Determination of Saffron Quality by High ... - ACS Publications

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Determination of Saffron Quality by High-Performance Liquid Chromatography M. Valle García-Rodríguez,† Jéssica Serrano-Díaz,†,‡ Petros A. Tarantilis,§ Horacio López-Córcoles,∥ Manuel Carmona,†,⊥ and Gonzalo L. Alonso*,† †

Cátedra de Química Agrícola, Escuela Técnica Superior de Ingenieros (ETSI) Agrónomos, Universidad de Castilla-La Mancha, Campus Universitario, 02071 Albacete, Spain ‡ Department of Food Technology, Nutrition and Food Science, Regional Campus of International Excellence “Campus Mare Nostrum”, Murcia University, CIBERobn, ISCIII, 30100 Murcia, Spain § Laboratory of Chemistry, Department of Food Science and Human Nutrition, School of Food, Biotechnology and Development, Agricultural University of Athens, Iera Odos 75, Athens 11855, Greece ∥ Instituto Técnico Agronómico Provincial (ITAP), S.A., Avenida Gregorio Arcos, s/n, 02006 Albacete, Spain ⊥ Albacete Science and Technology Park, Paseo de la Innovación 1, 02006 Albacete, Spain ABSTRACT: The aim of this work was to propose a high-performance liquid chromatography with diode array detection (HPLC−DAD) method for determining the three main compounds responsible for determining the quality of saffron (crocetin esters, picrocrocin, and safranal) by preparing an aqueous extract according to the ISO 3632 standard to solve the difficulty that this standard has for aroma and taste determination by ultraviolet−visible spectroscopy. Toward this aim, laboratory-isolated picrocrocin, a safranal standard with a purity of ≥88%, trans-crocetin di(β-D-gentiobiosyl) ester (trans-4-GG) and trans-crocetin (β-D-glucosyl)-(β-D-gentiobiosyl) ester (trans-3-Gg) standards, both with a purity of ≥99%, and 50 different saffron spice samples from Italy, Iran, Greece, and Spain were used in the intralaboratory validation of the HPLC method. The analytical method proposed was adequate in terms of linearity, selectivity, sensitivity, and accuracy for determining the three foremost parameters that define the quality of saffron using only a saffron solution prepared according to the ISO 3632 standard. KEYWORDS: Crocus sativus L., aqueous extract, trans-4-GG, trans-3-Gg, picrocrocin, safranal, HPLC−DAD

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INTRODUCTION Few spices are able to provide the combination of color, taste, and aroma to the foods and beverages to which they are added. The most popular of these is saffron.1,2 To hydrophilic foods, saffron leads to a yellow color because of its high crocin content (also called crocetin esters, which are glycosylated carotenoids derived from crocetin), bitter taste because of picrocrocin, and pleasant and unmistakable aroma owing to safranal. The first reference to crocin [crocetin di(β-gentiobiosyl) ester] was made in 1818 by Aschoff,3 who gave it its name. Pfander et al.4 were the first to isolate six glycosides of crocetin in saffron. In 1984, Speranza et al.5 identified their trans and cis isomers by high-performance liquid chromatography with ultraviolet−visible spectroscopy (HPLC−UV−vis). Tarantilis et al.6 identified a greater number of crocetin esters and their trans and cis isomers by high-performance liquid chromatography with diode array detection and mass spectrometry (HPLC−DAD−MS), and Carmona et al.7 identified four more by HPLC−DAD−MS. Thus far, the quantification of crocetin esters by HPLC has been carried out without high precision,8−10 and the use of commercial standards in calibration curves gives low correlation coefficients, owing to the phenomenon of aggregation that some authors have demonstrated that happens in highly concentrated aqueous solutions of crocetin esters.11,12 The kinetics of individual crocetin ester degradation in aqueous extracts of saffron were studied by Sánchez et al.8 to determine their stability. © 2014 American Chemical Society

Picrocrocin [4-(β-D-glucopyranosyloxy)-2,6,6-trimethyl-1-cyclohexene-1-carboxaldehyde] has been identified only in the genus Crocus, of which the only edible species is Crocus sativus L. For this reason, picrocrocin is a molecular marker of this spice that is able to give a unique taste that cannot be imitated from other spices or seasonings. This compound was isolated for the first time in the stigmas of C. sativus by Winterstein et al.13 Its molecular structure was defined in 1934 by Kuhn et al.14 Glycosidic compounds that are structurally related to picrocrocin have been identified. Tarantilis et al.6 identified three glycosidic compounds in 1995; the Winterhalter group15,16 recognized eight other glycosidic compounds between 1997 and 1998; and Carmona et al.17 identified four more by HPLC−DAD−MS in 2006, which were found in considerably lower amounts than picrocrocin. The picrocrocin stability was determined by Sánchez et al.,18 and picrocrocin is more stable than the crocetin esters. More than 40 related compounds with a saffron aroma have been identified,19−24 with the major aromatic compound being safranal (2,6,6-trimethyl-1,3-cyclohexadiene-1- carboxaldehyde). In the saffron with the highest quality in the world “Azafrán de La Mancha”, safranal represents over 65% of the Received: Revised: Accepted: Published: 8068

April 28, 2014 July 18, 2014 July 30, 2014 July 30, 2014 dx.doi.org/10.1021/jf5019356 | J. Agric. Food Chem. 2014, 62, 8068−8074

Journal of Agricultural and Food Chemistry

Article

total aroma, as determined by gas chromatography (GC).25 Safranal is a nonpolar compound, but its solubility in water has not been studied. Different analytical techniques have been developed for its quantification, such as gas chromatography− olfactometry (GC−O),26 gas chromatography/mass spectrometry (GC/MS),27 high-performance liquid chromatography (HPLC),28 or ultrasound-assisted extraction/ultraviolet−visible spectroscopy (USAE/UV−vis).29 The results obtained are quite different between these various methods. The first references related to the use of saffron regarded their therapeutic properties.30 In the first century, Dioscórides described a great number of therapeutic properties;30 in the tenth century, new properties were described by Avicena.31 The therapeutic uses of saffron declined greatly over the past decade. However, saffron has received much attention by scientists in recent years.32 Most of these properties are ascribed to the trans isomers33−35 and safranal.36−38 The quality of saffron in international commercial agreements is determined according to the ISO 363239 standard, which classifies it into three categories depending upon their physical and chemical characteristics. The three foremost parameters used to define the quality of saffron are color, taste, and aroma.17,28 These parameters are determined by UV−vis based on the study by Corradi et al.,40 that is, the E1% 1 cm at 440 nm (coloring strength), the E1% 1 cm at 257 nm (the wavelength of picrocrocin maximum absorbance), and the E1% 1 cm at 330 nm (the wavelength of safranal maximum absorbance). While the E1% 1 cm at 440 nm is directly correlated with the ability to transmit color to foodstuffs to which it is added, the E1% 1 cm at 257 nm and E1% at 330 nm do not give an accurate 1 cm measurement of picrocrocin and safranal because the trans isomers of the crocetin esters also absorb at 250 nm and the cis isomers of the crocetin esters absorb at 250 and 330 nm as well, causing interference in the measurements.7,28 Moreover, the UV−vis spectrophotometry does not provide the ratio between the trans and cis isomers of crocetin esters, which is important for their therapeutic use.33−35 Therefore, the purpose of this work was to validate a HPLC method for the independent determination of the crocetin esters, picrocrocin, and safranal using only a saffron solution prepared according to the ISO 3632.39

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was allowed to stand to separate the aqueous phase from the safranal, with the saturated aqueous solution of safranal on top of the flask. An aliquot of this saturated solution was taken, s, and five dilutions were carried out: s/2, s/4, s/8, s/16, and s/32. These were then analyzed by UV−vis. The water solubility was calculated as the mean of the concentration values at which the absorbance obeyed the Lambert− Beer law. These analyses were carried out in triplicate at 20 ± 2 °C. The safranal standard solutions were monitored by scanning from 200 to 400 nm using a PerkinElmer Lambda 25 spectrophotometer (Norwalk, CT) with UV WinLab 2.85.04 software (PerkinElmer). Their absorbance was determined at 330 nm. For the safranal quantification, two series of safranal standard solutions in water with concentrations of 3, 10, 20, 30, and 32 mg/L were prepared and analyzed in duplicate by UV−vis. A calibration curve was constructed for the safranal concentration, c (mg/L) as a function of its absorbance a, at 440 nm with the equation c = 22.899a − 0.3027 and a R value = 0.999. Saffron Extract Preparation. The saffron aqueous extracts were prepared according to ISO 3632.39 A total of 500 mg of powdered saffron, previously passed through a sieve of 0.5 mm pore diameter, was placed in a 1 L volumetric flask, and 900 mL of Milli-Q water as added. The solution was stirred using a magnetic stir bar at 1000 rpm for 1 h while being kept away from light. The flask was filled to the 1 L mark, and the solution was homogenized through agitation. The solution was filtered through a filter made of hydrophilic polytetrafluoroethylene (PTFE) with a pore size of 0.45 μm (Millipore, Bedford, MA). It was then transferred into a vial for HPLC−DAD analysis. The saffron extraction was checked by doing a second extraction of the vegetable material obtained by filtration of the first extraction of three different saffron extracts prepared according to ISO 3632.39 HPLC−DAD Analysis. A total of 20 μL of each sample (saffron aqueous extracts) were injected into an Agilent 1200 HPLC chromatograph (Palo Alto, CA) equipped with a 150 × 4.6 mm inner diameter, 5 μm Phenomenex (Le Pecq Cedex, France) Luna C18 column that was equilibrated at 30 °C. The eluents were water (A) and acetonitrile (B) with the following gradient: 20% B, 0−5 min; 20− 80% B, 5−15 min; and 80% B, 15−20 min. The flow rate was 0.8 mL/ min. The DAD detector (Hewlett-Packard, Waldbronn, Germany) was set at 250, 330, and 440 nm for picrocrocin, safranal, and crocetin ester detection, respectively. All of the analyses were performed in duplicate, and two measurements were taken for each replicate. The identifications of trans-4-GG and trans-3-Gg were carried out using the UV−vis spectrum, and the retention time was carried out by the HPLC−DAD method at 440 nm and the parameter %III/II41 of their standards. Their quantification was based on calibration curves. With the aim of avoiding the phenomenon of aggregation that some authors have described in carotenoids, such as crocetin,12,42 trans-4GG, and trans-3-Gg standard solutions, 10, 20, 30, 40, 50, 60 and 70% acetonitrile/water (v/v) solutions were prepared and analyzed in duplicate by UV−vis at 440 nm. Two series of trans-4-GG standard solutions in water and two other series in 50% acetonitrile/water (v/v) with concentrations of 100, 50, 25, 12.5, 6.25, 3.125, 1.6, and 0.8 mg/L were prepared and analyzed in duplicate by HPLC−DAD. Two series of trans-3-Gg standard solutions in water and two other series in 50% acetonitrile/water (v/v) with concentrations of 50, 25, 12.5, 6.25, 3.125, 1.6, and 0.8 mg/L concentration were prepared and analyzed in duplicate by HPLC−DAD. Isomeric standards for the cis-crocetin esters are not available on the market; therefore, they were identified using the UV−vis spectrum. The retention time by HPLC−DAD−MS at 440 nm was determined by this group7 and in other works by the parameter %III/II.41 Isomeric cis-crocetin ester quantification was performed by the molecular coefficient absorbance value (63 350), obtained by Speranza et al.5 Picrocrocin identification was carried out by combination of its UV−vis spectrum and its retention time by HPLC−DAD at 250 nm. For its quantification, picrocrocin was isolated as described by Sánchez et al.8 Two series of isolated picrocrocin solutions in water from 2 to 315 mg/L were prepared and analyzed in duplicate by HPLC−DAD.

MATERIALS AND METHODS

Samples and Reagents. Samples. A total of 50 samples of saffron from different countries were used during the same year of their harvest. These were obtained directly from the producers and packers with a guarantee of their origin and freedom from fraud. The samples were from Italy, Iran, Greece, and Spain. All of the samples were of Category I according to ISO 3632.39 Standards. Safranal with a purity of ≥88% was obtained from Sigma-Aldrich (Madrid, Spain), and crocetin esters, trans-crocetin di(β-D-gentiobiosyl) ester (trans-4-GG) and trans-crocetin (β-Dglucosyl)-(β-D-gentiobiosyl) ester (trans-3-Gg), with a purity of ≥99%, were obtained from Phytolab GmbH & Co. KG (Vestenbergsgreuth, Bavaria, Germany). Solvents. Acetonitrile was obtained from Panreac (Barcelona, Spain), while water was purified through a Milli-Q system (Millipore, Bedford, MA). Safranal Water Solubility. To prepare a saturated safranal aqueous solution, increasing aliquots of safranal standard were added to a known volume of water until it was observed that the aqueous solution was saturated with safranal. A volume of 20 μL of the safranal standard in 10 mL of Milli-Q water was enough to obtain a saturated safranal aqueous solution. This solution was stirred using a magnetic stir bar at 1000 rpm for 1 h while being kept away from light. Then, it 8069

dx.doi.org/10.1021/jf5019356 | J. Agric. Food Chem. 2014, 62, 8068−8074


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Safranal identification was carried out using the UV−vis spectrum and the retention time of the safranal standard by HPLC−DAD at 330 nm. Two series of safranal standard solutions in water with concentrations of 4, 2, 1, 0.5, 0.25, 0.125, 0.06, and 0.03 mg/L were prepared and analyzed in duplicate by HPLC−DAD. Ultrasound-Assisted Extraction Followed by Gas Chromatography/Mass Spectrometry (USAE/GC/MS). Safranal determination by USAE/GC/MS was carried out as described by Maggi et al.27 USAE was performed in a Super RK 255H ultrasound water bath (Sonorex, Berlin, Germany) at a fixed frequency of 35 kHz. The temperature of the sonicated water bath was 25 °C. The sample flask was charged with 4 g of saffron. The solvent system extractant was 40 mL of diethyl ether. Each saffron sample was sonicated twice for 15 min (two fractions per saffron sample). For each sonication, a new volume of the solvent system extractant was added to the sample flask. The organic extract (80 mL) was concentrated using a Laborata 4000 efficient rotary evaporator (Heidolph Instruments GmbH & Co. KG, Schwabach, Germany). The volume of the final aromatic extract in diethyl ether was 4 mL. The collected diethyl ether was analyzed by GC to check if there was a loss of saffron volatiles during the evaporation procedure, but no saffron volatiles were detected. A HP 5890 series II chromatograph (Hewlett-Packard, Palo Alto, CA) equipped with a HP 5972 mass-selective detector in electron impact mode (70 eV) and a HP-5 MSM capillary column (30 m, 0.25 mm inner diameter, and 0.25 μm film thickness) with helium as the carrier gas at a flow rate of 1 mL/min was used for the analysis of safranal. The column temperature was initially held for 3 min at 50 °C, then increased to 180 °C at a rate of 3 °C min−1, and finally increased to 250 °C at a rate of 15 °C min−1. The injector and detector temperatures were set at 200 and 290 °C, respectively. Aliquots of 1 μL were manually injected in the splitless mode. For the safranal quantification, the external standard method was applied. The calibration curve was established for the series of safranal standard solutions in diethyl ether, with eq 1.

mg of safranal/kg of saffron = 86.6area safranal

(R = 0.999)

The solubility of safranal in water has not been determined previously. The water solubility of safranal was calculated from the saturated safranal aqueous solutions and was found to be 673.07 ± 0.01 mg/L at 20 ± 2 °C. An aqueous extract prepared according to ISO 363239 contains 500 mg of saffron dissolved in 1 L of water, and its safranal content will be less than the solubility (673.07 mg/L); hence, safranal will be completely dissolved. This result suggests that a saffron aqueous extract according to ISO 363239 could be used for safranal determination by HPLC, with the same solution used for determining the crocetin esters and picrocrocin. GC−O,26 GC/ MS,27 HPLC,28 or USAE/UV−vis29 have been used to quantify safranal, with the results between these methods being quite different. HPLC is considered the most efficient analytical technique for the analysis of sensitive compounds in complex extracts of natural products.46 Some authors have used HPLC for determining safranal,47−49 but these authors carried out the extraction with organic solvents, such as ethanol or methanol, because of the nonpolar character of safranal and the ignorance of its water solubility. Calibration Graphs. Table 1 shows the calibration curves of the saffron components (trans-4-GG, trans-3-Gg, picrocrocin, and safranal) calculated by the least-squares method. Table 1. Linearity Data for Analysis by HPLC−DAD of trans-4-GG, trans-3-Gg, Picrocrocin, and Safranal

trans-4-GG trans-3-Gg picrocrocin safranal

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Analytical Method Validation. An intralaboratory method validation was carried out according to the Eurachem guideline.43 For the linearity study, calibration graphs were performed by injecting standard solutions of saffron components (trans-4-GG, trans-3-Gg, picrocrocin, and safranal) at different concentrations. Each level of concentration was analyzed in duplicate, and two measurements for each one were carried out, with a total of four replicates. The selectivity was determined by comparing the chromatograms obtained from the saffron samples to those of the standard solutions analyzed by HPLC−DAD. The sensitivity of the method was determined with regard to the limit of detection (LOD) and limit of quantification (LOQ), comparing the height of a sample peak and the height of a noise peak. The LOD was reached at a signal-to-noise ratio greater than 3, and the LOQ was reached at a signal-to-noise ratio greater than 10.44 The detection and quantification limits were determined by the empirical examination of the chromatograms from 50 saffron samples. The accuracy of the method was evaluated with the 50 samples of saffron from different countries. Each sample was analyzed in duplicate, and two measurements were also taken for each replicate. The standard deviation (square root of the arithmetic mean of the variances) was calculated to obtain the repeatability [percent relative standard deviation (% RSD)]. The standard deviation multiplied by the square root of 3 was taken as the reproducibility value if this value was higher than the repeatability. If not, this last value was also taken as the reproducibility.45

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linear range of concentration (mg/L)

slope

intercept

correlation coefficient (R2)

0.8−100 0.8−50 2−315 0.03−4

0.0094 0.0079 0.0290 0.0323

2.4861 0.6682 0.5194 0.0510

0.976 0.996 0.999 0.998

The absorbance at 440 nm of the trans-4-GG and trans-3-Gg standard solutions at different percentages of acetonitrile/water (v/v) increased until a ratio of 40% acetonitrile/water (v/v). From a ratio of 50% acetonitrile/water (v/v) and greater, the absorbance at 440 nm of the trans-4-GG and trans-3-Gg standard solutions remained stable. This result suggests that, in highly concentrated aqueous solutions of trans-4-GG and trans3-Gg standard in 40−50% acetonitrile/water (v/v) or greater, molecular aggregation does not occur. The calibration curves of trans-4-GG and trans-3-Gg in water gave correlation coefficients lower than those of the calibration curves of trans-4-GG and trans-3-Gg in 50% acetonitrile/water (v/v), probably because of the phenomenon of aggregation;12,42 hence, the calibration curves in 50% acetonitrile/water (v/v) were used. The chromatographic purity of the trans-4-GG was 99.43%. The calibration curve for the concentration of trans-4-GG, d (mg/L), as a function of its HPLC peak area, b, exhibited good linear regression in the range from 0.8 to 50 mg/L with the equation d = 0.0075b − 0.008 and a R value = 0.999 for a total of seven data points. The calibration curve of the trans-3-Gg concentration, f (mg/L), as a function of its HPLC peak area, g, showed good linear regression in the range from 0.8 to 25 mg/L with the equation f = 0.0071g − 0.0047 and a R value = 0.999 for a total of six data points. Its chromatographic purity was 99.19%. The molecular coefficient absorbance values for trans-4-GG and trans-3-Gg in 50% acetonitrile/water (v/v) were 106

RESULTS AND DISCUSSION

Safranal Water Solubility. Saffron contains metabolites of different polarity because crocetin esters and picrocrocin are highly water-soluble polar compounds and safranal is a nonpolar constituent that has low solubility in polar solvents. 8070



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Figure 1. (A) Chromatograms of the commercial trans-4-GG (440 nm), trans-3-Gg (440 nm), and safranal (330 nm) standards and the laboratoryisolated picrocrocina (250 nm) and their UV−vis spectrum. (B) Chromatograms of the saffron sample (440, 330, and 250 nm) and their UV−vis spectrum.

922.75 and 92 781.76 M−1 cm−1 (440 nm), respectively, which were close to the values obtained by other authors for trans-4GG: 89 000 M−1 cm−1,5 132 200 M−1 cm−1,50 133 750 M−1 cm−1,51 and 133 500 M−1 cm−1;52 these differences may be due to the use of different solvents. The quantification of the cis isomers was based on eq 2 ⎛ ⎛ ϵcis ⎞⎟ ϵcis ⎞⎟ Ccis = ⎜mtrans × areacis + ⎜ntrans × ⎝ ⎝ ϵtrans ⎠ ϵtrans ⎠

where Ccis is the concentration of the cis-crocetin esters in mg/ L, mtrans is the slope of the trans-crocetin ester, ϵcis/ϵtrans is the ratio cis/trans obtained by Speranza et al.,5 areacis is the ciscrocetin ester peak area, and ntrans is the intercept of the transcrocetin ester. The calibration curve of the picrocrocin concentration, j (mg/L), as a function of the picrocrocin peak area, k, showed good linear regression in the range from 2 to 315 mg/L with the equation j = 0.0290k + 0.5194 and a R value = 0.999 for a total of six data points. Its chromatographic purity was 96%.

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4-GG and 0.766 for trans-3-Gg; thus, in both cases, there was a positive correlation between both methods. The trans-3-Gg correlation is lower, possibly because of the use in the approximate equation of the molecular coefficient absorbance value calculated by Speranza et al.5 for trans-4-GG. In this work, we have demonstrated that there is a difference between the molecular absorbance coefficients for trans-4-GG and trans-3Gg. In summary, 50 saffron samples were analyzed by HPLC. The values obtained ranged from 182.648 to 82.827 g of trans4-GG/kg of saffron, from 84.093 to 58.819 g of trans-3-Gg/kg of saffron, from 73.501 to 36.802 g of picrocrocin/kg of saffron, and from 1.142 to 4.346 g of safranal/kg of saffron. The evaluation of the method, described in terms of its linearity, selectivity, sensitivity, and accuracy, showed that it was adequate for our objectives. In Table 1, we can see the linearity of the proposed method by means of the calibration graphs for the saffron components trans-4-GG, trans-3-Gg, picrocrocin, and safranal. The excellent linearity of this HPLC method is demonstrated by taking into account that the correlation coefficients were higher than 0.99 in the majority of the cases. The proposed method showed a good selectivity because the retention times of their corresponding peaks were practically the same for the standards and the saffron samples analyzed (Figure 1). The values of the detection and quantification limits, calculated as the concentration, gave a peak height 3 or 10 times the signal-to-noise ratio, as shown in Table 2. The accuracy of the experimental procedure was also evaluated by studying the reproducibility and repeatability (% RSD). To consider the reproducibility of a method to be acceptable, its value should be lower than 20%. As seen in the results, summarized in Table 2, this parameter ranged from 0.06% (safranal) to 3.33% (trans-3-Gg). The same limit (20%) was taken to represent good repeatability, which, in this case, ranged from 0.04% (safranal) to 1.93% (trans-3-Gg). Therefore, the method proposed showed very good repeatability and reproducibility parameters. Comparison of HPLC−DAD and USAE/GC/MS Methods for Determining Safranal. The safranal content of the 50 saffron samples was determined by USAE/GC/MS and HPLC−DAD from an aqueous extract to compare the results obtained with both analytical techniques. USAE/GC/MS is a method that has already been validated and published. The values obtained with this method were from 2.367 to 4.945 g of safranal/kg of saffron. The same sample analyzed by HPLC from a saffron aqueous extract gave values from 1.142 to 4.346 g of safranal/kg of saffron. The graph shown in Figure 3 represents the comparison of the safranal content obtained for the 50 saffron samples using HPLC−DAD and USAE/GC/MS. Pearson’s correlation coefficient (r) was 0.788, showing a positive correlation between both methods. In conclusion, the proposed HPLC−DAD method allows for the quantification of the three main compounds of saffron in an easy and fast way, using only a water solution,39 with good linearity, selectivity, sensitivity, and accuracy. This methodology is adequate for determining the three foremost parameters that define the quality of saffron by preparing an aqueous extract according to ISO 3632.39 Because HPLC−DAD is common in laboratories certified for the quality control of saffron, this method could even be included in the present ISO 3632.39

The chromatographic purity of safranal was 92.99%. The calibration curve of the safranal concentration, h (mg/L), as a function of the peak area for safranal, i, revealed good linear regression in the range from 0.03 to 4 mg/L with the equation h = 0.0323i + 0.051 and a R value = 0.998 for a total of eight data points. Saffron Extraction Conditions. Saffron extraction was carried out according to ISO 3632.39 Other authors40 showed that this method is suitable. In this work, these extraction conditions were checked again, and the results showed that the coloring strength in the second extraction was less than 2%; therefore, it is an efficient and eco-friendly extraction method. HPLC−DAD Conditions. For the HPLC method, we relied on the method proposed by the identification and quantification of the crocetin esters and picrocrocin.8,53 This method had not been validated previously. For the crocetin ester quantification, an approximate equation was used8,53 on the basis of the molecular weight of the crocetin ester, the coloring strength of the sample determined by UV−vis according to ISO 3632,39 the percentage peak area of the crocetin ester by HPLC−DAD at 440 nm, and the molecular coefficient absorbance value determined by Speranza et al.5 In this work, the quantification of the crocetin esters was carried out using the calibration curves of the standards of trans-4-GG and trans3-Gg. Figure 2 represents the comparison of the trans-4-GG and trans-3-Gg contents, respectively, obtained for 50 saffron samples using the approximate equation8 and their respective calibration curves. Pearson’s correlation (r) was 0.953 for trans-

Figure 2. Graphical representation of the (A) trans-4-GG and (B) trans-3-Gg concentrations calculated by the approximate equation of Sánchez et al.8 versus the (A) trans-4-GG and (B) trans-3-Gg concentrations calculated by the calibration curve for 50 saffron samples. 8072



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Table 2. Precision of the Experimental Procedure for Analysis by HPLC−DAD of trans-4-GG, trans-3-Gg, Picrocrocin, and Safranal (n = 4) LOD (g/kg of saffron)

LOQ (g/kg of saffron)

reproducibility (%)

RSD (%)

0.33 0.38 0.60 0.105

0.95 1.02 1.01 0.28

3.07 3.33 1.34 0.06

1.77 1.93 0.77 0.04

trans-4-GG trans-3-Gg picrocrocin safranal

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Figure 3. Graphical representation of the safranal content obtained by HPLC−DAD versus the safranal content obtained by USAE/GC/MS for 50 saffron samples.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +34-967-599310. Fax: +34-967-599238. E-mail: [email protected]. Funding

The authors thank the Verdú Cantó Saffron Spain S.L. Company (Novelda, Spain) for this collaboration. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Marco A. Garciá for technical assistance. REFERENCES

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