Application of the relative molar sensitivity method using GC-FID to quantify safranal in saffron (Crocus sativus L.).

Safranal is one flavor component of saffron, which is used as a spice, food additive, and crude drug. In ISO3632, safranal is defined as the compound that contributes to the quality of saffron, and many quantitative determination methods for safranal have been reported. However, safranal is volatile and degrades easily during storage, and an analytical standard with an exact known purity is not commercially available, making it difficult to quantify accurately the content of safranal in saffron. Here, we developed a method for quantifying safranal using relative molar sensitivity (RMS), called the RMS method, using a GC-flame ionization detector (GC-FID). We determined the RMS of safranal to 1,4-bis(trimethylsilyl)benzene-d4, a certified reference material commercially available, by a combination of quantitative NMR and chromatography. Using two GC-FID instruments made by different manufacturers to evaluate inter-instrument effect, the resultant RMS was 0.770, and the inter-instrument difference was 0.6%. The test solution, with a known safranal concentration, was measured by the RMS method, with an accuracy of 99.4-101%, repeatability of 0.81%, and reproducibility of 0.81-1.3%. Given the ease of degradation, high volatility, and uncertain purity of safranal reagents, the RMS method is a more accurate quantification approach compared to the calibration curve method and methods based on absorption spectrophotometry. Moreover, our findings revealed that the GC-FID makeup gas affected the RMS and quantitative values.

Neolignans and polyoxygenated seco-cyclohexenes from the stems and leaves of Piper suipigua Buch.-Ham. ex D. Don.

Five new compounds including, a neolignan, eupomatenoid-19 (1) and four polyoxygenated seco-cyclohexenes, artahongkongenes G-J (2-5), together with fifteen known compounds (6-20) were isolated from the stems and leaves of Piper suipigua Buch.-Ham. ex D. Don. Their structures were determined by spectroscopic evidence (IR, UV, 1H NMR, 13C NMR and 2 D NMR) as well as MS. The absolute configurations of polyoxygenated seco-cyclohexenes 2-8 were identified by NOESY data and by comparison of their experimental and calculated ECD spectral data. Neolignans, eupomatenoid-19 (1) and eupomatenoid-7 (10), displayed cytotoxicity against several cancer cell lines. In addition, eupomatenoid-7 (10) showed antibacterial activity against Bacillus cereus, Bacillus subtilis and Staphylococcus aureus.

Authentication of Iranian Saffron (Crocus sativus) Using Stable Isotopes δ13C and δ2H and Metabolites Quantification.

Saffron is a very high value-added ingredient used in the food supplement market and contains a high level of safranal. Adding synthetic safranal to saffron, which is significantly cheaper, and falsifying the origin of saffron may represent recurrent fraud. Saffron from different countries was analyzed to determine the stable isotope ratios δ13C and δ2H from safranal by gas chromatography coupled with isotope-ratio mass spectrometry (GC-C/P-IRMS) and the concentration of saffron metabolites with ultra-high performance liquid chromatography coupled with diode array detector (UHPLC-DAD). The isotopic analysis highlighted a higher ratio of δ2H in synthetic safranal than in natural safranal; the mean values were 36‱ (+/- 40) and -210‱ (+/- 35), respectively. The δ13C between Iranian, Spanish and other saffron was significantly different and represents median values of -28.62‱, -30.12‱ and -30.70‱, respectively. Moreover, linear and quadratic discriminant analyses (LDA and QDA) were computed using the two isotope ratios of safranal and the saffron metabolites. A first QDA showed that trans-crocetin and the δ13C of safranal, picrocrocin, and crocin C3 concentrations clearly differentiated Iranian saffron from other origins. A second model identified δ13C, trans-crocetin, crocin C2, crocin C3, and picrocrocin as good predictors to discriminate saffron samples from Iran, Spain, or other origins, with a total ability score classification matrix of 100% and a prediction matrix of 82.5%. This combined approach may be a useful tool to authenticate the origin of unknown saffron.

Determination of Saffron Volatiles by HS-SBSE-GC in Flavored Cured Ham.

At present, the development of new agri-food products, including flavored meat products presented in ready-to-eat vacuum packs, is encouraged. The addition of ingredients used as flavoring agents creates the need to be able to determine the volatile compounds responsible for their characteristic aroma. The aim of this study is to propose, develop, and validate a new method that uses headspace-stir bar sorptive extraction-gas chromatography/mass spectrometry (HS-SBSE-GC/MS) to determine the saffron aroma in cured ham flavored with this spice. Results showed that safranal was the main volatile compound that could be identified and quantified in cured ham flavored with saffron. This analytical method was adequate in terms of linearity, selectivity, sensitivity, and accuracy. To our knowledge, this is the first time that an HS-SBSE-GC/MS method for determining the saffron aroma of flavored cured ham has been developed and validated, and it is of interest to agri-food industries.

Nitric oxide induced carotenoid contents in Crocus sativus under salinity.

Crocus sativus possesses unique apocarotenoid compounds such as crocins and picrocrocein involved in color, taste, flavor and medicinal benefits of Saffron. Crocus sativus L. corms were treated with Nitric Oxide (NO) and salt. Crocins and picrocrocin contents were determined with high-performance liquid chromatography (HPLC) resulted a significant increase of crocins in treated plants with NO and salt that reveals the stimulating effect of NO in apocarotenoid biosynthesis besides the inductive role of salt stress. This raise can be attributed to expression of CsPDS, CsPSY, CsLYC, CsBCH, and CsCCD2 that were remarkably altered. Treating plants with NO caused more phenol production in oppose to less flavonoid content; however, salinity could increase both. Therefore, NO induced crocins and picrocrocin biosynthesis due to impressing gene expression. This increasing effect was enhanced when salinity was simultaneously imposed.

Impact of two different dehydration methods on saffron quality, concerning the prevalence of Saffron latent virus (SaLV) in Iran.

The dehydration process is a prerequisite to preserve saffron for a long time. According to this process, saffron shows differences in the main compounds responsible for its quality (colour, taste, aroma, and flavonol content). At present, the freeze-drying method obtains dried products with the highest quality. Viruses can modify the physiology and metabolism of plants, being able to affect the activities of several enzymes. For this reason, the main compounds of saffron have been analyzed under two different dehydrating processes, freeze-drying and dark-drying, considering their infection status with the Saffron latent virus (SaLV). Results showed that the picrocrocin and safranal content enables to differ dark-dried samples from freeze-dried ones. Besides, the kaempferol-3-O-sophoroside-7-O-glucoside content allows differentiating between SaLV-infected (SaLV+) and uninfected (SaLV-) saffron samples. Moreover, our data suggest that the freeze-drying would decrease crocins content, and dark-drying can nullify the adverse effect of SaLV on crocins content.

Intranasal administration of perillyl alcohol-loaded nanoemulsion and pharmacokinetic study of its metabolite perillic acid in plasma and brain of rats using ultra-performance liquid chromatography/tandem mass spectrometry.

Perillyl alcohol (POH) is a monocyclic terpene that has strong antitumor activity. Brain tumors are particularly difficult to treat with therapeutic agents, and clinical trials have shown their low tolerance through oral administration. We proposed the entrapment of POH into an oil-in-water chitosan nanoemulsion aiming its intranasal administration for brain targeting. An ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the quantitation of total metabolite perillic acid (PA) in plasma and brain of rats. The rat samples containing the metabolite were treated by liquid-liquid extraction with acetonitrile. The mobile phase was 0.1% formic acid in water (solvent A) and 0.1% formic acid in methanol (solvent B), at a flow rate of 0.3 mL min-1 in gradient elution. The chromatography was run for 10 min, and analytical curves were built in acetonitrile, plasma, and brain. The PA was detected in positive ion mode with multiple reaction monitoring. The method has shown high selectivity, sensitivity, and throughput. The low quantification limits of 162, 178, and 121 ng mL-1 for acetonitrile, brain, and plasma, respectively, indicate a good detectability of the method. The repeatability and precision observed were within the limits recommended in the literature. The accuracy of the method was verified through high recovery rates (106-118%). The validated method was successfully applied to the pharmacokinetic study of the metabolite PA after the intranasal administration of free or POH-loaded nanoemulsion in rats. The results showed that chitosan nanoemulsion improved the plasma and brain bioavailability of POH, representing a promising alternative to free POH treatment.

Cloning and functional characterization of a carotenoid cleavage dioxygenase 2 gene in safranal and crocin biosynthesis from Freesia hybrida.

Safranal and crocin, commonly derived from the oxidative cleavage reaction of zeaxanthin in plants, are two kinds of apocarotenoids with versatile functions, which were only found in limited number of plant species. In this study, both metabolites were detected and varied concomitantly with the expression of carotenoid cleavage dioxygenase (CCD) genes in Freesia hybrida, Red River® and Ambiance cultivars. The newly isolated CCD, denoted here as FhCCD2, was phylogenetically clustered with other reported saffron CCD2s. Besides, ten introns were also observed in the genomic DNA sequence of FhCCD2 and the presence of N-terminal transporter peptide suggested its plastidial sub-localization. Biochemical analysis showed that the FhCCD2 cleaved zeaxanthin at the 7, 8 and 7', 8' double bonds to generate intermediates prerequisite for the biosynthesis of safranal and crocin. Further, gene transient expression analysis showed that the promoter of FhCCD2 was functional in Ambiance as well as Red River® cultivars, even with slight variation in their promoter sequence. At present, CCD2 proteins have only been found in Freesia and Crocus genus of Iridaceae family. Phylogenetic and intron position analysis infer that CCD2 perhaps emerged after the intron loss during evolutionary process of CCD1 or their shared ancestry.

[Determination of Cyclohexene in workplace air by Solvent desorption gas chromatography].

Objective: To establish a solvent desorption gas chromatography method for determination of cyclohexene in workplace air. Methods: Cyclohexene in the air of workplace was collected with carbon tube and desorbed by carbon disulfide. The target toxicant was separated with the GC column and analyzed with FID detector, identified by retention time, and quantified by peak area. Results: The linear range of cyclohexene in the air of workplace was 0.77~4 050.00 µg/ml, with a correlation coefficient of 0.9999. The limit of detection was 0.23 µg/ml. The lower limit of quantification was 0.77 µg/ml. The minimum detectable concentration was 0.15 mg/m(3) under1.5 L sampling volume and 1.0 ml extraction solution volume. The within-run precision of different cyclohexene concentrations was 0.62%~1.9% and the between-run precisions was 1.5%~3.5%; The average extraction efficiency was 96.4%; Penetration capacity (100 mg of carbon tube) was 29.4 mg; The average collection efficiency was 100%; The samples could be stored for 7 days at room temperature. When placed in 4 ℃ refrigerator, the samples could be stored for 14 days. The potential coexistence of cyclohexane, hexane, benzene, toluene and ethylbenzene with cyclohexene in the air did not interfere with the results of determination. Conclusion: This method has high sensitivity, precision, accuracy and lower limit of detection and it is applicable for determination of cyclohexene in workplace air.

A rapid MALDI MS/MS based method for assessing saffron (Crocus sativus L.) adulteration.

We report on a sensitive and fast quantitative MALDI-MS/MS method used to assess saffron authenticity by direct analysis through the determination of picrocrocin as the saffron authenticity marker, and using curcumin as the non-isotopic isobaric internal standard. The internal standard curcumin yielded good linearity (R2 = 0.994), and with confidence intervals at 95% for intercept. The detectable maximum adulteration percentage (99.0%) was estimated interpolating the limit of detection (LOD) for the isobaric internal standard in linear regression. The LOD was 47.63 ppm, and LOQ was 56.53 ppm. Good accuracy and precision were obtained for all concentrations. The capability of the MS approach to monitor analytes in a specific, selective fashion was used to obtain a semi-quantitative adulteration percentage and to establish the adulterant by additional experiments. The detection of gardecin and its derivatives in commercial samples indicated that Gardenia jasminoides Ellis was used as the adulterant.

A streamlined workflow to study direct photodegradation kinetic and transformation products for persistence assessment of a fragrance ingredient in natural waters.

Photodegradation can be an important abiotic degradation process to consider for the fate and persistence assessment of chemical substances in the environment. In this work, using a fragrance ingredient (FI, (E)-4-(2,2,3,6-tetramethylcyclohexyl)but-3-en-2-one) as an example, we developed a streamlined workflow to investigate direct photodegradation of chemicals in the aquatic environment, including laboratory investigation of kinetics and transformation products and estimation of its aquatic environmental half-lives. Direct photodegradation was determined to be the dominant photodegradation process for FI with a quantum yield of 0.25, which was supported by photodegradation experiments conducted in natural sunlight. Accounting for light attenuation by dissolved organic matter in natural waters of different depths resulted in aquatic half-lives of <31 days even at polar latitudes. Photoisomerization was shown to be a major photodegradation pathway along with the formation and subsequent degradation of constitutional isomers and photooxidation products. These results contributed to FI being assessed as non-persistent in the environment.

Multi-residue analysis of captan, captafol, folpet, and iprodione in cereals using liquid chromatography with tandem mass spectrometry.

In this study, we propose an improved analytical method for the multiresidue analysis of captan (plus its metabolite, tetrahydrophthalimide), folpet (plus its metabolite, phthalimide), captafol, and iprodione in cereals using liquid chromatography tandem mass spectrometry (LC-MS/MS). As captan, captafol, and folpet are easily degraded during homogenisation and extraction, samples were comminuted with liquid nitrogen, and both QuEChERS and ethyl acetate-based extraction workflows provided a satisfactory method performance. The optimised LC-MS/MS procedure with electrospray ionisation did not degrade these compounds, and offered sufficient method selectivity by resolving and minimising co-eluting matrix-derived interferences. The method also resolved the problem of non-specific mass spectra that these compounds usually produce on GC-MS analysis involving electron ionisation. The method performance was satisfactory for all 6 compounds at 0.01 mg kg-1 and higher levels of fortification, and validated as per the SANTE/11813/2017 guidelines of analytical quality control in a wide range of cereals including rice, wheat, sorghum, and corn. The method provides special advantage of simultaneous analysis of captan, and folpet along with their metabolites (tetrahydrophthalimide, and phthalimide, respectively) in combination with captafol, and iprodione in a single chromatographic run. Although iprodione is known to degrade to 3,5-dichloroaniline, since this metabolite is not a part of the residue definition, it was not included in the scope of this method. As the method demonstrates satisfactory selectivity, sensitivity, accuracy, precision, and robustness in a wide range of cereal matrices, it is recommended for regulatory testing of these compounds in cereals.

Improved analysis of captan, tetrahydrophthalimide, captafol, folpet, phthalimide, and iprodione in fruits and vegetables by liquid chromatography tandem mass spectrometry.

An improved liquid chromatography tandem mass spectrometry method is reported for the determination of residues of captan (+tetrahydrophthalimide), captafol, folpet (+phthalimide), and iprodione in fruits and vegetables. The optimized electrospray ionization parameters (high cone gas flow, and a low desolvation temperature) did not result in degradation of target compounds, rather they provided a significant advantage over the conventional GC-MS/MS methods, which lack sensitivity and repeatability. Strategies for minimizing losses in recovery of these compounds during sample preparation included cryogenic comminution, extraction with acidified ethyl acetate or acetonitrile, and dilution of the final extract with acidified water prior to LC-MS/MS analysis. The method performance complied with the SANTE/11813/2017 guidelines, with recoveries in the range of 70-120% at the LOQ of 0.01 mg/kg across the tested matrices at various pHs. The efficiency of the method was reflected in its precision (RSDs < 10%) for incurred residues.

Evaluation of corm origin and climatic conditions on saffron (Crocus sativus L.) yield and quality.

BACKGROUND: Crocus sativus L. is an autumn-flowering geophyte belonging to the Iridaceae family, known for the medicinal and coloring uses of the spice from its dried stigmas. It is cultivated in countries with different pedoclimatic conditions. This paper reports on a two-year research project carried out in the Basilicata region of southern Italy on the question of how to obtain the highest performance from saffron. It considers corms from three different geographical origins - 'Sardinia', 'Abruzzo' and 'Kozani' at three different cultivation sites, namely Castelgrande (40° 46' N, 15° 26' E, 781 m a.s.l.), Genzano di Lucania (40° 50' N, 16° 08' E, 344 m a.s.l.), and Villa d'Agri (40° 22' N, 15° 48' E, 638 m a.s.l.). RESULTS: The highest yields were obtained in the second year by the combination of 'Genzano di L. × Sardinia' and 'Castelgrande × Abruzzo' with 28.1 and 23.9 kg ha-1 of dried stigma, respectively. Saffron quality was determined according to ISO 3632, which classifies the samples into three categories depending on the crocin, picrocrocin, and safranal content. Results highlighted that the highest values for coloring (242.1) and bitterness (97.7) were achieved in Genzano di L. during 2013. Moreover, the crocins were correlated positively with stigma yield and air mean temperature but negatively with safranal. CONCLUSION: This study demonstrated, through principal component analysis (PCA), that the cultivation site with higher air temperature and without excessive rain during the flowering period generated the best stigma yield with high-quality traits. © 2019 Society of Chemical Industry.

The effects of geographical origin and virus infection on the saffron (Crocus sativus L.) quality.

Saffron is appreciated by its colour, taste, and aroma. To examine the effect of abiotic and biotic stress on these main properties, in the span of 2014-2016, saffron stigmas were collected from major different saffron cultivation areas of Iran and saffron quality was estimated. The quality of saffron was assessed by ultraviolet-visible spectroscopy following the ISO3632:2011 standard. However, the composition and concentration of crocetin esters, picrocrocin, safranal, and kaempferols, the most critical compounds determining the properties and quality of saffron can vary with the geographical origin and virus effects, being more accurate High-Performance Liquid Chromatography and Diode Array Detection (HPLC-DAD) methods were used to analyze saffron quality. Using HPLC-DAD we analyzed saffron plants grown at various conditions (considering altitude, temperature, and precipitation/rainfall) and in presence/absence of virus infections; we found that edaphoclimatic and cultivation conditions significantly determine the quality of the spice and the presence of virus modifies the content of its metabolites.

Influence of the storage temperature on volatile emission, carotenoid content and chilling injury development in Star Ruby red grapefruit.

Grapefruits are sensitive to develop chilling injury (CI) on the peel upon postharvest storage at low temperature. We investigated the influence of the storage at 2 and 12 °C on CI, carotenoids, and emission of volatiles by intact fruit. CI symptoms at 12 °C were restricted to green fruit peel sectors but at 2 °C the CI severity was higher and distributed through the whole fruit surface. Fruit peel coloration and carotenes content increased at 12 °C whereas experienced minor changes at 2 °C. At 2 °C the emission of total volatiles and specific monoterpenes, mainly limonene, but also linalool and α-terpineol was enhanced, while storage at 12 °C resulted in higher emission and diversity of cyclic sesquiterpenes and aliphatic esters. Results indicate a selective emission of volatiles by intact red grapefruit that appears to be a specific response to the storage temperature or to the cold-induced damage.

Chemical Composition and Biological Activity of Five Essential Oils from the Ecuadorian Amazon Rain Forest.

The chemical composition and biological activity of essential oils isolated from the leaves of Siparuna aspera, Siparuna macrotepala, Piper leticianum, Piper augustum and the rhizome of Hedychium coronarium were evaluated. These species are used medicinally in different ways by the Amazonian communities that live near the Kutukú mountain range. Chemical studies revealed that the main components for the two Siparuna species were germacrene D, bicyclogermacrene, α-pinene, δ-cadinene, δ-elemene, α-copaene and ß-caryophyllene; for the two Piper species ß-caryophyllene, germacrene D, α-(E,E)-farnesene, ß-elemene, bicyclogermacrene, δ-cadinene and for H. coronarium 1,8-cineole, ß-pinene, α-pinene and α-terpineol. The antioxidant activity of all essential oils was evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), photochemiluminescence (PCL) quantitative assays, and DPPH and ABTS bioautographic profiles, with different results for each of them. Antimicrobial activity studies were carried out on three yeasts, six Gram positive and four Gram negative bacteria, by means of the disc diffusion method. The essential oil of H. coronarium showed the most relevant results on L. grayi, K. oxytoca and S. mutans, P. augustum and P. leticianum on S. mutans. An antibacterial bioautographic test for H. coronarium was also carried out and highlighted the potential activity of terpinen-4-ol and 1,8-cineole.

Effect of Different Drying Methods on the Physicochemical Properties and Bioactive Components of Saffron Powder.

Saffron is the most expensive spice in the world; so, determining optimum conditions for its processing is crucial. The most important processing stage of saffron is drying of its stigma, which should be optimized and there are no reports on reactance-window (RW) of saffron. In this research, drying of saffron with traditional, RW, and oven driers and at three temperatures of 60, 70 and 80 °C, as well as room temperature (25 °C) were studied. Regarding process duration, RW drier with 200 µm Mylar membrane and oven drier were the best methods with average drying time of 25.28 and 22.28 min, respectively. As far as the concentration of bioactive ingredients, i.e., picrocrocin, safranal, and crocin, of saffron was concerned, RW drier with Pyrex glass was better than other driers, resulting in 112.83 [Formula: see text] of picrocrocin, 51.79 [Formula: see text] of safranal, and 274.76 [Formula: see text] of crocin. The panelist most favored those saffron samples dried by RW with 300 µm Mylar membrane.

Chemical composition, antimicrobial and antioxidant activities of the essential oils of three Uzbek Lamiaceae species.

The chemical composition of the essential oil from the aerial parts of three Lamiaceae species from Uzbekistan was investigated by GC-MS analysis. ß-Linalool (26.6%), α-terpineol (10.0%), coumarin (8.9%) and 4,5,7,7α-tetrahydro-4,4,7α-trimethyl-2(6H)-benzofuranone (5.4%) resulted as the main components of Ajuga turkestanica essential oil, while camphene (17.1%), 1,8-cineole (15.9%), ß-cymene (7.9%) and limonene (7.4%) in Phlomis regelii. The essential oil of Thymus seravschanicus was dominated by thymol (37.5%), phellandral (26.0%), τ-terpinene (6.6%) and ß-cymene (5.2%). The essential oils had considerable antimicrobial activity against different bacterial strains and fungi. Among the tested samples of essential oils, P. regelii essential oil has the significant antioxidant activity with IC50 value of 117.8 ± 8.02 µg/mL.

Solid phase microextraction and gas chromatography mass spectrometry analysis of Zingiber officinale and Curcuma longa.

SPME analysis of Zingiber officinale Roscoe and Curcuma longa L. were performed by using a DVB/CARB/PDMS fiber. The SPME analysis of Zingiber officinale showed that the main components found were camphene (7.27%), geranial (8.37%), α-zingiberene (14.50%), α-farnesene (9.14%), ß-bisabolene (6.52%), and ß-sesquiphellandrene (9.92%). The SPME analysis of Curcuma longa showed that main components were p-cymene (12.96%) and ar-turmerone (12.08%). Other components were ß-phellandrene (7.86%), terpinolene (6.97%), ar-curcumene (8.53%), α-zingiberene (8.46%), and ß-sesquiphellandrene (7.37%).