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    • 专利摘要:
    The objective of the invention is the development of a procedure for the detection of adulterations of saffron with gardenia based on the determination of geniposide by high performance liquid chromatography with detection by mass spectrometry. The presence of geniposide in extracts of gardenia and its absence in saffron allows proposing said compound as a marker of adulteration of saffron with gardenia. The invention consists in the chromatographic separation of geniposides from the rest of the components of saffron samples using a c18 column and an isocratic elution with a mobile phase of water: acetonitrile (85:15 v/v) with 0.1% formic acid and detection by mass quadrupole-time-of-flight spectrometry. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2631834A1
    申请号:ES201600133
    申请日:2016-02-19
    公开日:2017-09-05
    发明作者:Miguel GUIJARRO DÍEZ；María CASTRO PUYANA；Antonio Luis Crego Navazo；María Luisa MARINA ALEGRE
    申请人:Universidad de Alcala de Henares UAH；
    IPC主号:
    专利说明:

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    PROCEDURE FOR THE DETECTION OF ADULTERATIONS PE AZAFRAN WITH GARDENIA BASED ON THE DETERM1NATION OF
    GENIPOSIDE
    DESCRIPTION
    The proposed analytical procedure allows the identification and quantification of geniposide in saffron samples using the technique of High Efficiency Liquid Chromatography with Mass Spectrometry detection. Taking into account that the presence of saffron geniposide reveals gardenia adulteration, this procedure is a great advance because of the possibility of using a single compound, geniposide, as a marker of saffron adulteration with gardenia in order to assess the quality of saffron as well as to avoid economic fraud.
    TECHNICAL SECTOR
    This invention makes it possible to detect saffron adulterations with gardenia, being the development of the analytical method for the determination of saffron geniposide of interest to the chemical sector and the procedure for the detection of saffron adulterations with gardenia is of interest to the food sector because of its potential to control the quality of saffron and avoid economic fraud.
    STATE OF THE TECHNIQUE
    Saffron is produced from the dry stigmas of the Crocus sativus L. plant, being the most expensive spice in the world due to the labor necessary for its cultivation, harvesting and handling, as well as having a limited production.
    The three main secondary metabolites in saffron are: crocins (responsible for color), a family of yellow pigments very soluble in water; picrocrocin (responsible for the bitter taste), a colorless glucoside; and the safranal (responsible for the smell), a volatile oil. In addition, saffron also contains flavonoids, proteins, sugars,
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    vitamins, amino acids, minerals and other chemical compounds [1]. Flavoring and coloring corao has long been used in food preparation, however, it is also known for a wide variety of health benefits (2), Recent studies show that saffron may offer some protection against coronary heart disease and cancer , as well as a high potential as a memory enhancer [3-61.
    Due to its high price, saffron has been subjected to various types of adultery over the centuries. It should be noted that a report has recently been published describing that saffron is, together with olive oil, milk and honey, one of the food ingredients that most commonly suffer from adulteration. | 7 | Saffron adulterations are carried out in order to increase their weight with foreign materials (usually plants with a certain external resemblance) and / or to improve their color with natural or synthetic dyes when saffron is old or to mask the addition of foreign matter. The most common fraudulent practices include the addition of different plant materials with similar color and morphology. Historically, the most frequent materials used in the adulteration of saffron have been stamens of the saffron plant itself (including corolla styles or strips), plant petals Carthamus tinctorius L. (cartamo) and Hemerocallis lilioasphodelus L., rhizomes of Curcuma longa L. (curcuma), flowers of the Calendula officinalis L. (calendula) and Arnica montana L. plants and seeds of Bixa orellana L. [8, 9 |. Recently, a new and sophisticated method of adultery of saffron has been discovered in the European inercado using extracts of the fruits of the Gardenia jasminoides Ellis L. (gardenia) [10], which is widely used in Asian countries in traditional medicine and as a natural dye due to the presence of a high number of flavonoids and crocins similar to those present in saffron (responsible for yellowing) [11-14], being banned from marketing in Europe. Given the morphological differences of gardenia against stigmas of saffron, this type of adulteration occurs when saffron is in ground form, being able to more easily hide gardenia extract [8, 15], On the other hand, the main secondary metabolites of the fruits of gardenia that differ from saffron are iridoid glucosides, which include: geniposide, gardenoside, genipin-1- P-gentiobioside, geniposidic acid, acetylgeniposide and gardosido] 16-18].
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    So far, saffron quality is certified in the international market through ISO 3632 [19, 20J. However, this regulation only characterizes saffron through the contents of picrocrocina, safranal and crocinas, as! as the possible presence of some of the artificial colors that may be toxic. Therefore, the methodology used in the ISO standard is a combination of spectrophotometric measurements of picrocrocin at 250 nm and safranal at 310 nm with chromatographic profiles of dyes and polar pigments (crocins) at 440 nm. This regulation is currently under revision due to its low reliability in the detection of plant materials with saffron-like color and morphology. In particular, it has recently been demonstrated that it is not possible to detect using adulthood an adulteration in saffron with amounts up to 20% of safflower, calendula or curcuma | 21 |. Therefore, in recent years several authors have proposed new analytical methods for the detection of adulterants in saffron using very different analysis techniques: UV-Vis Spectrophotometry [22-24J, Near Infrared Spectrophotometry (NIR) [25], Raman Spectroscopy and Nuclear Magnetic Resonance (NMR) [26], Capillary Electrophoresis (EC) | 27 | and High Efficiency Liquid Chromatography (HPLC) without and with Mass Spectrometry (MS) detection [21, 28-30 |. Likewise, it is worth highlighting the recent use of novel molecular methods to detect DNA markers of different plants (safflower and curcuma) in saffron which have allowed encouraging results, being able to detect up to 1% of adulteration [15, 3133].
    On the other hand, a metabolomic strategy based on the utilization of NMR has recently been developed and has been applied to the differentiation of authentic saffron from adulterated saffron with 20% of gardenia, curcuma, safflower and saffron stamens [34 |. However, there is a clear demand for sensitive methods that allow detecting low percentages of adulteration in saffron.
    Geniposide has been described as the majority iridoid glucoside in gardenia, so it could be used as a marker for saffron adulteration with gardenia. Although several methods have been developed to determine geniposide in the fruit of gardenia
    using HPLC and CE techniques [35-42], there is no method that allows the determination of geniposide in saffron.
    The objective of this invention is the development of a procedure by Liquid Chromatography 5 with detection by Quadruple Mass Spectrometry-Flight Time that allows to determine geniposide in saffron, in order to propose for the first time this compound as a marker of saffron adulterations with gardenia extracts.
    DESCRIPTION OF THE INVENTION
    10
    The procedure for the determination of geniposide by liquid chromatography is based on the use of a C18 column and an Agilent Technologies 1100 liquid chromatography equipment coupled to a quadrupole-time flight mass spectrometry (MS) detector (QTOF) Agilent Technologies 6530 through a source of 15 ionization by orthogonal electrospray with Jet Stream thermal focusing technology. The mass spectrometer is equipped with a Mass Hunter software from Agilent Technologies (B.040.00) for mass spectrometer control and data acquisition and analysis.
    20 Table 1. Summary of the procedure for the determination of geniposide in saffron by
    LC-MS
     Column  Ascentis Express Fused-core Cl 8 (100 mm x 2.1 mm, with 2.7 pm (0.5 pm thick stationary phase) "fused-core" film particles
     Mobile phase  Water: Acetonitrile (85:15 v / v) containing 0.1% formic acid
     Mobile phase fiujo  0.4 mL / min
     Injection volume  5 pL
     Temperature  40 ° C
     Quadruple Mass Spectrometry-Flight Time Detection  Ionization by Electrospray in negative mode (ESI -). Mass range of 100-1700 m / z (extended dynamic range) with an acquisition speed of 2 spectra / s. Conditions for the ionization source: electrospray potential, 3500 V with a nozzle voltage
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    500 V; drying gas conditions, 10 L / min and 350 ° C; nebulizer pressure, 1.7 bar; Surround gas, 7.5 L / min and 350 ° C. Other conditions: fragmenter, 150 V. Skimmer voltages (skimmer) and octopol 60 V and 750 V, respectively.
    Saffron samples are crushed in a mortar and weighed (0.3 g of sample). They are extracted with 25 mL of ethanobtampon borate solution at pH 9.0 (50:50, v / v) by solid-liquid extraction assisted by Ultrasound sample preparation for 15 min at room temperature. After centrifugation
    (15 min, 4000g and 25 ° C) the supernatant is diluted 1/50 with water and 4 mL of this solution is soaked to ultrafiltration through 3 kDa cut filters in order to eliminate carbohydrates and proteins.
    Preparation of patterns and samples
    The preparation of the standard geniposide solutions is made from an initial solution of geniposide I mg / mL in acetonitrile. This solution is stored at 4 ° C and different aliquots are taken that are diluted in water to the required concentration.
    The following sample treatment procedure is used: the saffron sample is crushed in a mortar and weighed (0.3 g of sample). They are extracted with 25 mL of ethanohtampon borate solution at pH 9.0 (50:50, v / v) by solid-liquid extraction assisted by ultrasound for 15 min at room temperature. After centrifugation (15 min, 4000g and 25 ° C) the supernatant is diluted 1/50 with water and 4 mL of this solution is subjected to ultra-filtration through 3kDa cut filters in order to eliminate carbohydrates and proteins.
    Analytical characteristics of the procedure
    The analytical characteristics evaluated for the process object of the invention are linearity, limits of detection and quantification, precision (instrumental and intermediate) and accuracy (Table 2).
    Linearity is checked in the range of working concentrations from 0.8 to 8 (ig / mL of geniposide injected in triplicate for three consecutive days.
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    The limit of detection is determined as the concentration of geniposide that gives rise to a value of the signal / noise ratio of 3. The limit of detection is 0.01 pg / mL which allows to detect 41.7 pg of geniposide per gram of analyzed sample, it is that is, up to 0.004%, which demonstrates the high sensitivity of the method developed for the determination of geniposide in saffron. For the calculation of the quantification limit, a signal / noise ratio of 10 is considered and a value of 0.03 pg / mL is obtained.
    Precision is evaluated as instrumental repeatability and intermediate precision. Instrumental repeatability is determined as the relative standard deviation (RSD) value in% for peak areas obtained when 3 consecutive injections of a standard geniposide solution at two concentration levels (0.8 and 8 pg / mL) are made and of a solution of a sample of authentic adulterated saffron with percentages of 10 and 90% gardenia extract. The values obtained for the RSD were less than 1.5%. The intermediate precision was determined from the RSD values of the peak areas obtained by injecting (in triplicate for two consecutive days) three replicates of a geniposide standard solution at two concentration levels (0.8 and 8 pg / mL) and of a solution of a sample of authentic adulterated saffron with percentages of 10 and 90% gardenia extract. The values obtained for RSD were below 1.8% for standard geniposide solutions and 2.9% for saffron samples.
    The procedure described in the present invention is applicable to the quantification of geniposide in saffron. Quantification is carried out using the external standard calibration method. This quantification method is used because it is verified, by comparing the confidence intervals of the slopes obtained by the calibration methods of the external pattern and of pattern additions, the absence of matrix effects, that is, that the analytical serial for geniposide It is not affected by the other components of the sample.
    The accuracy of the developed analytical method was evaluated as the recovery obtained for geniposide when an authentic saffron sample was enriched with geniposide.
    standard at a concentration of 1 pg / mL. The average recovery values obtained were 89 ± 14%.
    Table 2, Analytical characteristics of the procedure developed for the determination of geniposide in saffron LC-MS pair.
    Linear work interval *
    Pending ± t-sb
    0.8-8 pg / mL (1.91 ± 0.21) x 10 * * 6
     Ordered in origin ± tsa  (6.7 ± 9.5) x 10s
     R2 * *  0.997
     p-value of ANOVAb  0.078
     Matrixc Interferences  Sample Confidence Interval
     (n = 5)    (slope ± t sb)
     Geniposide pattern (1.91 ± 0.21) x 106
     Authentic saffron (2.06 ± 0.21) x 106
     Saffron / Gardenia Extract (50:50) (2.06 ± 0.22) x 106
     LOD * '  0.01 pg / mL (41.7 pg / g extract) 11
     LOQf  0.03 pg / mL (138.9 pg / g extract) '
     Accuracy  Aiiadide concentration (% mean ± t (n-l) * s / n '/ :)
     Recovery  1 pg / mL 89 ± 14
    10
    Precision
    Repeatability ^ (n = 3)
    Intermediate precision h (n = 6)
     Concentration level  RSD (%)
     0.8 pg / mL geniposide pattern  1.5
     8 pg / mL geniposide pattern  0.7
     90% saffron gardenia extract  0.8
     10% gardenia extract in saffron  0.6
     0.8 pg / mL geniposide pattern  1.8
     8 pg / mL geniposide pattern  1.6
     90% saffron gardenia extract  0.9
     10% gardenia extract in saffron  2.9
    “Five geniposide pattern solutions at different levels of concentration injected in triplicate
    for three consecutive days,
    6 p-value of the ANOVA to confirm that the experimental model fits a linear model.
    'Comparison of the slopes obtained by external standard methods and standard additions.
    '' LOD calculated as the geniposide concentration corresponding to an equal signal / noise (S / N) ratio
    to 3.
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    ‘LOQ calculated as the geniposide concentration corresponding to a signal / noise ratio (S / N) equal to 10.
    f Accuracy calculated from the recovery values obtained by adding 1 pg / mL of the geniposide pattern to three different solutions of authentic saffron injected in duplicate. g Repeatability expressed as RSD (%) of the areas obtained by injecting a solution of geniposide pattern at two levels of concentration in three days in a row and a solution of authentic adulterated saffron with percentages of 10 and 90% of gardenia extract .
    * Intermediate precision expressed as RSD (%) of the average value of the areas obtained by injecting three consecutive times for two consecutive days three replications of a standard solution of geniposide at two levels of concentration and a solution of authentic adulterated saffron with percentages of 10 and 90% gardenia extract.
    Main advantages of the procedure
    The main advantages of the process object of the present invention are the following:
    - The described procedure allows to determine the geniposide content in saffron.
    - The procedure developed allows the geniposide peak to be separated from the rest of the saffron components in less than 2 min.
    - The procedure developed allows the geniposide to be identified through its mass spectrum.
    - The procedure developed shows the absence of authentic saffron geniposide, being a valuable tool for quality control laboratories and for the food industry because geniposide can be used as a marker for saffron adulteration with gardenia.
    DESCRIPTION OF THE FIGURES
    Figure 1. Ion chromatograms extracted at a ratio m / z 433.1384 ± 50 ppm obtained by liquid chromatography with detection of quadrupole-flight time mass spectrometry, for four samples of saffron suspected of being adulterated. A), B), C) Saffron samples adulterated with gardenia. D) Saffron sample not adulterated with gardenia. The procedure for the preparation of the samples, the conditions used
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    for analysis by liquid chromatography and the conditions used for mass spectrometry detection have been detailed in Table 1.
    MODE OF REALIZATION
    The procedure described in the present invention applies to the detection of saffron adulterations with gardenia extracts. For this, authentic saffron samples and saffron samples suspected of having been adulterated are analyzed.
    Sample preparation is carried out by crushing saffron samples in a mortar and weighing 0.3 g of sample. They are extracted with 25 mL of ethanol solution: borate buffer at pH 9.0 (50:50, v / v) by ultrasound-assisted solid-liquid extraction for 15 min at room temperature. After centrifugation (15 min, 4000g and 25 ° C) the supernatant is diluted 1/50 with water and 4 mL of this solution is subjected to ultra-filtration through 3 kDa cut filters. 5 pL of the ultrafiltrate are injected into an Ascends Express Fused-core Cl8 column (100 mm x 2.1 mm, with 2.7 pm (0.5 pm thick stationary phase) “fused-core” film particles using an isocratic elution with a mobile water phase: acetonitrile (85:15 v / v) with 0.1% formic acid, a 0.4 mL / min mobile phase flow and a temperature of 40 ° C. Mass spectrometry detection is performed by Electrospray Ionization in Negative mode (ESI -) with a mass range of 100-1700 m / z (extended dynamic range) and an acquisition rate of 2 spectra / s The conditions for the ionization source are: a 3500 V electrospray potential with a nozzle voltage of 500 V, drying gas conditions of 10 L / min and 350 ° C, a nebulizer pressure of 1.7 bar and enveloping gas of 7.5 L / min and 350 ° C. The fragmenter is 150 V and the skimmer and octopol voltages are 60 V and 750 V, respectively.
    Figure 1 shows the ion chromatograms extracted at a ratio m / z 433.1384 ± 50 ppm for three of the samples adulterated with gardenia and for a sample suspected of adulteration for which it is confirmed that it has not been adulterated with gardenia. The peak of the geniposide is observed in the three samples adulterated with gardenia and the absence of the peak of the geniposide in the sample not adulterated with gardenia.
    For the determination of geniposide in the three samples adulterated with gardenia, the external standard calibration method is used using standard geniposide solutions. The determined amounts of geniposide in the three samples of adulterated saffron (sample 1, 2 and 3, respectively) are 5.90 ± 0.01, 13.2 ± 0.2 and 15.30 ± 0.01 5 mg / g of analyzed sample.
    The results of the analyzes confirm the absence of geniposide in the authentic saffron samples, so geniposide is proposed as a marker of saffron adulteration with gardenia. The results also confirm the presence of geniposide in some of the samples suspected of being adulterated demonstrating the new method of adultery of saffron with gardenia that has reached the European saffron market.
    INDUSTRIAL APPLICATION
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    Both the food industry and the administration are interested in the existence of sensitive analytical procedures that allow quality control and safety of saffron. The use of geniposide as a marker for adulteration of saffron with gardenia extracts allows for quality control of saffron and to detect 20 adulterations of saffron with gardenia in a sensitive and unequivocal way.
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    权利要求:
    Claims (9)
    [1]
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    RE1VINDICATIONS
    1. A procedure for the detection of adulteration of saffron with gardenia characterized in that it is based on the determination of geniposide in saffron by High Efficiency Liquid Chromatography with detection by Quadruple Mass Spectrometry-Flight Time.
    [2]
    2. The method according to claim I characterized in that it requires the following sample treatment: the saffron sample is crushed in a mortar and weighed 0.3 g, extracted with 25 mL of ethanol solution: 50:50 borate buffer, v / v , at pH 9.0 by solid-liquid extraction assisted by ultrasound for 15 min at room temperature, and after centrifugation for 15 min at 4000g and 25 ° C, the supernatant is diluted 1/50 with water and 4 mL of this solution is subjected to ultrafiltration through 3kDa cutting fdtros.
    [3]
    3. The method according to claim 1 characterized by performing the geniposide determination by high performance liquid chromatography coupled to mass spectrometry on a Cl8 column 100 mm long and 2.1 mm internal diameter, with film particles of "fused- technology" core ”of 2.7 pm and 0.5 pm of stationary phase thickness.
    [4]
    4. The method according to claim 3 characterized by the use of an isocratic elution with a mobile water phase: acetonitrile 85:15, v / v, with a formic acid content of 0.1%.
    [5]
    5. The method according to claims 3 and 4 characterized by the following instrumental parameters in liquid chromatography: mobile phase flow 0.4 mL / min, injection volume 5 pL, temperature 40 ° C.
    [6]
    6. The method according to claim 1 characterized in that the detection is carried out by means of a quadrupole-flight time mass spectrometry detector using the following instrumental parameters: nebulization pressure, 1.7 bar; drying gas flow, 10 L / min; envelope gas flow, 7.5 L / min;
    electrospray potential, 3500 V; drying gas temperature, 350 ° C, envelope gas temperature, 350 ° C; nozzle voltage, 500 V and fragmentation voltage, 150 V. Skimmer and octopus voltages 60 V and 750 V, respectively.
    5
    [7]
    7. The procedure according to revindication 6 characterized in that the identification of the geniposide peak in saffron samples is carried out through the mass spectrum obtained in the extracted ion chromatogram at a ratio m / z 433.1384 ± 50 ppm.
    10
    [8]
    8. The method according to the preceding claims characterized in that it allows the identification and quantitative determination of geniposide in saffron samples.
    The method according to the preceding claims characterized in that
    establishes the absence of geniposide in samples of authentic saffron.
    [10]
    10. The method according to claims 8 and 9 characterized by the proposal of geniposide as a marker of saffron adulterations with extracts of
    20 gardenia
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