Applications of Solid-phase Microextraction in Food Analysis
Extraction and Microextraction Techniques for the...
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Review DOI:10.13179/canchemtrans.2014.02.02.0097
Extraction and Microextraction Techniques for the
Determination of Compounds from Saffron
Somayeh Heydari* and Gholam Hossein Haghayegh
Faculty of Agriculture, Higher Education Complex of Torbat-e Jam, Torbat-e Jam, Iran
*Corresponding Author, E-mail: [email protected] Tel/Fax:+98 528 2237012
Received: February 23, 2014 Revised: March 25, 2014 Accepted: March 27, 2014 Published: March 28, 2014
Abstract: Saffron is the most expensive spice used in industry, with different uses as drug, textile dye and
culinary adjunct. It is mainly valued as a food additive for tasting, flavoring and coloring. Thus,
understanding the composition of Saffron matrix has gained increased attention in recent years. Since
Saffron matrix is very complex, the extraction, separation and quantitation of these chemicals are
challenging. The present review article focuses on the extraction and microextraction techniques used in
the determination of compounds (such as crocetin esters, picrocrocin and safranal, polycyclic aromatic
hydrocarbons (PAHs) include naphthalene, fluorene, anthracene and phenanthrene, inorganic
contaminants, the volatile compounds include isophorone related compounds and lipophilic carotenoids,
etc) from saffron samples. The extraction techniques include: solvent based extraction technique, steam
distillation, ultrasound-assisted extraction, membrane processes, supercritical fluid extraction and solid
phase extraction. The microextraction techniques include: solid phase microextraction (SPME), stir-bar
sorptive extraction (SBSE), and liquid phase microextraction (LPME). In the present review article have
been formulated future trends in extraction and microextraction for the determination of compounds from
saffron.
Keywords: Extraction; Microextraction; Saffron; Food Additive; Spice
1. INTRODUCTION
Since ancient times saffron (Crocus sativus L. stigma) is being used as a spice for flavoring and
coloring food preparations, as a perfume, dye or ink. Its traditional medicinal use has been reported [1–6].
Advanced pharmacological studies have verified its antitumor effects [7], free radical scavenging
properties [8], and hypolipaemic effects [9]. It is useful in neurodegenerative disorders accompanying
memory impairment [10]. All allies of genus Crocus are diploid. However, C. sativus is an exception. It is
triploid in genetic makeup (2n × 3X= 24). Meiosis is highly erratic, resulting in unbalanced or sterile
gametes with no seed formation [11]. This affects saffron cultivation, resulting in its high cost (the world’s
most expensive spice). Due to its high cost, saffron is also prone to adulteration by different means [12–
14].
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Chemical composition analyses have revealed a saffron composition of approximately 10%
moisture, 12% protein, 5% fat, 5% minerals, 5% crude fibre, and 63% sugars including starch, reducing
sugars, pentosans, gums, pectin, and dextrins (% w/w). Trace amounts of riboflavin and thiamine vitamins
have also been identified in saffron [15]. Ranges of all chemical constituents can vary greatly due to
growing conditions and country of origin.
Various analytical studies have been conducted to characterize the large number of potential
biologically active compounds found within saffron. The four major bioactive compounds in saffron are
crocin (mono-glycosyl or di-glycosyl polyene esters), crocetin (a natural carotenoid dicarboxylic acid
precursor of crocin), picrocrocin (monoterpene glycoside precursor of safranal and product of zeaxanthin
degradation), and safranal, all contributing not only to the sensory profile of saffron (color, color, taste,
and aroma, respectively), but also to the health-promoting properties [15].
Saffron's name is derived from the Arab word for yellow, a name reflecting the high concentration
of carotenoid pigments present in the saffron flowers' stigmas which contribute most to the color profile of
this spice. Both lipophilic carotenoids and hydrophilic carotenoids have been identified in saffron [16].
The lipophilic carotenoids, lycopene, α-, and β- carotene, and zeaxanthin have been reported in trace
amounts [17]. Of the carotenoids, the hydrophilic crocins constitutes approximately 6 to16% of saffron's
total dry matter depending upon the variety, growing conditions, and processing methods [18]. Crocin 1
(or α-crocin), a digentiobioside, is the most abundant crocin with a high solubility being attributed to these
sugar moieties. Crocin, typically deep red in color, quickly dissolves in water to form an orange colored
solution thereby making crocin widely used as a natural food colorant. In addition to being an excellent
colorant, crocin also acts as an antioxidant by quenching free radicals, protecting cells and tissues against
oxidation [19-21].
The actual taste of saffron is derived primarily from picrocrocin which is the second most
abundant component (by weight), accounting for approximately 1% to 13% of saffron's dry matter [16].
Natural de-glycosylation of picrocrocin will yield another important chemical component, safranal, which
is mainly responsible for the aroma of saffron.
Dehydration is not only important to the preservation of saffron but is actually critical in the
release of safranal from picrocrocin via enzymatic activity, the reaction yielding D-glucose and safranal,
the latter being the volatile oil in saffron. The six major volatile compounds in saffron are safranal,
isophorone, 2,2,6-trimethyl-1,4- cyclohexanedione, 4-ketoisophorone, 2-hydroxy-4,4,6-trimethyl-2,5-
cyclohexadien-1-one as well as 2,6,6-trimethyl-1,4-cyclohexadiene- 1-carboxaldehyde [22] but more than
160 additional volatile components have been identified [23]. Of these, safranal represents approximately
30 to 70% of essential oil and 0.001 to 0.006% of dry matter [22, 23]. Besides its typical spicy aromatic
note, safranal has also been shown to have high antioxidant potential [19, 24] as well as cytotoxicity
towards certain cancer cells in vitro [25].
The present standard of measurement of saffron quality and composition is ISO-3632 (2003) [26].
Other reported methods include thin layer chromatography [27, 28], gas chromatography (GC) [28, 29],
LC [30-34], fourier transform near infrared spectroscopy (FT-NIR) [35], thermal desorption gas
chromatography (TD-GC) [36, 37] and analysis of supercritical CO2 saffron extracts by GC and LC [38].
For the quality control of natural saffron, the Corradi et al. (1979) [39] method, and its modified version
(SOIVRE) [40] have been reported. Hydrolysis of picrocrocin by C-glucosidase enzyme, immobilized
onto nylon-hydrazide, in a continuous packed-bed reactor for LC quantification of total safranal in
commercial saffron samples [41] has also been attempted. Vibrational spectroscopic techniques (near-
infrared, NIR; mid-infrared, MIR), in combination with multivariate data analysis, are an alternative
option that does not require complex sample preparation and the spectral acquisition is fast. NIR
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spectroscopy has already found application in quality control and classification of saffron [42]. Mid-
infrared spectroscopy combined with multivariate analysis has recently been applied for the discrimination
of 250 saffron samples from Greece (40 samples), Iran (87 samples), Italy (60 samples) and Spain (63
samples) [43].
In order to obtain accurate information, the sample is prepared well which is the most challenging
step. In a typical analytical method, it is estimated that approximately 61% the time is spent on sample
processing (i.e. sample preparation) itself, and about 30% of the error relating to the analytical results is
due to sample preparation (Fig. 1) [44]. The sample preparation process generally consists of extraction,
cleanup, and concentration. For saffron samples, conventional extraction methods include solvent based
extraction technique, steam distillation (SD), ultrasonic extraction (USE), membrane processes (MP),
supercritical fluid extraction (SFE) and solid phase extraction (SPE). Table 1 gives a description of each
technique and lists the advantages and disadvantages of them. The whole process is time-consuming,
tedious, demanding a large amount of sample requiring, large volumes solvents which are hazardous in
nature. In recent years, the microextraction methods have attracted significant attention as these
techniques have high sensitivity, simple to use, with short sample pretreatment time and a high enrichment
factor, with low solvent usage, or somethimes solvent-free, and amenable to automation. Therefore, the
microextraction techniques become an attractive tool in the determination of compounds from saffron
matrixes. In this paper, the microextraction techniques used for the determination of compounds from
saffron samples are divided into two main categories: sorbent-based microextraction– solid phase
microextraction and stir-bar sorptive extraction, and solvent-based microextraction– dispersive liquid
liquid microextraction. Table 2 gives a description of each technique and lists the advantages and
disadvantages of them.
Figure 1. Proportions of time consumption and analytical error in chromatographic analysis. (A) The time
consumption in chromatographic analysis. (B) The error source in the analytical results of
chromatographic analysis. Chart is taken from [44].
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Table 1. Comparison of various extraction techniques used in the analysis of saffron metabolites [45].
Designation: SD – Steam distillation; USE – ultrasonic extraction; MP- Membrane processes; SFE – supercritical fluid extraction; SPE- solid
phase extraction
Table 2. Comparison of microextraction techniques used in the analysis of saffron metabolites [44].
SPME SBSE DLLME
Operation- Handling Easy Easy Easy
Enrichment factor High High High
Extraction time (min) 5−120 30−240 <1
Cost Moderate Moderate Low
Potential for automation High Moderate Low
Advantages Solvent- free, Great efficiency in preconcentration
Great efficiency in
preconcentration
High extraction efficiency,
Non-dependence on a commercial supplier
Disadvantages Dependence on a
commercial supplier,
Low robustness
Long equilibration,
Not suitable for polar compounds
Only suitable for liquid matrix sample,
Limitation in the choice of the proper solvent
Abbreviations: SPME, solid phase microextraction; SBSE, stir-bar sorptive extraction; DLLME, dispersive liquid liquid microextraction
The present review article focuses on the extraction and microextraction techniques used for the
determination of compounds from saffron samples (such as crocetin esters, picrocrocin and safranal,
polycyclic aromatic hydrocarbons (PAHs) include naphthalene, fluorene, anthracene and phenanthrene,
inorganic contaminants, the volatile compounds include isophorone related compounds and lipophilic
carotenoids).
Extraction Solvent based
extraction
SD USE MP SFE SPE
Cost Low Medium Low Low High Medium
Extraction time 6–48 h 1-12 h ˂30 min ˂ 60 min ˂ 60 min ˂ 30 min
Solvent use, mL 100–600 - ˂ 50 ˂ 10 ˂ 10 ˂ 5
Advantages Simplicity,
high recovery Quality Control,
Wide
Application to
immiscible
substances
Possibility of
extraction of many
samples
at once
Low solvent
consumption,
simplicity, high
selectivity
Low solvent volumes,
shortened extraction
time, ease of automation,
small sample size
needed, possibility of on-
line coupling with the
separation and
determination
techniques, high purity
and small volume of the
extract, high selectivity.
High selectivity, Wide
variety of sample
matrices, High
recoveries, good
reproducibility, ease of
automation, Low solvent
volumes
Disadvantages Long
extraction
time, large
consumption
of solvents
Substantial
energy
consumption,
Higher
Equipment and
Operating Cost,
thermal
decomposition of
substances
The need for
separation of the
extract from the
sample following
the extraction
Slowness, low
efficiency,
susceptibility to
membrane fouling by
solid impurities in
the donor phase with
sizes comparable to
the membrane pores.
High cost Perceived difficulty to
master its usage, More
steps and MD time
required, Greater cost per
sample
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2. Extraction Techniques for the Determination of Compounds from Saffron Samples
Numerous methods have been proposed for quality determination of saffron involving preparation
of saffron extract followed by measurement the quantity of saffron [46-48]. Preparation of extract has a
crucial impact on the accuracy of the results. It has been reported that the type of solvent, time and method
of extraction not only considerably affect the diffusion rate of the components across the cell wall but also
their stability [49]. Crocins have been shown to undergo degradation during prolonged extraction time in
aqueous media of high water activity. It has also been demonstrated that the use of alcohol or water-
alcohol results in higher extraction rate compared to water [49]. Accordingly, the methods of extraction
are being steadily revised and modified techniques proposed.
2.1 Solvent Based Extraction Technique
Aqueous methanol, ethanol, or water is commonly used for the extraction of many bioactive
constituents. The extraction and purification of crocins is well described in the literature [50-52]. After
defatting with diethyl ether, stigmas are commonly extracted 2 or 3 times using 70 – 90% methanol or
ethanol (~10 mL solvent/g saffron). Solvents containing extract are pooled, evaporated to dryness, and
then purified using silica gel column chromatography with ethanol–ethyl acetate–water (~6:3:1) as the
mobile phase. Crocin and crocetin esters are eluted separately [51].
Amin et al. (2012) evaluated therapeutic potential of systemically administered ethanolic and
aqueous extracts of saffron as well as its bioactive ingredients, safranal and crocin, in chronic constriction
injury (CCI)-induced neuropathic pain in rats [53]. The von Frey filaments, acetone drop, and radiant heat
test were performed to assess the degree of mechanical allodynia, thermal allodynia and thermal
hyperalgesia respectively, at different time intervals, i.e., one day before surgery and 3, 5, 7 and 10 days
post surgery. The results of this study suggested that ethanolic and aqueous extracts of saffron as well as
safranal could be useful in treatment of different kinds of neuropathic pains and as an adjuvant to
conventional medicines.
Amino acids are important in human nutrition and also influence the sensory traits of products.
Campo et al developed a rapid method for the extraction of free amino acids and ammonium ion from
saffron, thus permitting the differentiation of saffron samples by origin [54]. The best results for the free
amino acids and ammonium ion extraction were obtained using 0.1 N HCl during 60 min. Subsequently,
these compounds were derivatised using diethyl ethoxymethylenemalonate (DEEMM) and analysed by
HPLC. Using this method, 20 saffron samples from different countries (Spain, Italy, Greece and Iran) were
analysed. Alanine, proline and aspartic acid were the major amino acids in all the samples. Greek and
Iranian saffron presented the highest total free amino acid content, 0.50 ± 0.08 mg/100 mg and 0.55 ± 0.07
mg/100 mg, respectively.
The potential use of aqueous two-phase system (ATPS) for the extraction of crocins from C.
sativus stigmas was evaluated by Montalvo-Hernandez et al. (2012) [55]. The partitioning behavior of
crocins in different types of ATPS (polymer–polymer, polymer–salt, alcohol–salt and ionic liquid–salt)
was evaluated. Ethanol–potassium phosphate ATPS were selected based on their high top phase recovery
yield and low cost of system constituents. The results reported herein demonstrate the potential application
of ATPS, particularly ethanol–potassium phosphate systems, for the recovery of crocins from C. sativus
stigmas.
Serrano-Diaz et al. (2014) extracted floral bio-residues (FB) (tepals, stamens and styles) from
saffron spice [56]. Extracts of these bio-residues in water (W1), water: HCl (100:1, v/v) (W2), ethanol
(E3), ethanol: HCl (100:1, v/v) (E4), dichloromethane (D5) and hexane (H6) were prepared. Their
composition in flavonols and anthocyanins, and their effect on cell viability were determined. All extracts
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studied are not cytotoxic at concentrations lower than 900 μg/ml and W1 is proposed as the optimal for
food applications due to its greater contribution of phenolic compounds.
The influence of various steps of the extract preparation procedure on the absorbance readings
used for the commercial evaluation of the colouring strength of saffron was described by Orfanou et al.
(1996) [57]. The study was prompted by the diversity of extraction protocols found in the literature.
Extraction period, stage of the extract filtration, filter type and extraction solvents influenced the size and
the repeatability of the absorbance measurements. Long extraction periods (24 h) caused loss of the
colouring strength. Filtration through narrow pore filters yielded extracts with higher colouring strength.
The latter was significantly increased when the extract was filtered prior to final dilution. Alcoholic
extracts presented higher colouring strength when compared to aqueous extracts.
Soxhlet extraction is one of the oldest techniques for isolating metabolites from natural material.
The technique is used for the isolation and enrichment of analytes of medium and low volatility and
thermal stability (Fig 2) [58]. Picrocrocin can be effectively isolated from saffron stigma by successive,
exhaustive Soxhlet extraction using light petroleum, diethyl ether, and methanol to obtain three fractions
[32]. The diethyl ether phase containing picrocrocin and lipids is evaporated to dryness, defatted using
Soxhlet for picrocrocin purification, and then dissolved in methanol for filtration. The filtrate is then
analyzed using HPLC. Tarantilis and colleagues effectively analyzed picrocrocin using an HPLC
LiChroCART 125-4 Superspher 100 RP-18 column and 20–100% ACN in water linear gradient mobile
phase. Feizzadeh et al. (2008) evaluated the cytotoxic effect of aqueous extract of saffron on human
transitional cell carcinoma (TCC) and mouse non-neoplastic fibroblast cell lines [59]. Aqueous extract
was prepared with 15 g of its ground petal stigma and 400 mL of distilled water in a Soxhlet extractor for
18 hours. The prepared extract was concentrated to 100 mL with a rotatory evaporator in low pressure and
filtered through a 0.2-mm filter to be sterilized. The study showed that saffron aqueous extract has an in
vitro inhibitory effect on the proliferation of human TCC and mouse L929 cells which is dose dependent.
Also, the cytotoxity effect of the whole saffron (from the quantitative and morphologic point of
view) and the amount of NO production in HepG2 and Hep2 cells investigated by Aqueous extract in a
Soxhlet extractor [60]. Samarghandian et al. (2013) investigated the potential of saffron to induce
cytotoxic and apoptotic effects in lung cancer cells (A549) and examined the caspase-dependent pathways
activation of saffron-induced apoptosis against the A549 cells by aqueous extract with distilled water in a
Soxhlet extractor [61]. It has been reported that extracts of Crocus sativus prevent from scopolamine and
ethanol-induced memory impairment in Morris water maze and passive avoidance tests. It also protects
against ethanol-induced inhibition of hippocampal long-term potentiation (LTP) [62, 63]. In addition, it
has been reported that crocin counteracts the ethanol inhibition of NMDA receptor-mediated responses in
rat hippocampal neurons [64]. It has been also shown that saffron attenuates cerebral ischemia [65] and
reduces the extracellular hippocampal levels of glutamate and aspartate [66]. Saffron extracts or its active
constituents have other activities on the central nervous system including antidepressant [67, 68], and
anticonvulsant [69, 70]. Some actions of saffron on central nervous system have been attributed to its
effects on opioid system [69]. It has been also demonstrated that aqueous and ethanolic extracts of Crocus
sativus stigma and its constituent crocin can suppress morphine withdrawal syndrome [71].
2.2 Steam Distillation
Steam distillation is a valuable technique, allowing isolation from plants of volatile components, such as
the essential oils, some amines and organic acids, as well as other, relatively volatile compounds, insoluble
in water (Fig. 3) [72]. Among others, steam distillation has been used to isolate the essential-oil fraction
from either plant material or a previously prepared extract in a low-boiling solvent (petroleum ether or
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diethyl ether) [73]. Tarantilis et al. (1997) tested three distillation methods (steam distillation, micro-
simultaneous steam distillation–solvent extraction, vacuum head space distillation) for efficiency to
extract representative volatile profiles of saffron [74]. Also steam distillation was used to isolate the
volatile essential oil constituents responsible for aroma of saffron after γ-irradiation [75].
Figure 2. Experimental Soxhlet extraction apparatus. Reproduced with permission from ref [58],
Copyright 2006 Elsevier.
Figure 3. A schematic representation of a Steam distillation extractor [72].
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2.3 Ultrasound-assisted Extraction (UAE)
UAE involves application of high-intensity, high-frequency sound waves and their interaction with
materials. UAE is a potentially useful technology as it does not require complex instruments and is
relatively low-cost (Fig.4) [76]. It can be used both on small and large scale [77]. UAE involves ultrasonic
effects of acoustic cavitations. Under ultrasonic action solid and liquid particles are vibrated and
accelerated and, because of that solute quickly diffuses out from solid phase to solvent [78]. Several
probable mechanisms for ultrasonic enhancement of extraction, such as cell disruption, improved
penetration, and enhanced swelling, capillary effect, and hydration process have been proposed [79]. If the
intensity of ultrasound is increased in a liquid, then it reaches at a point at which the intramolecular forces
are not able to hold the molecular structure intact, so it breaks down and bubbles are created, this process
is called cavitation [80]. Collapse of bubbles can produce physical, chemical and mechanical effects which
result in the disruption of biological membranes to facilitate the release of extractable compounds and
enhance penetration of solvent into cellular materials and improve mass transfer [78, 81]. The beneficial
effects of sound waves on extraction are attributed to the formation and asymmetrical collapse of
microcavities in the vicinity of cell walls leading to the generation of microjets rupturing the cells. The
pulsation of bubbles is thought to cause acoustic streaming which improves mass transfer rate by
preventing the solvent layer surrounding the plant tissue from getting saturated and hence enhancement of
convection [82]. Skin of external glands of plant cell wall is very thin and can be easily destroyed by
sonication, and this facilitates release of essential oil contents into the extraction solvent, thus resulting in
reduced extraction time and increased extraction efficiency [83].
The active compounds of saffron were extracted using high power ultrasound at a constant
frequency of 30 kHz by Kadkhodaee et al. (2006) [82]. The effect of acoustic intensity, time and mode of
sonication on the extraction yield of three major constituents of saffron was investigated at 20 °C. The
efficiency of the process was compared with that of cold water extraction method proposed by ISO. The
results showed that ultrasound largely improved the extraction rate and incredibly reduced the process
time. It was also found that the use of pulsed ultrasound with short pulse intervals was more efficient than
continuous sonication.
The volatile components of Iranian saffron were extracted using ultrasonic solvent extraction
(USE) technique and then were separated and detected by gas chromatography–mass spectrometry (GC–
MS) by Jalali-Heravi et al. (2009) [84]. Variables affecting the extraction procedure were screened by
using a 25−1
fractional factorial design and among them; sample amount, solvent volume, solvent ratio and
extraction time were optimized by applying a rotatable central composite design (CCD). Forty
constituents were identified for Iranian saffron by GC–MS representing 90% of the total peak area. Some
new compounds were identified for the first time in saffron.
An ultrasound assisted extraction method is proposed for the recovery of bioactive glycosides (i.e.
crocins and picrocrocin) from Crocus sativus L. dry stigmas using aqueous methanol [85]. Response
surface methodology (RSM) was employed to optimize the extraction parameters. Ultrasound assisted
extraction speeded up further recovery of precious apocarotenoids. These findings for extraction
conditions are useful for both industrial and analytical applications and should be considered in a
forthcoming revision of the ISO 3632-2 technical standard.
Ultrasound-assisted extraction 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
by Maggi et al. (2011) [86]. Intra-laboratory validation of the optimised conditions and analysis by UV–
Vis spectrophotometry showed satisfactory results in linearity, repeatability, intermediate precision and
recovery.
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Figure 4. Schematic Representation of UAE Setup . Reproduced with permission from ref [76],
Copyright 2009 Elsevier.
2.4 Membrane Processes
Membrane techniques are finding ever-increasing application in the isolation of groups of
components from the plant extracts. A membrane is a selective barrier between two phases. The phase
from which mass transport takes place is called the donor phase while the receiving phase is called the
acceptor (permeation) phase. The general principle of separation of liquid mixture components is depicted
in Fig. 5 [87]. Membrane separation processes operate without heating and therefore use less energy than
conventional thermal separation processes such as distillation.
Membrane separation has been applied, for example, in the selective and tunable extraction as well
as the fingerprinting of saffron solutes [88], where the emulsion liquid membrane with span 80 as
surfactant and n-decane as membrane phase were used. Emulsion liquid membrane (ELM), invented by
Li. (1968) [89], is known as one of the most promising separation methods for trace extraction of metal
contaminants [90-92] and molecular species [93,94] owing to the high mass transfer rate, high selectively,
low solvent inventory and low equipment cost.
Figure 5. Separation of mixtures using membrane techniques. Reproduced with permission from ref [87],
Copyright 2007 Elsevier.
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The separation of crocin and picrocrocin from saffron cell culture broth was investigated by
macroporous resin adsorption methods [95]. Results showed that HPD-100A resin was the best resin to
separate crocin and picrocrocin from saffron cell culture broth among the investigated ones and
macroporous resin adsorption is feasible and holds promise for separating crocin and picrocrocin from
saffron cell culture broth.
2.5 Supercritical Fluid Extraction
SFE can be used to extract certain compounds from plants at temperature near to ambient, thus
preventing the substance from incurring in thermal denaturation. SFE is an old technique of solvent
extraction but its commercial application happened slowly due to the sophisticated and expensive high
pressure equipment and technology required [96]. SFE is currently a well established method for
extraction and separation because its design and operating criteria are now fully understood [97]. The
favourable transport properties of fluids near their critical points allow deeper penetration into solid plant
matrix and more efficient and faster extraction than with conventional organic solvents. The extraction is
carried out in high-pressure equipment in batch or continuous manner. In both cases, the supercritical
solvent is put in contact with the material from which a desirable product is to be separated. Generally
cylindrical extraction vessels are used for sample preparation [98]. In batch processing solid is placed into
extraction vessel and the supercritical solvent is fed in until the target extraction conditions are reached.
And in semi batch processing the supercritical solvent is fed continuously through a high pressure pump at
a fixed flow rate, to precipitate solute from supercritical solution one or more separation stages are used. A
supercritical fluid extraction (SFE) system is shown in Fig.6 [99]. Supercritical fluid technology is now
recognized as an effective analytical technique with efficiency comparable to existing chemical analysis
methods. SFE is favourably applicable for the qualitative and quantitative identification of constituents of
natural products, including heat-labile compounds [100].
The supercritical fluid extraction (SFE) method described by Lozano and colleagues extracted
safranal from 200 mg powdered saffron by holding supercritical fluid in the extraction chamber under
pressure with sample for 5 min before circulating supercritical fluid through the extraction unit. Safranal
was collected in 3 mL methanol for HPLC analysis, which revealed mainly safranal and a small amount of
4– hydroxy-2,6,6-trimethyl-1-carboxaldehyde-1-cyclohexane, a safranal precursor. The SFE method was
developed as a non-destructive method for isolating safranal as the major volatile component in the extract
[101].
A supercritical carbon dioxide extraction method to obtain selectively volatile compounds of
saffron without sample destruction has been developed. A decrease in supercritical fluid density was
shown to be a critical parameter for enhancing the extraction power of carbon dioxide. For all the assay
conditions, the extracts mainly contained safranal and HTCC, as demonstrated by gas chromatography and
high-performance liquid chromatography analyses [101].
Zougagh et al. (2006) developed a procedure allowing hydrolysis reactions to be conducted in a
dynamic supercritical-CO2 medium for quantifying total safranal (viz. free safranal present in the sample +
safranal resulting from picrocrocin hydrolysis), which are the main component of the essential oil and
responsible for the characteristic aroma of saffron [102]. The results obtained were compared with the
“safranal value” total index, which is widely used as a quality measure of saffron products. The
comparison revealed that the proposed method provides useful information not contained in the safranal
value, based on the fact that, some samples with a high “safranal index” contain low concentrations of
safranal. The proposed method is very useful for quality control in commercial saffron samples.
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Figure 6. Schematic diagram of a process-scale supercritical fluid extraction system [99]. Reproduced
with permission from ref [99], Copyright 2006 Elsevier.
2.6 SPE
Sample preparation often includes an extract enrichment step, wherein the analyte concentration is
increased above the determination limit of the final determination technique. Several enrichment
techniques are common, including gas, liquid and solid-phase extraction. One of the primary
considerations is the need for reduction of the amount of organic solvent used. This has resulted in
extensive use of solid-phase extraction (SPE). SPE involves adsorption of sample components on the
surface of a solid sorbent (aminopropyl or octadecyl stationary phases, bonded to silica gel, etc.), followed
by elution with a selected solvent. SPE is carried out in glass or polypropylene columns or on extraction
disks (Fig. 7) [103]. The steps of the solid phase extraction process are shown in Fig. 8 [104]. First, the
cartridge is equilibrated or conditioned with a solvent to wet the sorbent. Then the loading solution
containing the analyte is percolated through the solid phase. Ideally, the analyte and some impurities are
retained on the sorbent. The sorbent is then washed to remove impurities. The analyte is collected during
this elution step. An important advantage of SPE compared to liquid/ liquid extraction is a significant
reduction in volume of solvent used [105]. A variety of sorbents available on the market allows not only
the isolation of analytes but also the removal of interferences. Dry and wet modes of extraction were
compared and found to be equivalent. Due to its simplicity, SPE is finding ever-increasing fields of
application, including the isolation of proteins and peptides [106,107]. Nonpolar stationary phases are
used [107] and the technique has been automated and coupled to HPLC or electrophoresis [108,109].
The application of solid phase extraction (SPE) in the determination of picrocrocin by UV–Vis
spectrophotometry has been studied in order to develop a rapid and low cost method that can be used in
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the industry for routine quality control of saffron spice [110]. The results indicated the selectivity,
trueness, linearity, precision, good recovery (about 90%) and sensitivity (LOD = 0.30 mg L−1
; LOQ = 0.63
mg L−1
). The method also proved valid for overcoming the limitations of 257 nm due to crocetin
esters in the determination of picrocrocin.
Chryssanthi et al. (2011) developed an isocratic reversed-phase liquid chromatographic method for
the determination of crocetin levels in plasma of healthy human volunteers before and after consumption
of one cup of saffron tea and answered issues concerning the route and way of saffron administration, the
absorption and metabolism of saffron carotenoids in humans [111]. Samples were pre-treated by solid
phase extraction and were chromatographed with a mobile phase consisting of methanol–water–
trifluoroacetic acid
If the interfering substances and target analytes are transferred together in aqueous solution from
saffron matrix, which reduces the enrichment factor of the solid sorbent on the target chemicals will be
reduced which finally influences the sensitivity of SPE. To separate and co-extract the target chemicals,
and to improve the enrichment factor, novel solid sorbents such as molecularly imprinted polymer have
been developed and applied for the determination of crocin [112]. The new molecularly imprinted polymer
was prepared using gentiobiose (a glycoside moiety in crocin structure) as a template. Crocin binding to
gentiobiose imprinted polymer (Gent-MIP) was studied in comparison with a blank nonimprinted polymer
in aqueous media. Affinity of the Gent-MIP for the crocin was more than the nonimprinted polymer at all
concentrations. The Gent-MIP was then used as the sorbent in an SPE method for isolation and
purification of crocin from methanolic extract of saffron stigmas. The recovery of crocin, safranal and
picrocrocin was determined in washing and elution steps. The Gent-MIP had significantly higher affinity
for crocin than other compounds and enabled selective extraction of crocin with a high recovery (84%)
from a complex mixture. The results demonstrated the possibility of using a part of a big molecule in
preparing a molecularly imprinted polymer with a good selectivity for the main structure.
Figure 7. Typical SPE Tube and Disk [103].
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Figure 8. Solid Phase Extraction steps [104]
3. Microextraction Techniques for the Determination of Compounds from Saffron Samples
3.1 Sorbent-based Microextraction Technique
3.1.1. Solid Phase Microextraction (SPME)
Based on the sorption (adsorption-absorption) theory, solid phase microextraction (SPME) was
developed by Pawliszyn’s group in 1990 as a solvent-free technique [113]. Although it is an equilibrium
(non-exhaustive) technique, SPME was rapidly accepted as a simple, miniaturized, and green technique,
which combines sampling, extraction, concentration, cleanup and sample introduction in a single step.
Based on its advantages, SPME quickly became one of the 204 most widely used techniques in various
fields of analytical chemistry, including environmental and biological applications [113]. Analyte
enrichment by SPME involves two steps. In the first step, a fiber, coated with an adsorbent or stationary
liquid, is exposed to a liquid sample or the headspace above a sample and the analytes are sorbed on the
fiber. In the second step, the fiber is introduced into the injection chamber of a gas chromatograph, where
it is subjected to a high temperature, or it is introduced into the injector of a liquid chromatograph. The
released analytes are swept into the chromatographic column. Based on the position of the fiber coating in
the extraction system, the extraction methods are classified typically into two modes: direct immersion
(DI, the coating is completely immersed in the sample matrix) and headspace (HS, the coating is exposed
to the gas phase above the sample matrix) modes. Solid phase microextraction device and two modes of
operation are shown in Fig. 9 [114]. Solid-phase micro-extraction (SPME) is a sample preparation
technique best suited for gas chromatography, Although SPME has been successfully combined with
HPLC, this requires relatively complicated procedures and additional devices. Therefore, it can be
anticipated that the technique will be used mostly with gas chromatography. SPME–GC–MS analysis on
four samples of saffron from different areas of italy and from iran were performed by Auria et al. (2004)
[115]. In this work found 18 compounds never discovered in saffron. The analyses show that saffrons from
different cultivation sites have some peculiarities due to the presence of some unusual components.
Manzo et al. (2013) represent the first investigation of the quality of saffron product in alpine
areas evaluated by spectrophotometric analysis, and by solid-phase microextraction (SPME) followed by
gas chromatographic analysis combined with mass spectrometry (GC/MS) [116]. The samples, produced
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in two consecutive years (2012-2013), come from some areas of Valle Camonica (BS), and high Val
Trompia (BS) located at an altitude between 720 and 1200 m a.s.l. The analysis has showed a correlation
between the concentration of Crocin and the increasing of altitude. This correlation is a peculiarity of the
carotenoids; actually an increase of their concentration is related to the increasing of UV-radiation and
altitude. The SPME-GC/MS analysis evidences some differences about the aromatic profile of analyzed
samples, mainly in safranal concentration. Results show a correlation between the increase of the drying
temperature of the stigmas and safranal concentration.The effect of storage on the composition of saffron
aroma was studied by solid-phase microextraction–gas chromatography– mass spectrometry [117]. The
most important changes are in the presence of alcohols and aldehydes and oxidation products of the major
terpenoids components. Furthermore, the presence of safranal changes during the time, increases during 3
years, then decreases after 5 years.
Quantitative Structure-Retention Relationship (QSRR) studies were performed for predicting the
retention times of 43 constituents of saffron aroma, which were analyzed by solid-phase micro-extraction
gas chromatography–mass spectrometry (SPME-GC–MS) [118]. The chemical descriptors were calculated
from the molecular structures of the constituents of saffron aroma alone, and the linear and non-linear
QSRR models were constructed using the Best Multi-Linear Regression (BMLR) and Projection Pursuit
Regression (PPR) methods. The predicted results of the two approaches were in agreement with the
experimental data. The proposed models could also identify and provide some insights into structural
features that may play a role on the retention behaviors of the constituents of saffron aroma in the SPME-
GC–MS system. This study affords a simple but efficient approach for studying the retention behaviors of
other similar plants and herbs.
As the extraction efficiency of SPME is strongly dependent on the distribution constant of the
target chemical between the fiber coating and sample matrix, the fiber coatings is the most important and
key factor in SPME. Recently, a variety of commercially available fiber coatings have been used including
non-polar polydimethylsiloxane (PDMS), semi-polar polydimethyl siloxane-divinylbenzene (PDMS-
DVB), polar polyacrylate (PA), etc. These commercial fiber coatings have also been widely used to
determination volatile and semivolatile compounds (VOCs includes esters, alcohols, aldehydes,
hydrocarbons, ketones, terpenes, sesquiterpene, phenols, acids, etc [119-136] and pesticides includes
nicotinoids, carbamates, and fungicides [137]) from various samples including plant matrices (fruits [119-
125], vegetables [126-130,137], medicinal plants [131-136]) in the last five years.
To improve the stability of fibers, Sarafraz-Yazdi et al. (2012) [138] synthesized PDMS fiber, PEG
fiber and PEG/CNTs fiber by a sol-gel approach, for the determination of some polycyclic aromatic
hydrocarbons in aqueous saffron sample by direct immersion solid phase microextraction (SPME) and gas
chromatography. Among the three kinds of the fibers, the sol–gel-derived PEG/CNTs fiber has the best
affinity for PAHs due the special properties of MWCNTs.
3.1.2 Stir-bar Sorptive Extraction (SBSE)
Stir-bar sorptive extraction (SBSE) was developed by Baltussen et al. (1999) [139] in 1999 based
upon sorptive extraction, as well as SPME, SBSE is solventless sample preparation technique. The surface
area and amount of coating in SBSE are greater than those in the SPME fiber coating. Therefore, the
extraction efficiency was improved compared to SPME, and this is its main advantage [140]. However,
the extraction and desorption time of SBSE (more than 60 min) is usually longer than SPME, as the
analytes are distributed in the large volume of coating. After completing the extraction process, the stir-bar
is rinsed with distilled water and dried, and the sample is directly subjected to thermal desorption
chromatography analysis. The recovery of analytes ranges from 70% to 130%.
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Figure 9. Solid phase microextraction device and two modes of operation: (a) headspace, (b) direct
immersion . Reproduced with permission from ref [114], Copyright 2003 Royal Society of Chemistry.
A method for the simultaneous determination of 46 semi-volatile organic contaminants and
pollutants in saffron has been developed for the first time using a stir bar sorptive extraction technique and
thermal desorption in combination with gas chromatography–ion trap tandem mass spectrometry [141].
The analytical method proposed was easy, rapid and sensitive and showed good linearity, accuracy,
repeatability and reproducibility over the concentration range tested. Moreover, the correlation coefficients
were higher than 0.98 for all target compounds and detection limits were lower than 1 μg/kg except for
simazine.
For the first time saffron employed as environmental bioindicator to characterize the presence of a
complex polycyclic aromatic hydrocarbons (PAHs) mixture by Maggi et al. (2009) [142]. The objective of
this study was to provide quantitative information regarding PAHs contamination in saffron in order to
verify the possibility to use this spice as environmental biomarker and to determine the bioaccumulation
of these compounds. 27 saffron samples coming from different countries were analyzed by stir bar
sorptive extraction technique in combination with gas chromatography-ion trap tandem mass
spectrometry. The data show that the samples contained different levels of contaminants but some of them
have higher concentrations than the maximum residue limits (MRLs) of pollutants established by the
European Commission for spices.
3. 2. Solvent-based Microextraction Technique
Based on the distribution principle (i.e., distribution of analytes between the microextraction phase and
sample phase containing the analytes), micro-LLE extraction techniques, such as the liquid phase
microextraction technique (LPME) have been introduced [143-145]. Similar to SPME, LPME also
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integrates sampling, extraction, cleanup and concentration. LPME has some merits including lower cost,
simplicity, reduction in organic solvent use, and flexibility in selection of the extraction phase. Due to
these advantages, LPME techniques have been widely used in various samples, such as environmental
samples (wastewater, sewage water, river and tap water, and soil), and biological samples (urine and
blood), etc. Several review papers on LPME [146-151] have been published in the past five years.
3.2.1. Dispersive Liquid-Liquid Microextraction (DLLME)
Based on the LLE principle, dispersive liquid liquid microextraction (DLLME) was first
introduced in 2006 by Rezaee et al. (2006) [152]. In this technique, a mixture of the extraction phase (low
solubility in water, density different to that of water, and has the ability to extract target analytes) and the
dispersive solvent (miscible with both water and the extraction phase, such as acetonitrile, acetone, and
methanol) is rapidly injected using a syringe into the aqueous sample, and tiny droplets are formed in the
aqueous sample, which provides a vast interphase contact and accelerates the mass transfer of analytes
(Fig. 9) [153]. The major benefits of DLLME are that it is fast (extraction time less than 5 min), and
inexpensive, and more importantly DLLME can achieve quantitative extraction. Based on the extraction
principle, DLLME is only suitable for liquid matrix samples. Thus, DLLME is primarily applied in
aqueous samples. For solid matrices such as plant samples, the target compounds must be transferred to an
aqueous phase from the matrix for DLLME analysis.
Sereshti et al applied a combined extraction method of ultrasound-assisted extraction (UAE) in
conjunction with dispersive liquid–liquid microextraction (DLLME) to isolation and enrichment of saffron
volatiles [154]. The extracted components of the saffron were separated and determined by gas
chromatography–mass spectrometry (GC–MS) technique. The mixture of methanol/acetonitrile was
chosen for the extraction of the compounds and chloroform was used at the preconcentration stage. The
important parameters, such as composition of extraction solvent, volume of preconcentration solvent,
ultrasonic applying time, and salt concentration were optimised by using a half-fraction factorial central
composite design (CCD).
Solidification of floating organic drop microextraction (SFODME) is the new microextraction
technique introduced in 2007 [155]. In SFODME method a droplet of an immiscible solvent with a
melting point of 10–30 ◦C is floated in the surface of an aqueous sample containing the analytes. The
mixture is agitated to maximize contact area between the two solutions. The sample vial is then placed in
an ice bath to solidify the droplet which is easily removed and allowed to melt for determination of
analyte. This method had been used for the extraction of organic compounds [155] and metal ions [156–
158] from water samples. In 2008, Leong and Huang [159] reported a novel variation of SFODME called
DLLME-SFO; this method is based on the principle of DLLME and SODME, i.e. instead of maintaining
one droplet of the extraction solvent in the sample, a dispersion of fine droplet is produced by injection of
a mixed solution of the extraction and dispersive solvent into the sample. This produces a vast contact area
between the extraction solvent and the sample which resulted in faster mass transfer and shorter extraction
time. Like SFODME, the DLLME-SFO has the advantages of speed, simplicity, high efficiency, low cost,
simple extraction apparatus and consumption of very small amount of low-toxic organic solvent. In
addition, the extraction time of DLLME-SFO is even shorter than SFODME. Recently, a liquid–liquid
microextraction method based on solidification of floating organic drop (DLLME-SFO) was successfully
used for determination of cadmium ions in saffron samples [160].
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Figure 10. Different steps in dispersive liquid-liquid microextraction . Reproduced with permission from
ref [153], Copyright 2007 Elsevier.
4. Future Trends
4.1. Extraction Techniques
The need to extract compounds from saffron samples prompts continued searching for
economically and ecologically feasible extraction technologies. Traditional solid–liquid extraction
methods require a large quantity of solvent and are time consuming. The large amount of solvent used not
only increases operating costs but also causes additional environmental problems. Several novel extraction
techniques such as ultrasound-assisted extraction, membrane processes, supercritical fluid extraction and
solid phase extraction have been developed as an alternative to conventional extraction methods, offering
advantages with respect to extraction time, solvent consumption, extraction yields and reproducibility.
However, most of these novel extraction techniques are still conducted successfully at the laboratory or
bench-scale although several industrial applications of supercritical fluid extraction can be found. More
research is needed to exploit industrial applications of the novel extraction techniques. Novel extraction
processes are complex thermodynamic systems with higher capital costs. The engineering design of novel
extraction systems requires knowledge of the thermodynamic constraints of solubility and selectivity, and
kinetic constraints of mass transfer rate. Modeling of novel extraction processes can provide a better
understanding of the extraction mechanisms and be used to quickly optimize extraction conditions and
scale-up any design. High-pressure and microwave energy can be combined with conventional Soxhlet
extraction leading to a new design of high-pressure and microwave-assisted Soxhlet extractors. Also
supercritical CO2 solvent can be used in the Soxhlet extraction. Ultrasound can be used to enhance the
mass transfer in supercritical fluid extraction [161]. Supercritical fluid extractions can be coupled with
silica gels to improve the overall extraction selectivity and on-line fractionations. These combined
techniques retain the advantages of conventional Soxhlet, while overcoming the limitations of the novel
extraction techniques such as ultrasound-assisted, microwave assisted, supercritical fluid and accelerated
solvent extractions.
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4.2. Microextraction techniques
To improve the performance of microextraction methods, automation needs to be introduced into
sample preparation, and will be a major research topic in the future. Automation may result in better
reproducibility compared to manual methods. At present, several automated techniques have been
developed in plant samples, including direct immersion and headspace SPME [162-166], and SPME
coupled with chromatographic analysis [167], etc. In the future, microchannel SPME will be applied in
automation as the coating could be placed on the inner surface of the microchannel and only a small
amount of sample will be required. However, full automation of microextraction techniques appears to be
very difficult for plant samples. This is due to the fact that certain sample pretreatments are necessary
prior to microextraction of plant samples at present, and that automation of these sample pretreatment
processes (blending, homogenization, filtration, and centrifugation) is very difficult. In order to improve
automation, future work will focus on direct microextraction analysis of plant samples such as saffron
without pretreatment steps.
5. CONCLUSION
i. The need to extract compounds from saffron prompts continued searching for economically and
ecologically feasible extraction technologies.
ii. For saffron samples, conventional extraction methods include solvent based extraction technique,
steam distillation (SD), ultrasonic extraction (USE), membrane processes (MP), supercritical
fluid extraction (SFE) and solid phase extraction (SPE).
iii. Traditional extraction methods such as Soxhlet extraction require a large quantity of solvent and
are time consuming. The large amount of solvent used not only increases operating costs but also
causes additional environmental problems.
iv. Novel extraction techniques including ultrasound assisted extraction, supercritical fluid extraction
and solid phase extraction have been developed for the extraction of compounds from saffron in
order to shorten the extraction time, decrease the solvent consumption, increase the extraction
yield and enhance the quality of extracts.
v. In recent years, the microextraction methods have attracted significant attention as these
techniques have high sensitivity, simple to use, with short sample pretreatment time and a high
enrichment factor, with low solvent usage, or somethimes solvent-free, and amenable to
automation. Therefore, the microextraction techniques become an attractive tool in the
determination of compounds from saffron matrixes.
vi. The microextraction techniques used for the determination of compounds from saffron samples
are divided into two main categories: sorbent-based microextraction– solid phase microextraction
and stir-bar sorptive extraction, and solvent-based microextraction– dispersive liquid liquid
microextraction.
vii. The present review article focuses on the extraction and microextraction techniques used for the
determination of compounds from saffron samples. This paper gives a description of each
technique and lists the advantages and disadvantages of them.
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The authors declare no conflict of interest
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