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Rapid determination of safranal in the quality control of saffron spice (Crocus sativus L.)
Food Chemistry 127 (2011) 369–373
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
Rapid determination of safranal in the quality control of saffron spice (Crocus sativus L.) Luana Maggi a, Ana M. Sánchez a, Manuel Carmona a, Charalabos D. Kanakis b, Eirini Anastasaki b, Petros A. Tarantilis b, Moschos G. Polissiou b, Gonzalo L. Alonso a,⇑ a b
Cátedra de Química Agrícola, E.T.S.I. Agrónomos, Universidad de Castilla-La Mancha, Campus Universitario, 02071 Albacete, Spain Laboratory of Chemistry, Department of Science, Agricultural University of Athens, Iera Odos 75, 118 55 Athens, Greece
a r t i c l e
i n f o
Article history: Received 26 July 2010 Received in revised form 21 December 2010 Accepted 5 January 2011 Available online 9 January 2011 Keywords: Safranal Saffron Crocus sativus L. UV–Vis spectrophotometry Gas-chromatography Aroma
a b s t r a c t A method to determine safranal content based on non-polar solvent extraction followed by UV–Vis analysis available in the industry for quality control of saffron spice has been studied. Ultrasound-assisted extraction of safranal was carried out and optimised with respect to the solvent: diethyl ether, hexane and chloroform; the time of extraction; and the concentration of saffron in each organic solvent. Best extraction conditions were obtained when 20 g L1 of saffron was extracted with chloroform for 15 min. Intra-laboratory validation of the optimised conditions and analysis by UV–Vis spectrophotometry showed satisfactory results in linearity, repeatability, intermediate precision and recovery. The limit of detection was 1 mg safranal kg1 saffron and the limit of quantification was 3 mg safranal kg1 saffron. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction The red dried stigmas of Crocus sativus L. is considered as one of the most expensive spice used in food industry. Hence, it is important to monitor the quality of saffron available in the market. The quality of saffron is determined according to the ISO 3632 (2003) that classifies it into three categories depending on their physical and chemical characteristics. The three foremost parameters used to define the saffron quality are colour, taste and aroma (Carmona et al., 2006; Tarantilis, Polissiou, & Manfait, 1994). With regard to the saffron aroma, safranal (2,6,6-trimethyl-1,3cyclohexadiene-1-carboxaldehyde) is the major compound (Carmona, Zalacain, Salinas, & Alonso, 2007; Tarantilis, Beljebar, Manfait, & Polissiou, 1998; Tarantilis, Tsoupras, & Polissiou, 1995; Tarantilis et al., 1994). Safranal content is measured by the absorbance of aqueous saffron extract at 330 nm according to the ISO 3632 (2003). This determination is not the most adequate for safranal since it is not very soluble in water and also the cis-crocetin esters isomers (colour compounds) adsorb at 330 nm (Alonso, Salinas, Esteban-Infantes, & Sánchez-Fernández, 1996; Hadizadeh et al., 2007; Tarantilis et al., 1994), interfering on its determination. Various extraction methods have been used for the extraction of volatiles from plant material such as microsimultaneous hydrodi⇑ Corresponding author. Tel.: +34 967 599310; fax: +34 967 599238. E-mail address: [email protected] (G.L. Alonso). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.01.028
stillation–extraction (Kanakis, Daferera, Tarantilis, & Polissiou, 2004), vacuum headspace (Tarantilis & Polissiou, 1997), supercritical fluid extraction (Lozano, Delgado, Gómez, Rubio, & Iborra, 2000), thermal desorption (Alonso et al., 1996), liquid extraction with organic solvents (Sujata, Ravishankar, & Venkataraman, 1992; Tarantilis et al., 1994) and the ultrasound-assisted extraction (USAE) (Alissandrakis, Tarantilis, Harizanis, & Polissiou, 2007; Anastasaki et al., 2009; Kanakis et al., 2004), followed by gas chromatography (GC) (Alonso et al., 1996; Maggi et al., 2009; Sujata et al. 1992) or liquid chromatography (HPLC) (Loskutov, Beninger, Hosfield, & Sink, 2000; Lozano et al., 2000; Tarantilis & Polissiou, 1997; Tarantilis et al., 1994; Tarantilis et al., 1995). Various chromatographic analytical techniques, such as GC (Kanakis et al., 2004; Maggi et al., 2009) or HPLC (Sujata et al. 1992; Tarantilis et al., 1994), have been used to quantify safranal but these techniques can hardly be used for routine industrial purposes to monitor raw materials, production processes or end products since they are time consuming and require equipment that is seldom found in small or medium-size companies that process and package saffron spice. Thus, there is a real interest in the development of rapid method for routine quality control of saffron using UV–Vis spectral information (Sánchez, Carmona, Del Campo, & Alonso, 2009; Sánchez et al., 2008; Zalacain et al., 2005a, 2005b). In a routine method a suitable solvent that can completely solubilise safranal and also be used to detect non-polar colourants that can be used to adulterate saffron spice is required.
1.26–1.38 0.68–0.80 1.42–1.95 1.13–2.04 0.45–0.77 0.29–0.36 0.46–0.96 0.34–0.80 0.88–0.96 0.49–0.56 0.99–1.36 0.79–1.42 0.39–0.50 0.20–0.24 0.39–0.62 0.29–0.52 I (3) II (4) I (8) I (9) 194.6–248.4 164.8–173.0 272.9–297.0 226.4–266.5 34.8–36.7 34.9–39.2 30.3–37.0 32.6–44.4
() Number of samples analysed for each country.
194.6–241.2 139.3–177.1 193.4–228.1 149.6–200.2 7.28–8.26 7.97–8.25 7.58–9.32 5.49–6.73
1.25–2.07
In chloroform In hexane
0.35–1.00 0.87–1.44 0.23–0.65
Range of E1% 1 cm 310 nm in chloroform Range of E1% 1 cm 305 nm in hexane ISO category
I (16) 193.5–267.7 34.8–49.7 149.1–186.9 7.01–9.96
(7) (9) (3) (4) (8) (5) (4) Italy (8) Spain (9)
A Hewlett Packard 5890 Series II chromatograph (Palo Alto, CA, USA) equipped with a flame ionisation detector (FID) and a HP-
Iran (7)
2.4. Gas chromatographic analysis
2006 2005 2006 2005 2006 2006 2005
Spectral characteristics of aqueous and organic saffron extracts were monitored by scanning from 200 to 700 nm using a Perkin-Elmer Lambda 25 spectrophotometer (Norwalk, CT, USA) with UV WinLab 2.85.04 software (Perkin-Elmer). Colouring strength (E1% 1 cm 1% 440 nm), (E1% 1 cm 257 nm and (E1 cm 330 nm of saffron aqueous extracts were determined according to ISO 3632 (2003). (E1% 1 cm 310 nm and (E1% 1 cm 305 nm were assessed from the absorbance at 310 and 305 nm of the chloroform and hexane extracts, respectively. Quantification was carried out by calibration curves of safranal standard solutions in the range of the six levels (2.0 and 60.0 mg L1) in hexane and chloroform.
Greece (16)
2.3. UV–Vis spectrophotometric analysis
Range of E1% 1 cm 440 nm
Diethyl ether extracts were prepared following the method reported by Maggi et al. (2009). Four millilitres of chloroform or hexane were added to ground saffron used as reference (0.1, 0.2 or 0.3 g depending on the assay) and the mixture was submitted to USAE with an ultrasound water bath (Sonorex, Super RK 255H type, Berlin, Germany) at the fixed-frequency of 35 kHz. During the extraction procedure the temperature was kept below 25 °C. The organic extract was filtered through a syringe filter of polyester with a porosity of 0.45 lm and 25 mm of diameter (Millipore, Bedford, MA, USA). The filtrate was adjusted to a volume of 5 mL and homogenised before measuring. The safranal extraction was optimised using reference saffron with respect to the solvent: hexane and chloroform; the time of extraction: 15, 30 and 60 min; and the concentration of saffron in each organic solvent: 20, 40 and 60 g L1. The selected extraction conditions were obtained with chloroform, 20 g L1 of saffron and 15 min extraction time.
Range of E1% 1 cm 330 nm
2.2. Safranal extraction procedure
Range of E1% 1 cm 257 nm
A saffron sample coming from Greece, harvested in 2006 and belonging to commercial category I according to ISO 3632 (2003) was used as reference. This sample was used for the optimisation of the method, as well as for its validation. Forty samples of saffron spice in filaments were obtained directly from producers and packers with a guarantee of their origin and freedom from fraud (Table 1). They came from Greece (16 samples), Iran (7 samples), Italy (8 samples) and Spain (9 samples). Seventeen samples were harvested during the year 2005 and the rest during 2006. Saffron samples were ground, passed through a sieve of 0.5 mm pore diameter and kept at 4 °C in absence of light until their analysis. Safranal with purity of 88% was supplied by Sigma–Aldrich (Madrid, Spain). Analytical grade solvents: diethyl ether, chloroform and hexane were purchased from Panreac (Barcelona, Spain). Water was purified through a Milli-Q System (Millipore, Bedford, MA, USA).
Moisture and volatile matter content (%)
2.1. Samples and chemicals
Harvest
2. Materials and methods
Origin
The objective of this work was to study non-polar solvent extractions to determine safranal by an UV–Vis spectrophotometric method that can be used in the industry for quality control of saffron spice.
Range of safranal content (g kg1) by GC
L. Maggi et al. / Food Chemistry 127 (2011) 369–373 Table 1 1% 1 Origin, harvest and quality characteristics of the saffron samples according to ISO 3632 (2003). The values of E1% ) measured 1 cm 305 nm and E1 cm 310 nm determined by the proposed USAE/UV–Vis method and the safranal content (g kg by GC.
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2.5. Validation of USAE/UV–Vis method Intra-laboratory method validation was carried out according to Eurachem Guidelines (Eurachem, 1998). Accuracy was studied as two components: trueness and precision. Trueness was assessed as the closeness of agreement between the average content of safranal for the same samples obtained after USAE/UV–Vis, and the reference value of safranal content determined by direct injection of saffron extracts in the GC/MS chromatograph. With regard to precision, two parameters were determined: repeatability and intermediate precision and they were stated in terms of relative standard deviation (RSD). Triplicate measurements were analysed by the same analyst within the same day with the proposed method to determine repeatability. The repeatability limit, Lr, at the 95% confidence level was calculated as:
Lr ¼ 1:96
pffiffiffi 2 rr
ð1Þ
where rr is the standard deviation measured under repeatability conditions. Intermediate precision was determined for the saffron used as reference that was used to calculate reproducibility. The sample of reference was analysed by different analysts on two separate days. The reproducibility limit, LR, at the 95% confidence level was calculated as:
LR ¼ 1:96
pffiffiffi 2 rR
ð2Þ
where rR is the standard deviation measured under reproducibility conditions. Besides the same sample was used in the recovery study of safranal. The recovery was determined extracting 100 mg of the saffron sample (reference) in chloroform (5 mL) following the optimised procedure and then its absorbance was recorded at 310 nm. Afterwards, three known amounts of safranal: 13.6, 27.2 and 42.5 lg were added in 100 mg of saffron reference. The absorbance of saffron reference at 310 nm was subtracted from that of the spiked samples and the amount of the added safranal was calculated using the corresponding calibration curve. In this case a series of safranal standards solutions in chloroform in the range between 0.02 and 20.00 mg safranal L1 were prepared, and the absorbance of safranal at 310 nm was recorded and the limit of detection (LOD) was determined by visual evaluation method. The limit of quantification (LOQ) was calculated using the equation LOQ = 3 LOD. Linearity was determined by plotting signal response versus safranal concentration in chloroform and hexane in the range of 2.0–60.0 mg L1 using six levels of calibration.
2.6. Statistical analysis Evaluation of the statistical significance of differences was performed using analysis of variance (ANOVA) and the Student t test (a = 0.05) with the aid of SPSS 17.0 for Windows (SPSS Inc., Chicago, USA) statistical program. A significant difference was considered at a level of p < 0.05. 3. Results and discussion 3.1. Saffron samples characterisation The saffron sample used as reference was analysed and results showed that it belonged to commercial category I according to ISO 3632 (2003) with the following characteristics: moisture and volatile matter content, 8.02%; colouring strength (E1% 1 cm 440 nm), 1% 247.3; E1% 1 cm 257 nm, 178.3; E1 cm 330 nm, 43.5. As can be seen in Table 1 the moisture and volatile matter content of all saffron samples ranged from 5.49 to 9.96%, values lower than 12%, maximum limits established by ISO 3632 (2003) for Crocus sativus L. to be considered as saffron spice. Colouring strength ranged from 164.8 to 297.0; (E1% 1 cm 257 nm ranged from 139.3 to 228.1 and (E1% 330 nm ranged from 30.3 to 49.7. Ninety percent 1 cm of analysed samples were belonged to category I according to ISO 3632 (2003). 3.2. Comparison of UV–Vis and USAE/GC/MS method The graph reported in Fig. 1 represents the comparison of safranal content obtained for 40 saffron samples using the UV–Vis according to ISO 3632 (2003) and already published method (Maggi et al., 2009). There was no correlation between the safranal content obtained by ISO 3632 (2003) and validated GC method, since there are other substances present in saffron absorbing at 330 nm, such as cis-crocetin esters isomers. 3.3. Optimisation of safranal extraction 3.3.1. Selection of solvent The three solvents have been evaluated by GC analysis and the following equations were obtained: y = 86.6 106 As (R2 = 0.999) for diethyl ether, y = (159.1 106 As) + 0.3 (R2 = 0.996) for hexane and y = (188.7 106 As) 2.8 (R2 = 0.980) for chloroform, where y is the concentration of safranal in the extract expressed as mg of safranal L1; As is the safranal’s peak area in GC chromatograms. The three solvents showed a good linearity with R2 P 0.98. Chloroform showed the most efficient safranal extraction, followed by hexane in the majority of the cases. These two
Safranal content (mg/kg)
5 ms capillary column (30 m, 0.25 mm i.d., 0.25 lm film thickness) with helium as carrier gas at 1 mL min1 was used for the analysis of saffron organic extracts. Column temperature was initially kept for 3 min at 50 °C, then gradually increased to 180 °C with a rate of 3 °C min1, and finally increased to 250 °C with a rate of 15 °C min1 and held isothermally for 5 min. The injector and detector temperatures were set at 220 and 290 °C, respectively. One microlitre of saffron organic extract was injected manually in the splitless mode. For quantification of safranal, the external standard method was applied. Six different levels of safranal standard solution in hexane and chloroform were prepared to build the calibration curves. The GC/MS analysis was performed under the same conditions as GC/FID, using a Hewlett–Packard 5890 series II chromatograph equipped with a 5972 series mass selective detector (MSD) in the electron impact mode (70 eV). Identification was carried out using the NIST library, the MS spectrum and the retention time of the safranal standard.
900 800 700 600 500 400 300 200 100 0
0
10
20
30 E330 nm
40
50
60
Fig. 1. Comparison between the safranal content obtained at 330 nm according to current ISO 3632 (2003) and the quantification performed by USAE/GC analysis (N = 40) (Maggi et al., 2009).
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solvents were selected to test the rest of conditions for determining safranal in quality control of saffron. Hexane and chloroform were checked in the range between 2.0 and 60.0 mg L1 by UV–Vis analysis and the following equations were obtained: y = (21.5 Abs) 0.5 (R2 = 0.999) for hexane and y = (19.9 Abs) 0.8 (R2 = 0.992) for chloroform, where y is the concentration of safranal in the extract expressed as mg of safranal L1; Abs is the safranal’s absorbance at 305 or 310 nm in UV–Vis determinations in hexane and chloroform, respectively. Satisfactory results were found for both solvents, with R2 > 0.99. 3.3.2. Extraction time Different extraction times were tested (15, 30 and 60 min) at temperature lower than 25 °C during the extraction process to avoid secondary reactions. Neither in hexane nor in chloroform extracts, significant differences were found in the concentration of safranal obtained after USAE for 15, 30 and 60 min by UV–Vis spectrophotometry (Table 2). It seemed that studied times did not influence the efficiency of ultrasound extraction. Consequently, 15 min was set as the appropriate extraction time for further studies. 3.3.3. Concentration of saffron in the organic solvent Different concentrations of ground saffron stigmas (20, 40 and 60 mg mL1) were tested in both solvents. The extraction of safranal was not proportional with the increment of saffron concentration when hexane or chloroform was used. In case of hexane, no significant differences were observed between 20 and 40 g L1 making differences with chloroform (Table 2). With 60 g L1 of saffron, there were significant differences in the content of extracted safranal in both solvents, but it was not three times the concentration of saffron (20 g L1) or 1.5 times as much as that of 40 g L1. Therefore, the lower concentration of saffron (20 g L1) was selected as the best one, with the subsequent saving of sample in the quality control methods. In Fig. 2A and B were reported the
UV–Vis spectra ranging from 200 to 700 nm of hexane and chloroform extract of saffron (20 mg mL1) used as reference.
3.4. Comparison of proposed UV–Vis and USAE/GC/MS methods 1% In Table 1 the values of (E1% 1 cm 305 nm and (E1 cm 310 nm for 40 1% samples were reported. (E1 cm 305 nm ranged between 0.23 and 0.65 for saffron samples belonging to category I and between 0.20 and 0.24 for saffron belonging to category II. Regarding the extraction with chloroform, (E1% 1 cm 310 nm ranged between 0.79 and 1.44 for samples belonging to category I and between 0.49 and 0.56 for category II. These values could be a first approximation, although further research should be done including various samples of the different categories. The results obtained by the proposed USAE/UV–Vis and USAE/ GC/MS methods for the forty saffron samples were compared. The data were plotted and the two following equations were obtained: y = (1.1 AGC) + 123.6 (R2 = 0.928) for hexane and y = (0.8 AGC) + 598.8 (R2 = 0.936) for chloroform; where y was the content of safranal by UV–Vis, expressed as mg kg1 of saffron, and AGC is safranal content obtained by GC, expressed as mg kg1 of saffron. The correlation coefficients are good for both solvents, slightly better for chloroform. From these results, it can conclude that both solvents can be used for the extraction and quantification of safranal in the saffron samples. In addition to the determination of safranal, the possibility of using the same extract for detecting adulterations with colourants would be of great interest in the unification of methods to determine the purity and quality of saffron. Consequently, preliminary trials (data not shown) were carried out to value the use of hexane and chloroform for detection of non-polar colourants in saffron quality control. Chloroform showed the best characteristics to be applied for determining safranal and with the capability to be at the same time used for detection of non-polar colourants in saffron quality control. The validation of proposed method was performed using chloroform as solvent extraction.
Table 2 Saffron concentration (mg mL1), time extraction (min) and concentration of safranal (mg mL1) in hexane and chloroform, respectively. Conc. saffron (mg mL1)
Time extraction (min) 15
30
60
Conc. safranal (mg mL1) Hexane
20.0 40.0 60.0
0.030a ± 0.005 0.034a ± 0.002 0.038b ± 0.002
0.030a ± 0.004 0.036a ± 0.003 0.048b ± 0.002
0.031a ± 0.005 0.037a ± 0.004 0.050b ± 0.004
Chloroform
20.0 40.0 60.0
0.034a ± 0.005 0.042b ± 0.002 0.057c ± 0.001
0.034a ± 0.004 0.057b ± 0.003 0.070c ± 0.002
0.040a ± 0.005 0.048b ± 0.004 0.051c ± 0.004
Different letters between rows indicate significant differences at 0.05% level.
2
2
(A)
1 0.5 0 200
(B)
1.5 Abs
Abs
1.5
1 0.5
400 Wavelength[nm]
600
700
0 200
400 Wavelength[nm]
Fig. 2. UV–Vis spectra of saffron extracts (20 g L1) in hexane (A) and chloroform (B).
600
700
L. Maggi et al. / Food Chemistry 127 (2011) 369–373
3.5. Validation of proposed USAE/UV–Vis method The proposed method for the determination of safranal content in saffron has been validated. The method showed a good linearity with R2 > 0.99 in the range between 2.0 and 60.0 mg L1. This method also showed a good sensitivity, the LOD was 1 mg safranal kg1 saffron and the LOQ was 3 mg safranal kg1 saffron. The repeatability of the procedure was satisfactory since the values of RDS obtained (2.1, 2.6 and 1.5%). From the repeatability standard deviation it was useful to calculate the Lr by Eq. (1), which enabled the analyst to decide whether the difference between replicate analyses of a sample, determined under repeatability conditions, was significant. Their values were 115, 100 and 109 expressed as mg safranal kg1 saffron. The values of reproducibility, expressed as % RSD, were lower than 3.4%, so it demonstrated the good reproducibility of the method. Intermediate precision determined by different analysts on three different days was also found satisfactory (RSD = 6.5%). The LR was calculated according to Eq. (2) and indicated the same as Lr but under reproducibility conditions. Its value was 263.7 expressed as mg safranal kg1 saffron. With regard to the recovery, the results ranged between 83% and 93%. By the comparison of means with the Student t test for checking the trueness resulted significant differences in the content of safranal determined by UV–Vis and GC methods in the samples studied, although the results from the two methods showed a good correlation coefficient with a light overestimation of safranal content. These data confirmed the efficacy of the proposed USAE/UV–Vis method for the determination of safranal content in quality control. 4. Conclusion The selected extraction conditions were the following: 100 mg of ground saffron were extracted using 5 mL of chloroform (20 mg mL1) for 15 min at temperature lower than 25 °C. Satisfactory results were obtained in linearity, repeatability, reproducibility and recovery and also good values of LOD and LOQ, although the trueness of the method should be improved. This analytical method is easy, fast and adequate to determine safranal content in the industry for routine quality control of saffron spice. Acknowledgements This work has been co-financed by EC 6th Framework Programme for Research as a Research Project for the benefit of SMEs Associations (SAFFIC COLL-CT-2006-Contract No. 030195-2). We thank the whole Project Consortium and the Project Officer Mr. Valcárcel (e-mail: [email protected]) for their support and collaboration and Amaya Zalacain for her support with analysis. References Alissandrakis, E., Tarantilis, P. A., Harizanis, P., & Polissiou, M. (2007). Comparison of the volatile composition in thyme honeys from several origins in Greece. Journal of Agricultural and Food Chemistry, 55, 8152–8157.
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
Rapid determination of safranal in the quality control of saffron spice (Crocus sativus L.) Luana Maggi a, Ana M. Sánchez a, Manuel Carmona a, Charalabos D. Kanakis b, Eirini Anastasaki b, Petros A. Tarantilis b, Moschos G. Polissiou b, Gonzalo L. Alonso a,⇑ a b
Cátedra de Química Agrícola, E.T.S.I. Agrónomos, Universidad de Castilla-La Mancha, Campus Universitario, 02071 Albacete, Spain Laboratory of Chemistry, Department of Science, Agricultural University of Athens, Iera Odos 75, 118 55 Athens, Greece
a r t i c l e
i n f o
Article history: Received 26 July 2010 Received in revised form 21 December 2010 Accepted 5 January 2011 Available online 9 January 2011 Keywords: Safranal Saffron Crocus sativus L. UV–Vis spectrophotometry Gas-chromatography Aroma
a b s t r a c t A method to determine safranal content based on non-polar solvent extraction followed by UV–Vis analysis available in the industry for quality control of saffron spice has been studied. Ultrasound-assisted extraction of safranal was carried out and optimised with respect to the solvent: diethyl ether, hexane and chloroform; the time of extraction; and the concentration of saffron in each organic solvent. Best extraction conditions were obtained when 20 g L1 of saffron was extracted with chloroform for 15 min. Intra-laboratory validation of the optimised conditions and analysis by UV–Vis spectrophotometry showed satisfactory results in linearity, repeatability, intermediate precision and recovery. The limit of detection was 1 mg safranal kg1 saffron and the limit of quantification was 3 mg safranal kg1 saffron. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction The red dried stigmas of Crocus sativus L. is considered as one of the most expensive spice used in food industry. Hence, it is important to monitor the quality of saffron available in the market. The quality of saffron is determined according to the ISO 3632 (2003) that classifies it into three categories depending on their physical and chemical characteristics. The three foremost parameters used to define the saffron quality are colour, taste and aroma (Carmona et al., 2006; Tarantilis, Polissiou, & Manfait, 1994). With regard to the saffron aroma, safranal (2,6,6-trimethyl-1,3cyclohexadiene-1-carboxaldehyde) is the major compound (Carmona, Zalacain, Salinas, & Alonso, 2007; Tarantilis, Beljebar, Manfait, & Polissiou, 1998; Tarantilis, Tsoupras, & Polissiou, 1995; Tarantilis et al., 1994). Safranal content is measured by the absorbance of aqueous saffron extract at 330 nm according to the ISO 3632 (2003). This determination is not the most adequate for safranal since it is not very soluble in water and also the cis-crocetin esters isomers (colour compounds) adsorb at 330 nm (Alonso, Salinas, Esteban-Infantes, & Sánchez-Fernández, 1996; Hadizadeh et al., 2007; Tarantilis et al., 1994), interfering on its determination. Various extraction methods have been used for the extraction of volatiles from plant material such as microsimultaneous hydrodi⇑ Corresponding author. Tel.: +34 967 599310; fax: +34 967 599238. E-mail address: [email protected] (G.L. Alonso). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.01.028
stillation–extraction (Kanakis, Daferera, Tarantilis, & Polissiou, 2004), vacuum headspace (Tarantilis & Polissiou, 1997), supercritical fluid extraction (Lozano, Delgado, Gómez, Rubio, & Iborra, 2000), thermal desorption (Alonso et al., 1996), liquid extraction with organic solvents (Sujata, Ravishankar, & Venkataraman, 1992; Tarantilis et al., 1994) and the ultrasound-assisted extraction (USAE) (Alissandrakis, Tarantilis, Harizanis, & Polissiou, 2007; Anastasaki et al., 2009; Kanakis et al., 2004), followed by gas chromatography (GC) (Alonso et al., 1996; Maggi et al., 2009; Sujata et al. 1992) or liquid chromatography (HPLC) (Loskutov, Beninger, Hosfield, & Sink, 2000; Lozano et al., 2000; Tarantilis & Polissiou, 1997; Tarantilis et al., 1994; Tarantilis et al., 1995). Various chromatographic analytical techniques, such as GC (Kanakis et al., 2004; Maggi et al., 2009) or HPLC (Sujata et al. 1992; Tarantilis et al., 1994), have been used to quantify safranal but these techniques can hardly be used for routine industrial purposes to monitor raw materials, production processes or end products since they are time consuming and require equipment that is seldom found in small or medium-size companies that process and package saffron spice. Thus, there is a real interest in the development of rapid method for routine quality control of saffron using UV–Vis spectral information (Sánchez, Carmona, Del Campo, & Alonso, 2009; Sánchez et al., 2008; Zalacain et al., 2005a, 2005b). In a routine method a suitable solvent that can completely solubilise safranal and also be used to detect non-polar colourants that can be used to adulterate saffron spice is required.
1.26–1.38 0.68–0.80 1.42–1.95 1.13–2.04 0.45–0.77 0.29–0.36 0.46–0.96 0.34–0.80 0.88–0.96 0.49–0.56 0.99–1.36 0.79–1.42 0.39–0.50 0.20–0.24 0.39–0.62 0.29–0.52 I (3) II (4) I (8) I (9) 194.6–248.4 164.8–173.0 272.9–297.0 226.4–266.5 34.8–36.7 34.9–39.2 30.3–37.0 32.6–44.4
() Number of samples analysed for each country.
194.6–241.2 139.3–177.1 193.4–228.1 149.6–200.2 7.28–8.26 7.97–8.25 7.58–9.32 5.49–6.73
1.25–2.07
In chloroform In hexane
0.35–1.00 0.87–1.44 0.23–0.65
Range of E1% 1 cm 310 nm in chloroform Range of E1% 1 cm 305 nm in hexane ISO category
I (16) 193.5–267.7 34.8–49.7 149.1–186.9 7.01–9.96
(7) (9) (3) (4) (8) (5) (4) Italy (8) Spain (9)
A Hewlett Packard 5890 Series II chromatograph (Palo Alto, CA, USA) equipped with a flame ionisation detector (FID) and a HP-
Iran (7)
2.4. Gas chromatographic analysis
2006 2005 2006 2005 2006 2006 2005
Spectral characteristics of aqueous and organic saffron extracts were monitored by scanning from 200 to 700 nm using a Perkin-Elmer Lambda 25 spectrophotometer (Norwalk, CT, USA) with UV WinLab 2.85.04 software (Perkin-Elmer). Colouring strength (E1% 1 cm 1% 440 nm), (E1% 1 cm 257 nm and (E1 cm 330 nm of saffron aqueous extracts were determined according to ISO 3632 (2003). (E1% 1 cm 310 nm and (E1% 1 cm 305 nm were assessed from the absorbance at 310 and 305 nm of the chloroform and hexane extracts, respectively. Quantification was carried out by calibration curves of safranal standard solutions in the range of the six levels (2.0 and 60.0 mg L1) in hexane and chloroform.
Greece (16)
2.3. UV–Vis spectrophotometric analysis
Range of E1% 1 cm 440 nm
Diethyl ether extracts were prepared following the method reported by Maggi et al. (2009). Four millilitres of chloroform or hexane were added to ground saffron used as reference (0.1, 0.2 or 0.3 g depending on the assay) and the mixture was submitted to USAE with an ultrasound water bath (Sonorex, Super RK 255H type, Berlin, Germany) at the fixed-frequency of 35 kHz. During the extraction procedure the temperature was kept below 25 °C. The organic extract was filtered through a syringe filter of polyester with a porosity of 0.45 lm and 25 mm of diameter (Millipore, Bedford, MA, USA). The filtrate was adjusted to a volume of 5 mL and homogenised before measuring. The safranal extraction was optimised using reference saffron with respect to the solvent: hexane and chloroform; the time of extraction: 15, 30 and 60 min; and the concentration of saffron in each organic solvent: 20, 40 and 60 g L1. The selected extraction conditions were obtained with chloroform, 20 g L1 of saffron and 15 min extraction time.
Range of E1% 1 cm 330 nm
2.2. Safranal extraction procedure
Range of E1% 1 cm 257 nm
A saffron sample coming from Greece, harvested in 2006 and belonging to commercial category I according to ISO 3632 (2003) was used as reference. This sample was used for the optimisation of the method, as well as for its validation. Forty samples of saffron spice in filaments were obtained directly from producers and packers with a guarantee of their origin and freedom from fraud (Table 1). They came from Greece (16 samples), Iran (7 samples), Italy (8 samples) and Spain (9 samples). Seventeen samples were harvested during the year 2005 and the rest during 2006. Saffron samples were ground, passed through a sieve of 0.5 mm pore diameter and kept at 4 °C in absence of light until their analysis. Safranal with purity of 88% was supplied by Sigma–Aldrich (Madrid, Spain). Analytical grade solvents: diethyl ether, chloroform and hexane were purchased from Panreac (Barcelona, Spain). Water was purified through a Milli-Q System (Millipore, Bedford, MA, USA).
Moisture and volatile matter content (%)
2.1. Samples and chemicals
Harvest
2. Materials and methods
Origin
The objective of this work was to study non-polar solvent extractions to determine safranal by an UV–Vis spectrophotometric method that can be used in the industry for quality control of saffron spice.
Range of safranal content (g kg1) by GC
L. Maggi et al. / Food Chemistry 127 (2011) 369–373 Table 1 1% 1 Origin, harvest and quality characteristics of the saffron samples according to ISO 3632 (2003). The values of E1% ) measured 1 cm 305 nm and E1 cm 310 nm determined by the proposed USAE/UV–Vis method and the safranal content (g kg by GC.
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2.5. Validation of USAE/UV–Vis method Intra-laboratory method validation was carried out according to Eurachem Guidelines (Eurachem, 1998). Accuracy was studied as two components: trueness and precision. Trueness was assessed as the closeness of agreement between the average content of safranal for the same samples obtained after USAE/UV–Vis, and the reference value of safranal content determined by direct injection of saffron extracts in the GC/MS chromatograph. With regard to precision, two parameters were determined: repeatability and intermediate precision and they were stated in terms of relative standard deviation (RSD). Triplicate measurements were analysed by the same analyst within the same day with the proposed method to determine repeatability. The repeatability limit, Lr, at the 95% confidence level was calculated as:
Lr ¼ 1:96
pffiffiffi 2 rr
ð1Þ
where rr is the standard deviation measured under repeatability conditions. Intermediate precision was determined for the saffron used as reference that was used to calculate reproducibility. The sample of reference was analysed by different analysts on two separate days. The reproducibility limit, LR, at the 95% confidence level was calculated as:
LR ¼ 1:96
pffiffiffi 2 rR
ð2Þ
where rR is the standard deviation measured under reproducibility conditions. Besides the same sample was used in the recovery study of safranal. The recovery was determined extracting 100 mg of the saffron sample (reference) in chloroform (5 mL) following the optimised procedure and then its absorbance was recorded at 310 nm. Afterwards, three known amounts of safranal: 13.6, 27.2 and 42.5 lg were added in 100 mg of saffron reference. The absorbance of saffron reference at 310 nm was subtracted from that of the spiked samples and the amount of the added safranal was calculated using the corresponding calibration curve. In this case a series of safranal standards solutions in chloroform in the range between 0.02 and 20.00 mg safranal L1 were prepared, and the absorbance of safranal at 310 nm was recorded and the limit of detection (LOD) was determined by visual evaluation method. The limit of quantification (LOQ) was calculated using the equation LOQ = 3 LOD. Linearity was determined by plotting signal response versus safranal concentration in chloroform and hexane in the range of 2.0–60.0 mg L1 using six levels of calibration.
2.6. Statistical analysis Evaluation of the statistical significance of differences was performed using analysis of variance (ANOVA) and the Student t test (a = 0.05) with the aid of SPSS 17.0 for Windows (SPSS Inc., Chicago, USA) statistical program. A significant difference was considered at a level of p < 0.05. 3. Results and discussion 3.1. Saffron samples characterisation The saffron sample used as reference was analysed and results showed that it belonged to commercial category I according to ISO 3632 (2003) with the following characteristics: moisture and volatile matter content, 8.02%; colouring strength (E1% 1 cm 440 nm), 1% 247.3; E1% 1 cm 257 nm, 178.3; E1 cm 330 nm, 43.5. As can be seen in Table 1 the moisture and volatile matter content of all saffron samples ranged from 5.49 to 9.96%, values lower than 12%, maximum limits established by ISO 3632 (2003) for Crocus sativus L. to be considered as saffron spice. Colouring strength ranged from 164.8 to 297.0; (E1% 1 cm 257 nm ranged from 139.3 to 228.1 and (E1% 330 nm ranged from 30.3 to 49.7. Ninety percent 1 cm of analysed samples were belonged to category I according to ISO 3632 (2003). 3.2. Comparison of UV–Vis and USAE/GC/MS method The graph reported in Fig. 1 represents the comparison of safranal content obtained for 40 saffron samples using the UV–Vis according to ISO 3632 (2003) and already published method (Maggi et al., 2009). There was no correlation between the safranal content obtained by ISO 3632 (2003) and validated GC method, since there are other substances present in saffron absorbing at 330 nm, such as cis-crocetin esters isomers. 3.3. Optimisation of safranal extraction 3.3.1. Selection of solvent The three solvents have been evaluated by GC analysis and the following equations were obtained: y = 86.6 106 As (R2 = 0.999) for diethyl ether, y = (159.1 106 As) + 0.3 (R2 = 0.996) for hexane and y = (188.7 106 As) 2.8 (R2 = 0.980) for chloroform, where y is the concentration of safranal in the extract expressed as mg of safranal L1; As is the safranal’s peak area in GC chromatograms. The three solvents showed a good linearity with R2 P 0.98. Chloroform showed the most efficient safranal extraction, followed by hexane in the majority of the cases. These two
Safranal content (mg/kg)
5 ms capillary column (30 m, 0.25 mm i.d., 0.25 lm film thickness) with helium as carrier gas at 1 mL min1 was used for the analysis of saffron organic extracts. Column temperature was initially kept for 3 min at 50 °C, then gradually increased to 180 °C with a rate of 3 °C min1, and finally increased to 250 °C with a rate of 15 °C min1 and held isothermally for 5 min. The injector and detector temperatures were set at 220 and 290 °C, respectively. One microlitre of saffron organic extract was injected manually in the splitless mode. For quantification of safranal, the external standard method was applied. Six different levels of safranal standard solution in hexane and chloroform were prepared to build the calibration curves. The GC/MS analysis was performed under the same conditions as GC/FID, using a Hewlett–Packard 5890 series II chromatograph equipped with a 5972 series mass selective detector (MSD) in the electron impact mode (70 eV). Identification was carried out using the NIST library, the MS spectrum and the retention time of the safranal standard.
900 800 700 600 500 400 300 200 100 0
0
10
20
30 E330 nm
40
50
60
Fig. 1. Comparison between the safranal content obtained at 330 nm according to current ISO 3632 (2003) and the quantification performed by USAE/GC analysis (N = 40) (Maggi et al., 2009).
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solvents were selected to test the rest of conditions for determining safranal in quality control of saffron. Hexane and chloroform were checked in the range between 2.0 and 60.0 mg L1 by UV–Vis analysis and the following equations were obtained: y = (21.5 Abs) 0.5 (R2 = 0.999) for hexane and y = (19.9 Abs) 0.8 (R2 = 0.992) for chloroform, where y is the concentration of safranal in the extract expressed as mg of safranal L1; Abs is the safranal’s absorbance at 305 or 310 nm in UV–Vis determinations in hexane and chloroform, respectively. Satisfactory results were found for both solvents, with R2 > 0.99. 3.3.2. Extraction time Different extraction times were tested (15, 30 and 60 min) at temperature lower than 25 °C during the extraction process to avoid secondary reactions. Neither in hexane nor in chloroform extracts, significant differences were found in the concentration of safranal obtained after USAE for 15, 30 and 60 min by UV–Vis spectrophotometry (Table 2). It seemed that studied times did not influence the efficiency of ultrasound extraction. Consequently, 15 min was set as the appropriate extraction time for further studies. 3.3.3. Concentration of saffron in the organic solvent Different concentrations of ground saffron stigmas (20, 40 and 60 mg mL1) were tested in both solvents. The extraction of safranal was not proportional with the increment of saffron concentration when hexane or chloroform was used. In case of hexane, no significant differences were observed between 20 and 40 g L1 making differences with chloroform (Table 2). With 60 g L1 of saffron, there were significant differences in the content of extracted safranal in both solvents, but it was not three times the concentration of saffron (20 g L1) or 1.5 times as much as that of 40 g L1. Therefore, the lower concentration of saffron (20 g L1) was selected as the best one, with the subsequent saving of sample in the quality control methods. In Fig. 2A and B were reported the
UV–Vis spectra ranging from 200 to 700 nm of hexane and chloroform extract of saffron (20 mg mL1) used as reference.
3.4. Comparison of proposed UV–Vis and USAE/GC/MS methods 1% In Table 1 the values of (E1% 1 cm 305 nm and (E1 cm 310 nm for 40 1% samples were reported. (E1 cm 305 nm ranged between 0.23 and 0.65 for saffron samples belonging to category I and between 0.20 and 0.24 for saffron belonging to category II. Regarding the extraction with chloroform, (E1% 1 cm 310 nm ranged between 0.79 and 1.44 for samples belonging to category I and between 0.49 and 0.56 for category II. These values could be a first approximation, although further research should be done including various samples of the different categories. The results obtained by the proposed USAE/UV–Vis and USAE/ GC/MS methods for the forty saffron samples were compared. The data were plotted and the two following equations were obtained: y = (1.1 AGC) + 123.6 (R2 = 0.928) for hexane and y = (0.8 AGC) + 598.8 (R2 = 0.936) for chloroform; where y was the content of safranal by UV–Vis, expressed as mg kg1 of saffron, and AGC is safranal content obtained by GC, expressed as mg kg1 of saffron. The correlation coefficients are good for both solvents, slightly better for chloroform. From these results, it can conclude that both solvents can be used for the extraction and quantification of safranal in the saffron samples. In addition to the determination of safranal, the possibility of using the same extract for detecting adulterations with colourants would be of great interest in the unification of methods to determine the purity and quality of saffron. Consequently, preliminary trials (data not shown) were carried out to value the use of hexane and chloroform for detection of non-polar colourants in saffron quality control. Chloroform showed the best characteristics to be applied for determining safranal and with the capability to be at the same time used for detection of non-polar colourants in saffron quality control. The validation of proposed method was performed using chloroform as solvent extraction.
Table 2 Saffron concentration (mg mL1), time extraction (min) and concentration of safranal (mg mL1) in hexane and chloroform, respectively. Conc. saffron (mg mL1)
Time extraction (min) 15
30
60
Conc. safranal (mg mL1) Hexane
20.0 40.0 60.0
0.030a ± 0.005 0.034a ± 0.002 0.038b ± 0.002
0.030a ± 0.004 0.036a ± 0.003 0.048b ± 0.002
0.031a ± 0.005 0.037a ± 0.004 0.050b ± 0.004
Chloroform
20.0 40.0 60.0
0.034a ± 0.005 0.042b ± 0.002 0.057c ± 0.001
0.034a ± 0.004 0.057b ± 0.003 0.070c ± 0.002
0.040a ± 0.005 0.048b ± 0.004 0.051c ± 0.004
Different letters between rows indicate significant differences at 0.05% level.
2
2
(A)
1 0.5 0 200
(B)
1.5 Abs
Abs
1.5
1 0.5
400 Wavelength[nm]
600
700
0 200
400 Wavelength[nm]
Fig. 2. UV–Vis spectra of saffron extracts (20 g L1) in hexane (A) and chloroform (B).
600
700
L. Maggi et al. / Food Chemistry 127 (2011) 369–373
3.5. Validation of proposed USAE/UV–Vis method The proposed method for the determination of safranal content in saffron has been validated. The method showed a good linearity with R2 > 0.99 in the range between 2.0 and 60.0 mg L1. This method also showed a good sensitivity, the LOD was 1 mg safranal kg1 saffron and the LOQ was 3 mg safranal kg1 saffron. The repeatability of the procedure was satisfactory since the values of RDS obtained (2.1, 2.6 and 1.5%). From the repeatability standard deviation it was useful to calculate the Lr by Eq. (1), which enabled the analyst to decide whether the difference between replicate analyses of a sample, determined under repeatability conditions, was significant. Their values were 115, 100 and 109 expressed as mg safranal kg1 saffron. The values of reproducibility, expressed as % RSD, were lower than 3.4%, so it demonstrated the good reproducibility of the method. Intermediate precision determined by different analysts on three different days was also found satisfactory (RSD = 6.5%). The LR was calculated according to Eq. (2) and indicated the same as Lr but under reproducibility conditions. Its value was 263.7 expressed as mg safranal kg1 saffron. With regard to the recovery, the results ranged between 83% and 93%. By the comparison of means with the Student t test for checking the trueness resulted significant differences in the content of safranal determined by UV–Vis and GC methods in the samples studied, although the results from the two methods showed a good correlation coefficient with a light overestimation of safranal content. These data confirmed the efficacy of the proposed USAE/UV–Vis method for the determination of safranal content in quality control. 4. Conclusion The selected extraction conditions were the following: 100 mg of ground saffron were extracted using 5 mL of chloroform (20 mg mL1) for 15 min at temperature lower than 25 °C. Satisfactory results were obtained in linearity, repeatability, reproducibility and recovery and also good values of LOD and LOQ, although the trueness of the method should be improved. This analytical method is easy, fast and adequate to determine safranal content in the industry for routine quality control of saffron spice. Acknowledgements This work has been co-financed by EC 6th Framework Programme for Research as a Research Project for the benefit of SMEs Associations (SAFFIC COLL-CT-2006-Contract No. 030195-2). We thank the whole Project Consortium and the Project Officer Mr. Valcárcel (e-mail: [email protected]) for their support and collaboration and Amaya Zalacain for her support with analysis. References Alissandrakis, E., Tarantilis, P. A., Harizanis, P., & Polissiou, M. (2007). Comparison of the volatile composition in thyme honeys from several origins in Greece. Journal of Agricultural and Food Chemistry, 55, 8152–8157.
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