Food Chemistry 176 (2015) 17–21
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Short communication
Gas chromatography of safranal as preferable method for the commercial grading of saffron (Crocus sativus L.) Monica Bononi a,⇑, Paola Milella b, Fernando Tateo a a b
Department of Agricultural and Environmental Sciences, University of Milan, Via Celoria, 2, 20133 Milan, Italy Department of Veterinary Medicine, University of Bari, S.P. per Casamassima, 70010 Valenzano, BA, Italy
a r t i c l e
i n f o
Article history: Received 14 April 2014 Received in revised form 1 December 2014 Accepted 13 December 2014 Available online 23 December 2014 Keywords: Saffron Safranal UV GC
a b s t r a c t We present a new extraction protocol, using ethyl alcohol as a solvent, to evaluate safranal by gas chromatography (GC). A linear response was obtained with R2 = 0.995 and a reproducibility standard deviation of 4.7–6.0%. The limit of detection and limit of quantitation were 0.05 and 0.25 g kg 1, respectively. The GC data for several samples of powdered saffron from different origins were compared to specific absorbance values measured according to the ISO Normative 3632-1:2011 method. The aroma strength of saffron samples quantitated by GC and the specific absorbance values of safranal by the UV method did not correlate. Quantitative evaluation of safranal by GC appears to be more specific and useful for commercial comparisons of saffron quality. Ó 2014 Published by Elsevier Ltd.
1. Introduction Crocus sativus L. (saffron) is one of the most expensive spices used in the food industry due to its aroma, flavour, and colour. The world’s saffron sectors have classified saffron by its aroma strength, flavour strength, and colouring strength using the ISO Normative 3632-1:2011 (ISO, 2011) method, which is a spectrophotometric method that produces three chemical specifications representing: aroma strength (expressed as safranal), flavour or bitterness strength (expressed as picrocrocin), and colouring strength (expressed as crocin). The saffron aroma is produced after a thermal process (Carmona, Zalacain, Salinas, & Alonso, 2007; Raina, Agarwal, Bhatia, & Gaur, 1996; Sharafzadeh, 2011; Tarantilis & Polissiou, 1997) and safranal is considered the most important key compound. The bitter taste is currently identified as flavour; while, the colour exhibits various shades of yellow or red depending on the country of origin (Anastasaki et al., 2009, 2010; Cossignani et al., 2014; Luykx & Van Ruth, 2008). The commercial classification of saffron is determined by the ISO Normative method, which considers three categories according to spectrophotometric parameters, but this non-specific analysis does not provide quality grading, which is useful in an international market (Anastasaki et al., 2009, 2010; Cullerè, San-Juan, & Cacho, 2011; Gregory, Menary, & Davies, 2005; Jalali-Heravi, Parastar, & Ebrahimi-Najafabadi, 2009; Lech, Witowska-Jarosz, & ⇑ Corresponding author. Tel.: +39 02 50316540; fax: +39 02 50316539. E-mail address: [email protected] (M. Bononi). http://dx.doi.org/10.1016/j.foodchem.2014.12.047 0308-8146/Ó 2014 Published by Elsevier Ltd.
Jarosz, 2009; Maggi et al., 2011). In addition, the ISO method cannot adequately distinguish between genuine and adulterated saffron (Lozano, Castellar, Simancas, & Iborra, 1999; Zougagh, Rios, & Valcarcel, 2005). Various authors have proposed more specific analytical methods for saffron authentication (Alonso, Salinas, & Garijo, 1998; Kanti, Sushma, & Ghandi, 2011; Marieschi, Torelli, & Bruni, 2012; Saffron in Europe & 15, 2013; Torelli, Marieschi, & Bruni, 2014; Wintherhalter & Straubinger, 2000) and for determination of single parameters (Caballero-Ortega et al., 2004; Cullerè et al., 2011; Gregory et al., 2005; Hadizadeh et al., 2007; Jalali-Heravi et al., 2009; Kanakis, Daferera, Tarantilis, & Polissiou, 2004; Lage et al., 2010; Lozano et al., 1999; Luykx & Van Ruth, 2008; Tarantilis & Polissiou, 1997; Vignolini et al., 2008). Many papers have been published on the volatile composition of saffron, and many other compounds in addition to safranal contribute to its complete aroma (Carmona et al., 2007; Jalali-Heravi et al., 2009). Yet, safranal is considered the more influential sensorial compound and accurate quantification of safranal may represent a very useful index of commercial quality. The chemical specification expressed as ‘‘safranal’’ using the ISO 6323-2:2010 method (ISO, 2010) is not useful as a quantitative specific marker for quality control and the classification used in the ISO 63231:2011 method (ISO, 2011) considers the same range (20–50) to be a characteristic of the three categories. Thus, the safranal index adopted by ISO Normative is not able to differentiate between commercial samples because safranal (Fig. 1) is difficult to solubilise in water, the solvent used in the method for UV determination at k = 330 nm (Gregory et al., 2005; Lech et al., 2009; Maggi et al.,
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M. Bononi et al. / Food Chemistry 176 (2015) 17–21
2011). In addition, it has been reported that the cis-crocetin ester isomers interfere at the same wavelength (Hadizadeh et al., 2007). Recently Valle Garcia-Rodriguez et al. (2014) validated a HPLC method for the independent determination of crocetin esters, picrocrocin and safranal, considering the interferences in the measurements made in UV–vis based method. The aim of the present paper was to demonstrate that the results obtained by the ISO Normative method for safranal in saffron are not consistent with the quantity determined by gas chromatography (GC), and the data produced by specific GC analysis are more useful as a quality control. We compared data, from 76 powdered saffron samples, obtained by the ISO 6323-2:2010 Normative (ISO, 2010) method with data derived from quantitative (g kg 1) GC evaluation. We also identified ethyl alcohol as the preferred safranal-extracting solvent after comparing various solvents and several extraction methods previously used (Kanakis et al., 2004; Maggi et al., 2011). We obtained a high safranal extraction yield (P99.5%) using ethyl alcohol and ultrasonic-assisted extraction (USE). Our results demonstrated that quantitative GC data, which represent a measure of the quality of aroma strength, can be used to determine the commercial chemical quality. 2. Materials and methods 2.1. Chemicals and reagents Ethyl alcohol (P99.5%) and safranal (P88%) were purchased from Sigma–Aldrich (Milan, Italy). Hydrophilic Polypropylene Membrane Filters (0.45 lm, GH Polypro, Pall) were purchased from VWR International (Milan, Italy). 2.2. Samples A total of 76 samples of powdered saffron were collected during the quality evaluation, from the import/export activities of various European companies and distribution by large-scale retail channels.
2.4. Safranal extraction Approximately 20 mL of ethyl alcohol was added to 50–60 mg ground saffron in a 25 mL flask. The mixture was vortexed for 1 min and underwent USE for 60 min (T = 25 °C) at the fixed frequency of 35 kHz. The organic extract was adjusted to a volume of 25 mL and filtered through a polypropylene membrane filter with 0.45 lm porosity and a 25 mm diameter. The filtrate was mixed well and the injection volume were 2 lL. Experiments were performed in a previous phase to verify the extraction kinetics of safranal and define the preferable extraction ratio and extraction time, which was identified as 60 min.
2.5. Analytical quality assurance Quantification was performed using a calibration curve of safranal standards in ethyl alcohol across the range 5–150 mg L 1, and 2 lL samples were injected in triplicate. The calibration curve of safranal in clean solvent showed a linear response with a correlation coefficient (R2) of 0.995. Following NMKL Procedure No. 5 (Nordic Committee on Food Analysis, 1997), the reproducibility standard deviation (RSD) ranged from 4.7% to 6.0%. The limit of detection (LOD) was in the order of 0.05 g kg 1, and the limit of quantitation (LOQ) was assumed to be 0.25 g kg 1 (i.e., five times the LOD). The recovery yield of safranal at low concentration was estimated by spiking sample 2, containing 3.61 g kg 1 safranal (mean of two determinations), with 2.00 g kg 1 to reach a calculated concentration of 5.61 g kg 1. The measured concentration was 5.41 g kg 1 (mean of two determinations), corresponding to a 96.4% recovery. For higher concentrations, sample 52, containing 6.52 g kg 1 (mean of two determinations), was spiked with 2.00 g kg 1 to reach a calculated concentration of 8.52 g kg 1. The measured concentration was 8.38 g kg 1 (mean of two determinations), corresponding to a 98.3% recovery.
3. Results and discussion 2.3. Analytical methods Samples were analysed by UV–vis spectrophotometry and GC. The chemical specifications A1% 1cm considered for safranal in ISO 3632-2:2010 (ISO, 2010) are reported in Table 1 in increasing order. The results are expressed as specific absorbance at k = 330 nm. GC analyses were carried out with a Shimadzu 2010 Plus gas chromatograph (Shimadzu, Italy). Hydrogen was used as the carrier gas at a flow rate of 1.5 mL min 1. Safranal was quantified using a Supelco SPB column (5% diphenyl–95% dimethylpolysiloxane; 60 m 0.25 mm, 0.25 lm i.d. film thickness). The oven temperature programme was 60 °C (held for 1 min) and increased to 240 °C at a rate of 3 °C min 1 (held for 1 min). The injector temperature was 220 °C and the split injector mode (1:20) used. The detector temperature (FID) was 240 °C. The GC data, expressed as g kg 1, are reported in Table 1.
O H3C
H CH3 CH3
Fig. 1. Molecular structure of safranal (2,6,6-trimethyl-1,3-cyclohexadiene-1-carboxaldehyde) – CAS number: 116-26-7 (C10H14O) M.W. 150.22.
The UV specific absorbance values at k = 330 nm are reported in increasing order in Table 1 for the 76 saffron samples measured following the ISO method 3632-2:2010. The safranal contents determined by the GC method are also reported. Fig. 2 is a graphic representation of the results in Table 1. The values derived from the ISO method for safranal did not exhibit the same trend as the values determined by the GC method; thus, the ISO data do not show the same differences between samples as the GC data. The samples 28 and 29, for example, showing the same specific absorbance value 36, contain 5.43 and 9.16 g kg 1 of safranal, respectively. The same observations apply the pairs of samples: 16 and 17, 21 and 22, 31 and 32, 35 and 36, 46 and 47, 68 and 69, and others. The lack of correlation is also demonstrated when we compare pairs of samples having very similar safranal concentrations and very different specific absorbance values; for example, samples 4 and 49, have specific absorbance values of 27 and 42, respectively, and very similar safranal content (6.49 and 6.46 g kg 1, respectively). The same observation applies to the sample pairs: 2 and 67, 5 and 65, 11 and 74, 27 and 63, 26 and 47, and others. Example UV traces for samples 24, 76, 1, and 2 are shown in Fig. 3, which shows obvious interferences on the measures of safranal absorbances, which correspond to the data in Table 1. In particular, the specific absorbance values for samples 1, 2, and 24 are not consistent with values determined by GC (1.35, 3.61, and 7.72 g kg 1, respectively). Sample 76 has the highest specific absorbance value, but the lowest safranal content (0.54 g kg 1). The GC
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M. Bononi et al. / Food Chemistry 176 (2015) 17–21 Table 1 Chemical specification of safranal (specific absorbance at k = 330 nm) by the ISO 3632-2:2010 method and the corresponding GC data.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
Samples
Origin
ISO A1% 1cm
GC (g kg
Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Italy Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Italy Swiss Raw mat. Raw mat. Raw mat. Raw mat. Italy Raw mat. Raw mat. Raw mat. Italy Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Italy Raw mat. Raw mat. Raw mat. Raw mat.
China Spain Iran Iran Iran n.d. Iran Iran Iran Iran Iran Iran Iran Iran n.d. Spain Iran Iran Iran Spain n.d. Greece Iran Iran n.d. Iran Iran Iran Iran Iran Iran Greece Iran n.d. Iran Iran Iran Iran
19 26 26 27 28 29 30 31 32 32 32 33 33 33 34 34 34 34 34 35 35 35 35 36 36 36 36 36 36 37 37 37 37 38 38 38 38 38
1.35 3.61 6.44 6.49 4.99 6.54 5.22 7.29 6.04 6.56 7.68 5.40 6.30 5.86 6.90 4.46 7.40 10.58 7.34 6.76 5.13 9.48 7.54 7.72 7.70 8.50 6.80 5.43 9.16 5.93 8.73 6.86 6.35 5.10 4.16 7.97 7.43 7.97
1
) 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76
Samples
Origin
ISO A1% 1cm
GC (g kg
Raw mat. Raw mat. Raw mat. Raw mat. Italy Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Italy Italy Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Italy Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Italy Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Swiss
Iran Iran Greece Iran n.d. Greece Greece Iran Iran Greece Iran Iran Iran n.d. n.d. Iran Iran Iran Iran India Iran Iran Iran n.d. Greece Iran Iran Greece India India Greece n.d. Greece Iran Iran Iran Greece Spain
38 39 40 40 40 40 41 42 42 42 42 42 42 42 42 43 43 43 43 43 44 44 44 44 44 45 46 46 47 47 47 48 48 48 50 50 50 75
5.87 6.63 5.67 7.86 6.27 10.56 9.23 6.06 9.68 7.01 6.46 5.86 6.14 6.25 8.82 6.20 5.24 4.52 6.68 6.43 6.76 7.07 6.28 6.22 6.87 8.41 5.04 10.7 3.65 4.19 10.38 8.65 9.32 7.76 5.59 7.48 6.37 0.54
1
)
n.d.: origin not declared on label.
(a)
10 g kg-1
5 0 0
10
20
30
40
50
60
70
40
50
60
70
(b)
80 60 40 20 0 0
10
20
30 samples
Fig. 2. (a) Safranal content (g kg
) determined by the GC method and by (b) specific absorbance A1% 1cm using the ISO method for the 76 saffron samples identified in Table 1.
1
traces corresponding to the same four samples of Fig. 3 are reported in Fig. 4. The absence of correlation between the UV trace and the specific safranal content raises concrete doubts about the UV trace representing a link between the ‘‘aroma strength’’ parameter and safranal content. The safranal data depicted in Fig. 2a does not correspond to the data reported in Fig. 2b; although, all samples (excluding the first and the last) have the same chemical specification for safranal (range 20–50) following the ISO 6323-2:2011 classification.
4. Conclusion The chemical specification ‘‘safranal’’ obtained by the ISO 63232:2010 method (ISO, 2010) does not indicate differences between saffron samples of varying qualities and origins and does not correlate with the quantitative determination by GC. The results of the present study indicate that the UV evaluation of specific absorbance does not lead to differentiation between safranal content ranged from 3.61 to 7.48 g kg 1 (all included for the three categories in UV range 20–50). More, for the 76 samples in the present
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M. Bononi et al. / Food Chemistry 176 (2015) 17–21
24)
76)
330 330
1)
2)
330
330
Fig. 3. Examples of UV traces produced following the ISO 6323-2:2010 method from samples 24, 76, 1, and 2 (see Table 1), showing obvious interferences in the safranal absorbance values at k = 330 nm in aqueous extracts. In particular, the specific absorbance values for samples 1, 2, and 24 are not consistent with values determined by GC (1.35, 3.61, and 7.72 g kg 1, respectively). Sample 76 has the highest specific absorbance value, but the lowest safranal content (0.54 g kg 1).
Fig. 4. GC traces of safranal produced by samples 24, 76, 1, and 2 (see Table 1) analysed as reported in Sections 2.3 and 2.4.
study, the absorbance were 19–75, but no correlation was found between the two data series, as reported in Table 1 and in Fig. 2. It appears that substances that interfere with the UV evaluation do not permit an effective comparison of the safranal content in saffron. As affirmed by Hadizadeh et al. (2007), the interference of cis-crocetin ester isomers has been evidenced at the wavelength of safranal maximum absorbance. More, it may be useful to consider the paper of Valle Garcia-Rodriguez et al. (2014) where the UV spectrum of safranal is reported vs UV spectra of two transcrocetin esters. These UV spectra demonstrate that, on the low
absorbance value due to the low safranal concentration, the higher absorbance value of more represented trans-crocetin esters produces a clear interference. Thus, GC is preferable for determining the effective safranal content for the purposes of commercial saffron grading. References Alonso, G. L., Salinas, M. R., & Garijo, J. (1998). Method to determine the authenticity of aroma of Saffron (Crocus sativus L.). Journal of Food Protection, 61, 1525–1528.
M. Bononi et al. / Food Chemistry 176 (2015) 17–21 Anastasaki, E., Kanakis, C., Pappas, C., Maggi, L., del Campo, C. P., Carmona, M., Alonso, G. L., & Polissiou, M. G. (2009). Geographical differentiation of saffron by GC–MS/FID and chemometrics. European Food Research and Technology, 229, 899–905. Anastasaki, E., Kanakis, C., Pappas, C., Maggi, L., del Campo, C. P., Carmona, M., Alonso, G. L., & Polissiou, M. G. (2010). Differentiation of saffron from four countries by mid-infrared spectroscopy and multivariate analysis. European Food Research Technology, 230, 571–577. Caballero-Ortega, H., Pereda-Miranda, R., Riveron-Negrete, L., Hernandez, J. M., Medecigo-Rios, M., Castillo-Villanueva, A., & Abdullaev, F. I. (2004). Chemical composition of saffron (Crocus sativus L.) from four countries. Acta Horticulturae, 650, 321–326. Carmona, M., Zalacain, A., Salinas, M. R., & Alonso, G. L. (2007). A new approach to saffron aroma. Critical Reviews in Food Science and Nutrition, 47, 145–159. Cossignani, L., Urbani, E., Simonetti, M. S., Maurizi, A., Chiesi, C., & Blasi, F. (2014). Characterisation of secondary metabolites in saffron from central Italy (Cascia, Umbria). Food Chemistry, 143, 446–451. Cullerè, L., San-Juan, F., & Cacho, J. (2011). Characterization of aroma active compounds of Spanish saffron by gas chromatography-olfactometry: Quantitative evaluation of the most relevant aromatic compounds. Food Chemistry, 127, 1866–1871. Gregory, M. J., Menary, R. C., & Davies, N. W. (2005). Effect of drying temperature and air flow on the production and retention of secondary metabolites in saffron. Journal of Agricultural and Food Chemistry, 53, 5969–5975. Hadizadeh, F., Mahdavi, M., Emami, S. A., Khashayarmanesh, Z., Hassanzadeh, M., Asili, J., Seifi, M., Nassirli, H., Shariatimoghadam, A., & Noorbakhsh, R. (2007). Evaluation of ISO method in saffron qualification. Acta Horticulturae, 739, 405–410. ISO 3632-2 Technical Specification. (2010). Saffron (Crocus sativus L.). Part 2 (test methods). Geneva, Switzerland: International Organization for Standardization. ISO 3632-1 Technical Specification. (2011). Saffron (Crocus sativus L.). Part 1 (specification). Geneva, Switzerland: International Organization for Standardization. Jalali-Heravi, M., Parastar, H., & Ebrahimi-Najafabadi, H. (2009). Characterization of volatile components of Iranian saffron using factorial-based response surface modeling of ultrasonic extraction combined with gas chromatography–mass spectrometry analysis. Journal of Chromatography A, 1216, 6088–6097. Kanakis, C. D., Daferera, D. J., Tarantilis, P. A., & Polissiou, M. G. (2004). Qualitative determination of volatile compounds and qualitative evaluation of safranal and 4-hydroxy-2,6,6-trimethyl-1-cyclohexene-1-carboxaldehyde (HTCC) in Greek saffron. Journal of Agricultural and Food Chemistry, 52, 4515–4521. Kanti, R., Sushma, K., & Ghandi, R. (2011). Adulteration and marketing of saffron (Crocus sativus L.). Journal of Tropical Medicinal Plants, 12, 135–140.
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Lage, M., Gaboun, F., Bakhy, K., Dakak, H., Zouahri, A., & Cantrell, C. L. (2010). Sustainable production of high quality saffron (Crocus sativus L.) in some Moroccan areas. Acta Horticulturae, 850, 235–238. Lech, K., Witowska-Jarosz, J., & Jarosz, M. (2009). Saffron yellow: Characterization of carotenoids by high performance liquid chromatography with electrospray mass spectrometric detection. Journal of Mass Spectrometry, 44, 1661–1667. Lozano, P., Castellar, M. R., Simancas, M. J., & Iborra, J. L. (1999). Quantitative high performance liquid chromatographic method to analyse commercial saffron (Crocus sativus L.) products. Journal of Chromatography A, 830, 477–483. Luykx, D. M. A. M., & Van Ruth, S. M. (2008). An overview of analytical methods for determining the geographical origin of food products. Food Chemistry, 107, 897–911. Maggi, L., Sanchez, A. M., Carmona, M., Kanakis, C. D., Anastasaki, E., Tarantilis, P. A., Polissiou, M. G., & Alonso, G. L. (2011). Rapid determination of safranal in the quality control of saffron spice. Food Chemistry, 127, 369–373. Marieschi, M., Torelli, A., & Bruni, R. (2012). Quality control of saffron (Crocus sativus L.): Development of SCAR markers for the detection of plant adulterants used as bulking agents. Journal of Agricultural and Food Chemistry, 60, 10998–11004. Nordic Committee on Food Analysis. (1997). Estimation and expression of measurement uncertainty in chemical analysis. NMKL Procedure No. 5. Raina, B. L., Agarwal, S. G., Bhatia, A. K., & Gaur, G. S. (1996). Changes in pigments and volatiles of saffron (Crocus sativus L.) during processing and storage. Journal of the Science of Food and Agriculture, 71, 27–32. Saffron in Europe. (2013). White Book. http://www.europeansaffron.eu/archivos/ White book english.pdf Accessed 15.04.13. Sharafzadeh, S. (2011). Saffron: A concise review of researches. Advances in Environmental Biology, 5, 1617–1621. Tarantilis, P. A., & Polissiou, M. G. (1997). Isolation and identification of the aroma components from saffron (Crocus sativus). Journal of Agriculture and Food Chemistry, 45, 459–462. Torelli, A., Marieschi, M., & Bruni, R. (2014). Authentication of saffron (Crocus sativus L.) in different processed, retail products by means of SCAR markers. Food Control, 36, 126–131. Valle Garcia-Rodriguez, M., Serrano-Diaz, J., Tarantilis, P. A., López-Córcoles, H., Carmona, M., & Alonso, G. L. (2014). Determination of saffron quality by highperformance liquid chromatography. Journal of Agriculture and Food Chemistry, 62, 8068–8074. Vignolini, P., Heimler, D., Pinelli, P., Ieri, F., Sciullo, A., & Romani, A. (2008). Characterization of by-products of saffron (Crocus sativus L.) production. Natural Product Communications, 3, 1959–1962. Wintherhalter, P., & Straubinger, M. (2000). Saffron-renewed interest in an ancient spice. Food Reviews International, 16, 39–59. Zougagh, M., Rios, A., & Valcarcel, M. (2005). An automated screening method for the fast, simple discrimination between natural and artificial colorants in commercial saffron products. Analytica Chimica Acta, 535, 133–138.
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Short communication
Gas chromatography of safranal as preferable method for the commercial grading of saffron (Crocus sativus L.) Monica Bononi a,⇑, Paola Milella b, Fernando Tateo a a b
Department of Agricultural and Environmental Sciences, University of Milan, Via Celoria, 2, 20133 Milan, Italy Department of Veterinary Medicine, University of Bari, S.P. per Casamassima, 70010 Valenzano, BA, Italy
a r t i c l e
i n f o
Article history: Received 14 April 2014 Received in revised form 1 December 2014 Accepted 13 December 2014 Available online 23 December 2014 Keywords: Saffron Safranal UV GC
a b s t r a c t We present a new extraction protocol, using ethyl alcohol as a solvent, to evaluate safranal by gas chromatography (GC). A linear response was obtained with R2 = 0.995 and a reproducibility standard deviation of 4.7–6.0%. The limit of detection and limit of quantitation were 0.05 and 0.25 g kg 1, respectively. The GC data for several samples of powdered saffron from different origins were compared to specific absorbance values measured according to the ISO Normative 3632-1:2011 method. The aroma strength of saffron samples quantitated by GC and the specific absorbance values of safranal by the UV method did not correlate. Quantitative evaluation of safranal by GC appears to be more specific and useful for commercial comparisons of saffron quality. Ó 2014 Published by Elsevier Ltd.
1. Introduction Crocus sativus L. (saffron) is one of the most expensive spices used in the food industry due to its aroma, flavour, and colour. The world’s saffron sectors have classified saffron by its aroma strength, flavour strength, and colouring strength using the ISO Normative 3632-1:2011 (ISO, 2011) method, which is a spectrophotometric method that produces three chemical specifications representing: aroma strength (expressed as safranal), flavour or bitterness strength (expressed as picrocrocin), and colouring strength (expressed as crocin). The saffron aroma is produced after a thermal process (Carmona, Zalacain, Salinas, & Alonso, 2007; Raina, Agarwal, Bhatia, & Gaur, 1996; Sharafzadeh, 2011; Tarantilis & Polissiou, 1997) and safranal is considered the most important key compound. The bitter taste is currently identified as flavour; while, the colour exhibits various shades of yellow or red depending on the country of origin (Anastasaki et al., 2009, 2010; Cossignani et al., 2014; Luykx & Van Ruth, 2008). The commercial classification of saffron is determined by the ISO Normative method, which considers three categories according to spectrophotometric parameters, but this non-specific analysis does not provide quality grading, which is useful in an international market (Anastasaki et al., 2009, 2010; Cullerè, San-Juan, & Cacho, 2011; Gregory, Menary, & Davies, 2005; Jalali-Heravi, Parastar, & Ebrahimi-Najafabadi, 2009; Lech, Witowska-Jarosz, & ⇑ Corresponding author. Tel.: +39 02 50316540; fax: +39 02 50316539. E-mail address: [email protected] (M. Bononi). http://dx.doi.org/10.1016/j.foodchem.2014.12.047 0308-8146/Ó 2014 Published by Elsevier Ltd.
Jarosz, 2009; Maggi et al., 2011). In addition, the ISO method cannot adequately distinguish between genuine and adulterated saffron (Lozano, Castellar, Simancas, & Iborra, 1999; Zougagh, Rios, & Valcarcel, 2005). Various authors have proposed more specific analytical methods for saffron authentication (Alonso, Salinas, & Garijo, 1998; Kanti, Sushma, & Ghandi, 2011; Marieschi, Torelli, & Bruni, 2012; Saffron in Europe & 15, 2013; Torelli, Marieschi, & Bruni, 2014; Wintherhalter & Straubinger, 2000) and for determination of single parameters (Caballero-Ortega et al., 2004; Cullerè et al., 2011; Gregory et al., 2005; Hadizadeh et al., 2007; Jalali-Heravi et al., 2009; Kanakis, Daferera, Tarantilis, & Polissiou, 2004; Lage et al., 2010; Lozano et al., 1999; Luykx & Van Ruth, 2008; Tarantilis & Polissiou, 1997; Vignolini et al., 2008). Many papers have been published on the volatile composition of saffron, and many other compounds in addition to safranal contribute to its complete aroma (Carmona et al., 2007; Jalali-Heravi et al., 2009). Yet, safranal is considered the more influential sensorial compound and accurate quantification of safranal may represent a very useful index of commercial quality. The chemical specification expressed as ‘‘safranal’’ using the ISO 6323-2:2010 method (ISO, 2010) is not useful as a quantitative specific marker for quality control and the classification used in the ISO 63231:2011 method (ISO, 2011) considers the same range (20–50) to be a characteristic of the three categories. Thus, the safranal index adopted by ISO Normative is not able to differentiate between commercial samples because safranal (Fig. 1) is difficult to solubilise in water, the solvent used in the method for UV determination at k = 330 nm (Gregory et al., 2005; Lech et al., 2009; Maggi et al.,
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M. Bononi et al. / Food Chemistry 176 (2015) 17–21
2011). In addition, it has been reported that the cis-crocetin ester isomers interfere at the same wavelength (Hadizadeh et al., 2007). Recently Valle Garcia-Rodriguez et al. (2014) validated a HPLC method for the independent determination of crocetin esters, picrocrocin and safranal, considering the interferences in the measurements made in UV–vis based method. The aim of the present paper was to demonstrate that the results obtained by the ISO Normative method for safranal in saffron are not consistent with the quantity determined by gas chromatography (GC), and the data produced by specific GC analysis are more useful as a quality control. We compared data, from 76 powdered saffron samples, obtained by the ISO 6323-2:2010 Normative (ISO, 2010) method with data derived from quantitative (g kg 1) GC evaluation. We also identified ethyl alcohol as the preferred safranal-extracting solvent after comparing various solvents and several extraction methods previously used (Kanakis et al., 2004; Maggi et al., 2011). We obtained a high safranal extraction yield (P99.5%) using ethyl alcohol and ultrasonic-assisted extraction (USE). Our results demonstrated that quantitative GC data, which represent a measure of the quality of aroma strength, can be used to determine the commercial chemical quality. 2. Materials and methods 2.1. Chemicals and reagents Ethyl alcohol (P99.5%) and safranal (P88%) were purchased from Sigma–Aldrich (Milan, Italy). Hydrophilic Polypropylene Membrane Filters (0.45 lm, GH Polypro, Pall) were purchased from VWR International (Milan, Italy). 2.2. Samples A total of 76 samples of powdered saffron were collected during the quality evaluation, from the import/export activities of various European companies and distribution by large-scale retail channels.
2.4. Safranal extraction Approximately 20 mL of ethyl alcohol was added to 50–60 mg ground saffron in a 25 mL flask. The mixture was vortexed for 1 min and underwent USE for 60 min (T = 25 °C) at the fixed frequency of 35 kHz. The organic extract was adjusted to a volume of 25 mL and filtered through a polypropylene membrane filter with 0.45 lm porosity and a 25 mm diameter. The filtrate was mixed well and the injection volume were 2 lL. Experiments were performed in a previous phase to verify the extraction kinetics of safranal and define the preferable extraction ratio and extraction time, which was identified as 60 min.
2.5. Analytical quality assurance Quantification was performed using a calibration curve of safranal standards in ethyl alcohol across the range 5–150 mg L 1, and 2 lL samples were injected in triplicate. The calibration curve of safranal in clean solvent showed a linear response with a correlation coefficient (R2) of 0.995. Following NMKL Procedure No. 5 (Nordic Committee on Food Analysis, 1997), the reproducibility standard deviation (RSD) ranged from 4.7% to 6.0%. The limit of detection (LOD) was in the order of 0.05 g kg 1, and the limit of quantitation (LOQ) was assumed to be 0.25 g kg 1 (i.e., five times the LOD). The recovery yield of safranal at low concentration was estimated by spiking sample 2, containing 3.61 g kg 1 safranal (mean of two determinations), with 2.00 g kg 1 to reach a calculated concentration of 5.61 g kg 1. The measured concentration was 5.41 g kg 1 (mean of two determinations), corresponding to a 96.4% recovery. For higher concentrations, sample 52, containing 6.52 g kg 1 (mean of two determinations), was spiked with 2.00 g kg 1 to reach a calculated concentration of 8.52 g kg 1. The measured concentration was 8.38 g kg 1 (mean of two determinations), corresponding to a 98.3% recovery.
3. Results and discussion 2.3. Analytical methods Samples were analysed by UV–vis spectrophotometry and GC. The chemical specifications A1% 1cm considered for safranal in ISO 3632-2:2010 (ISO, 2010) are reported in Table 1 in increasing order. The results are expressed as specific absorbance at k = 330 nm. GC analyses were carried out with a Shimadzu 2010 Plus gas chromatograph (Shimadzu, Italy). Hydrogen was used as the carrier gas at a flow rate of 1.5 mL min 1. Safranal was quantified using a Supelco SPB column (5% diphenyl–95% dimethylpolysiloxane; 60 m 0.25 mm, 0.25 lm i.d. film thickness). The oven temperature programme was 60 °C (held for 1 min) and increased to 240 °C at a rate of 3 °C min 1 (held for 1 min). The injector temperature was 220 °C and the split injector mode (1:20) used. The detector temperature (FID) was 240 °C. The GC data, expressed as g kg 1, are reported in Table 1.
O H3C
H CH3 CH3
Fig. 1. Molecular structure of safranal (2,6,6-trimethyl-1,3-cyclohexadiene-1-carboxaldehyde) – CAS number: 116-26-7 (C10H14O) M.W. 150.22.
The UV specific absorbance values at k = 330 nm are reported in increasing order in Table 1 for the 76 saffron samples measured following the ISO method 3632-2:2010. The safranal contents determined by the GC method are also reported. Fig. 2 is a graphic representation of the results in Table 1. The values derived from the ISO method for safranal did not exhibit the same trend as the values determined by the GC method; thus, the ISO data do not show the same differences between samples as the GC data. The samples 28 and 29, for example, showing the same specific absorbance value 36, contain 5.43 and 9.16 g kg 1 of safranal, respectively. The same observations apply the pairs of samples: 16 and 17, 21 and 22, 31 and 32, 35 and 36, 46 and 47, 68 and 69, and others. The lack of correlation is also demonstrated when we compare pairs of samples having very similar safranal concentrations and very different specific absorbance values; for example, samples 4 and 49, have specific absorbance values of 27 and 42, respectively, and very similar safranal content (6.49 and 6.46 g kg 1, respectively). The same observation applies to the sample pairs: 2 and 67, 5 and 65, 11 and 74, 27 and 63, 26 and 47, and others. Example UV traces for samples 24, 76, 1, and 2 are shown in Fig. 3, which shows obvious interferences on the measures of safranal absorbances, which correspond to the data in Table 1. In particular, the specific absorbance values for samples 1, 2, and 24 are not consistent with values determined by GC (1.35, 3.61, and 7.72 g kg 1, respectively). Sample 76 has the highest specific absorbance value, but the lowest safranal content (0.54 g kg 1). The GC
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M. Bononi et al. / Food Chemistry 176 (2015) 17–21 Table 1 Chemical specification of safranal (specific absorbance at k = 330 nm) by the ISO 3632-2:2010 method and the corresponding GC data.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
Samples
Origin
ISO A1% 1cm
GC (g kg
Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Italy Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Italy Swiss Raw mat. Raw mat. Raw mat. Raw mat. Italy Raw mat. Raw mat. Raw mat. Italy Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Italy Raw mat. Raw mat. Raw mat. Raw mat.
China Spain Iran Iran Iran n.d. Iran Iran Iran Iran Iran Iran Iran Iran n.d. Spain Iran Iran Iran Spain n.d. Greece Iran Iran n.d. Iran Iran Iran Iran Iran Iran Greece Iran n.d. Iran Iran Iran Iran
19 26 26 27 28 29 30 31 32 32 32 33 33 33 34 34 34 34 34 35 35 35 35 36 36 36 36 36 36 37 37 37 37 38 38 38 38 38
1.35 3.61 6.44 6.49 4.99 6.54 5.22 7.29 6.04 6.56 7.68 5.40 6.30 5.86 6.90 4.46 7.40 10.58 7.34 6.76 5.13 9.48 7.54 7.72 7.70 8.50 6.80 5.43 9.16 5.93 8.73 6.86 6.35 5.10 4.16 7.97 7.43 7.97
1
) 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76
Samples
Origin
ISO A1% 1cm
GC (g kg
Raw mat. Raw mat. Raw mat. Raw mat. Italy Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Italy Italy Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Italy Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Italy Raw mat. Raw mat. Raw mat. Raw mat. Raw mat. Swiss
Iran Iran Greece Iran n.d. Greece Greece Iran Iran Greece Iran Iran Iran n.d. n.d. Iran Iran Iran Iran India Iran Iran Iran n.d. Greece Iran Iran Greece India India Greece n.d. Greece Iran Iran Iran Greece Spain
38 39 40 40 40 40 41 42 42 42 42 42 42 42 42 43 43 43 43 43 44 44 44 44 44 45 46 46 47 47 47 48 48 48 50 50 50 75
5.87 6.63 5.67 7.86 6.27 10.56 9.23 6.06 9.68 7.01 6.46 5.86 6.14 6.25 8.82 6.20 5.24 4.52 6.68 6.43 6.76 7.07 6.28 6.22 6.87 8.41 5.04 10.7 3.65 4.19 10.38 8.65 9.32 7.76 5.59 7.48 6.37 0.54
1
)
n.d.: origin not declared on label.
(a)
10 g kg-1
5 0 0
10
20
30
40
50
60
70
40
50
60
70
(b)
80 60 40 20 0 0
10
20
30 samples
Fig. 2. (a) Safranal content (g kg
) determined by the GC method and by (b) specific absorbance A1% 1cm using the ISO method for the 76 saffron samples identified in Table 1.
1
traces corresponding to the same four samples of Fig. 3 are reported in Fig. 4. The absence of correlation between the UV trace and the specific safranal content raises concrete doubts about the UV trace representing a link between the ‘‘aroma strength’’ parameter and safranal content. The safranal data depicted in Fig. 2a does not correspond to the data reported in Fig. 2b; although, all samples (excluding the first and the last) have the same chemical specification for safranal (range 20–50) following the ISO 6323-2:2011 classification.
4. Conclusion The chemical specification ‘‘safranal’’ obtained by the ISO 63232:2010 method (ISO, 2010) does not indicate differences between saffron samples of varying qualities and origins and does not correlate with the quantitative determination by GC. The results of the present study indicate that the UV evaluation of specific absorbance does not lead to differentiation between safranal content ranged from 3.61 to 7.48 g kg 1 (all included for the three categories in UV range 20–50). More, for the 76 samples in the present
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M. Bononi et al. / Food Chemistry 176 (2015) 17–21
24)
76)
330 330
1)
2)
330
330
Fig. 3. Examples of UV traces produced following the ISO 6323-2:2010 method from samples 24, 76, 1, and 2 (see Table 1), showing obvious interferences in the safranal absorbance values at k = 330 nm in aqueous extracts. In particular, the specific absorbance values for samples 1, 2, and 24 are not consistent with values determined by GC (1.35, 3.61, and 7.72 g kg 1, respectively). Sample 76 has the highest specific absorbance value, but the lowest safranal content (0.54 g kg 1).
Fig. 4. GC traces of safranal produced by samples 24, 76, 1, and 2 (see Table 1) analysed as reported in Sections 2.3 and 2.4.
study, the absorbance were 19–75, but no correlation was found between the two data series, as reported in Table 1 and in Fig. 2. It appears that substances that interfere with the UV evaluation do not permit an effective comparison of the safranal content in saffron. As affirmed by Hadizadeh et al. (2007), the interference of cis-crocetin ester isomers has been evidenced at the wavelength of safranal maximum absorbance. More, it may be useful to consider the paper of Valle Garcia-Rodriguez et al. (2014) where the UV spectrum of safranal is reported vs UV spectra of two transcrocetin esters. These UV spectra demonstrate that, on the low
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