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Borderless Science Publishing 221 Canadian Chemical Transactions Year 2014 | Volume 2 | Issue 2 | Page 221-247 ISSN 2291-6458 (Print), ISSN 2291-6466 (Online) 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 [ 16]. 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 [1214].

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Page 1: Extraction and Microextraction Techniques for the ......spectroscopy has already found application in quality control and classification of saffron [42]. Mid-infrared spectroscopy

Ca

Borderless Science Publishing 221

Canadian Chemical Transactions Year 2014 | Volume 2 | Issue 2 | Page 221-247

ISSN 2291-6458 (Print), ISSN 2291-6466 (Online)

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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