,05599.499.699.999.9999.5

aRC = [Mass concentration (‘i’th compound) / mass concentration (sum) at a given exp day] s100bValues below detection limit (BDL) are underlined (calculated as method detection limit)

Table 3. Relationship between relative composition (RP) and odor activity values (OAV) of the major VOCs (n = 53) emitted from strawberry.
Table 3. Relationship between relative composition (RP) and odor activity values (OAV) of the major VOCs (n = 53) emitted from strawberry.
OrderCompoundsOAV (concentration/ threshold)Relative proportion * (RP, %)Odor type / descriptor

A. Ester
1Methyl acetate0.640.6311.522.55E-041.22E-044.93E-038.98E-030.461.30E-041.66E-03
2Ethyl acetate1.821.6351.30.054.49E- fruita, grapea, contact gluei, fruityl, pinapplel
3Methyl propionate0.420.520.241.78E-033.21E-
4S-Methyl thioacetate58.680.40.451.15sulfurousa, cheesya
5Isopropyl acetate0.
6Ethyl propionate7.537.650.060.30
7Propyl acetate0.197.54E-040.013.85E-04
8Methyl butyrate16819512.50.071.302.790.500.03applea, fruityb, c, d, i, cheesei
9Methyl 2-methylbutanoate21821713210.861.683.105.255.54green applea, fruitya, sweeta
10Isobutyl acetate0., light fruityi, floweryi
11Methyl isovalerate24.925.93.540.190.370.14fruitya, applea, pineapplea
12Ethyl butyrate6,1604,5766866.0047.565.427.23.06fruitya, b, c, d, e, i, sweeta, b, i, pineapplea, ester-likeb, strawberrye, cheesei, fruity sweetm, p, r, u
13Butyl acetate0., bananaa, applei, gluei
14Isopropyl butyrate4.954.180.040.06fruitya, sweeta, pineapplea
15Ethyl 2-methylbutyrate94235699346.97.265.0839.323.9soura, cheesya, sweatya, fruityd
16Ethyl isovalerate1,5927554057.8812.310.816.04.02fruitya, d, applea, pineapplea, sweet fruite
17Isoamyl acetate21.418.243.50.320.160.261.720.16
18Ethyl valerate21.
19Methyl hexanoate30.111.90.730.230.170.03fruitya, b, i, pineapplea, ester-likeb
20trans-2-Hexenyl acetate0.900.040.010.001
21Ethyl hexanoate3,608683103.70.1427.89.774.110.07fruitya, b, i, sweeta, pineapplea, ester-likeb, green appleb, fruit gumi
22Hexyl acetate54.712.72.340.420.180.09fruitya, i, green applea, bananaa, i, applei, peari
23Methyl octanoate0.160.021.23E-033.45E-04Sweet fruitye

ΣConcentration (ester)12,9176,9422,45372.24.61E-0399.699.397.236.80.06

B. Alcohol
1Ethyl alcohol0.011.690.054.25E-040.860.67
2Isopropyl Alcohol3.37E-041.76E-041.74E-033.57E-030.012.60E-062.52E-066.90E-051.82E-030.14fruityl
3n-Propyl alcohol0. (candy)l
4Isobutyl alcohol0.041140.381.52E-0358.35.22Plasticl, badl
5Isopropenylethyl alcohol9.64E-041.79E-034.92E-040.02
63-Methyl-1-butanol0., harshe, k, Chemicalk, stalek, Alcoholicr, greenr, Fusel oils, pomaces
72-Methyl-1-butanol0.230.480.126.49Alcoholics, greens, Fusel oilt, pomacet
10n-Hexanol0.010.033.45E-030.029.16E-051.05E-031.76E-030.34winey-fruityf, Greeni, k, r, heavyi, nutsi, grassk, freshk
11Linalool27.427.00.210.39citrusa, fruitya, e, florala, floweryb, k, sweetb, lemonc, floral-lavenderf, rosek

ΣConcentration or its RC (alcohol)27.427.11.221171.940.210.390.0559.626.3

C. Aldehyde
1Acetaldehyde8.708.8556.93.922.906.70E- applea, fruityj, pungentj, chemicalk, alcoholk, aldehydek, acetaldehydek, pungentk, v, greenn, o, t, sweetv
2Allyl aldehyde (Acrolein)
3Methylacryl aldehyde0.210.390.115.31
4Isobutyraldehyde0.010.093.01E-031.27greenl, maltyl, floralt
5Isovaleraldehyde0.461.090.2314.9greenn, o, maltyn, o
ΣConcentration or its RC (aldehyde)8.708.8556.94.634.526.70E-
D. Ketone
1Acetone0.060.075.34E-031.75E-046.68E-044.61E-041.07E-032.12E-048.93E-059.06E-03aldehydicg, wood pulpl, hayl
2Methyl ethyl ketone1.48E-041.73E-047.56E-052.35E-03Butterh, sweeth, chocolateh, Butterscotchl
3Methyl n-propyl ketone0.030.032.69E-044.34E-04thinnerb, acetoneb, kerosenei, solventsi, orange peelq, sweetw, x, fruityw, x
4Methyl Isobutyl Ketone0.010.012.06E-036.63E-052.00E-048.17E-05
5Methyl amyl ketone0.040.053.60E-034.34E-033.08E-046.61E-041.83E-030.06meatyb, danish blue cheesei, greent, Animalsr, bluer, cheeser
ΣConcentration or its RC (ketone)0.1430.1657.40E-033.92E-035.18E-031.10E-032.36E-032.93E-042.00E-030.07
E. Fatty acid
1Acetic acid17.3713.2312.151.450.601.34E-010.190.480.748.08sourd, vinagere, i, k
22-PROPYNOIC ACID0.745.71E-03
ΣConcentration or its RC (fatty acid)
2Ethyl ether2.12E-031.23E-034.39E-038.40E-056.26E-040.06
ΣConcentration or its RC (etc)0.730.340.560.900.310.
ΣConcentration or its RC (all)12,9726,9922,5241967.37100100100100100

*RP = [OAV (n) / OAV (sum of main VOCs) at the exp day] x 100References:aDu et al. [1],bUlrich et al. [2],cSchulbach et al. [28],dSchieberle and Hofmann [29],eAznar et al. [30],fBuchbauer et al. [31],gCai et al. [32],hClausen et al. [33],iLarsen and Poll [34],jSemmelroch and Grosch (1995) [35],kKomes et al. [36],lArora et al. [37],mCarpino and Mallia [38],nKubck ova and Grosch [39],oKubícková and Grosch [40],pLe Quéré et al. [41],qMoio and Addeo [42],rMoio et al. [43],sMoio et al. [44],tRychlik and Bosset [45],uChristensen and Reineccius [46],vMilo and Reineccius [47],wPreininger and Grosch [48],xPreininger et al. [49]

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