Abstract Sample preparation is the crucial first step inthe analysis of herbs. In recent years there has been in-creasing interest worldwide in the use of alternative/herbalmedicine for the prevention and treatment of various ill-nesses. Currently, however, quality-related problems (lackof consistency, safety, and efficacy) seem to be overshad-owing the potential genuine health benefits of various herbalproducts. Thus, the development of “modern” sample-prep-aration techniques with significant advantages over con-ventional methods for the extraction and analysis of me-dicinal plants is likely to play an important role in theoverall effort of ensuring and providing high-quality herbalproducts to consumers worldwide. In this article, recentdevelopments and applications of modern sample-prepa-ration techniques for the extraction, clean-up, and concen-tration of analytes from medicinal plants or herbal materi-als are reviewed. These modern techniques include solid-phase microextraction, supercritical-fluid extraction, pres-surized-liquid extraction, microwave-assisted extraction,solid-phase extraction, and surfactant-mediated extrac-tion.

Keywords Sample preparation · Extraction · Qualitycontrol · Medicinal plants · Herbs

Introduction

Plants are naturally gifted at the synthesis of medicinalcompounds. The extraction and characterization of activecompounds from medicinal plants have resulted in thediscovery of new drugs with high therapeutic value [1, 2].A classic example is aspirin, which was initially discov-ered as salicylic acid in willow bark and leaves [1]; an-other noted example is taxol, recently proven to be effec-

tive against breast and ovarian cancers, which was ini-tially discovered in bark of yew trees [2].

The use of medicinal plants (herbs) has a long historythroughout the world and herbal preparations, includingherbal extracts, can be found in the pharmacopoeias ofnumerous countries [3]. In recent years there have been arenaissance of interest in natural or herbal remediesworldwide, partly because of the realization that modernmedicine is not capable of providing a “cure-all” solutionagainst human diseases and that the presence of unwantedside-effects is almost unavoidable. Unlike modern drugsthat invariably comprise a single active species, herb ex-tracts and/or prescriptions contain multiple active con-stituents. Interestingly, natural compounds contained inthese “herbal cocktails” can act in a synergistic mannerwithin the human body, and can provide unique therapeu-tic properties with minimal or no undesirable side-effects[4].

A key factor in the widespread acceptance of natural oralternative therapies by the international community in-volves the “modernization” of herbal medicine. In otherwords, the standardization and quality control of herbalmaterials by use of modern science and technology is crit-ical. At present, however, quality-related problems (lackof consistency, safety, and efficacy) seem to be overshad-owing the potential genuine health benefits of variousherbal products, and a major cause of these problemsseems to be related to the lack of simple and reliable ana-lytical techniques and methodologies for the chemicalanalysis of herbal materials [5, 6].

Sample preparation is the crucial first step in the analy-sis of herbs, because it is necessary to extract the desiredchemical components from the herbal materials for fur-ther separation and characterization. Thus, the develop-ment of “modern” sample-preparation techniques withsignificant advantages over conventional methods (e.g.reduction in organic solvent consumption and in sampledegradation, elimination of additional sample clean-upand concentration steps before chromatographic analysis,improvement in extraction efficiency, selectivity, and/orkinetics, ease of automation, etc.) for the extraction and

Carmen W. Huie

A review of modern sample-preparation techniques for the extraction and analysis of medicinal plants

Anal Bioanal Chem (2002) 373 :23–30DOI 10.1007/s00216-002-1265-3

Received: 12 November 2001 / Revised: 12 February 2002 / Accepted: 14 February 2002 / Published online: 3 April 2002

SPECIAL ISSUE PAPER

C.W. Huie (✉)Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong e-mail: cwhuie@net1.hkbu.edu.hk

© Springer-Verlag 2002

analysis of medicinal plants is likely to play an importantrole in the overall effort of ensuring and providing high-quality herbal products to consumers worldwide.

In this article recent developments and applications ofmodern sample-preparation techniques for the extraction,clean-up, and concentration of analytes from medicinalplants or herbal materials are reviewed. These moderntechniques include solid-phase microextraction, supercrit-ical-fluid extraction, pressurized-liquid extraction, mi-crowave- assisted extraction, solid-phase extraction, andsurfactant-mediated extraction. Emphasis is placed onbrief description of the unique capabilities and advantagesand disadvantages of each modern sample-preparationtechniques and of how these techniques were exploited toimprove the extraction and analysis of a variety of medic-inal plants. More detailed description of the basic princi-ples of these modern sample-preparation techniques forthe extraction of solid materials in general is available innumber of excellent review articles recently appeared inthe literature [7, 8, 9, 10].

Modern techniques for sample preparation in the analysis of medicinal plants

Headspace analysis

The medicinal properties of plants can be related in part tothe presence of volatile constituents (e.g. essential oils) inthe plant matrix, and gas chromatography (GC) is fre-quently used for determination of the volatile compositionof plant materials. Because the sample to be injectedshould be free from non-volatile components, a fractiona-tion step is necessary before GC analysis. The disadvan-tages of commonly used sample-preparation techniquessuch as distillation and liquid solvent extraction are thatthey usually require large amounts of organic solvents andmanpower; these methods also tend to be destructive innature (i.e. significant artifact formation can occur owingto sample decomposition at high temperatures) [11].

Headspace (HS) sampling is well suited for the frac-tionation of volatile compounds from complex solid ma-trices such as plant materials. Sanz and co-workers [12]have shown that reproducible and rapid identification ofvolatile compounds in aromatic plants can be achievedwhen static HS sampling is coupled with GC–mass spec-trometry (MS), with the advantages of eliminating the ex-traction or fractionation step, reducing artifact formation,and providing on-line capacity. More recently the sameresearch group has extended the capabilities of automatedHS sampling in the GC–MS analysis of volatile com-pounds from Origanum vulgare [13].

Another example of herb analysis by automatic HS–GCis recent work by Stuppner and Ganzera [14] on determi-nation of the safrole content from different Asarum spe-cies from China and Europe. These Asarum species con-tain up to 5.5% essential oils with methyleugenol as themajor constituent and several minor components, e.g. saf-role, which is known to have mutagenic and carcinogenic

effects. The HS–GC results for safrole were found to be ingood agreement with those obtained by “classical” GCanalysis, i.e., using an organic solvent (dichloromethane)for extraction.

A novel and effective approach, known as solid-phasemicroextraction (SPME), was recently developed by Paw-liszyn and co-workers [15] as a solvent-free samplingtechnique. In this approach, the analytes from the sampleare adsorbed directly onto an adsorbent coated fused-sil-ica fiber (which fits inside a syringe needle) and then ei-ther thermally desorbed directly into a GC injection portor high-performance liquid chromatography (HPLC) in-jection valve. By placing the fiber in head space at equi-librium with a sample (i.e. the technique of HS-SPME), arapid and inexpensive method for isolation of volatile or-ganic analytes for subsequent GC–MS analysis has beensuccessfully demonstrated for the quality control of herbalmedicine and other formulations containing herb extracts,such as terpenoids, peppermint, rosemary, sage, and thyme[16]. In addition to endogenous active components in me-dicinal plants, the usefulness of SPME as a sampling toolhas also been recently demonstrated by Hwang and Lee[17] for the GC–MS analysis of toxic contaminants, i.e.,organochlorine pesticide residues, present in Chinese herbalmaterials.

In a recent paper Pawliszyn and co-workers [18] re-ported successful demonstration of the feasibility andunique advantages of SPME for the characterization andquantification of the biogenic volatile organic compounds(e.g. isoprene and terpenoid compounds) emitted by liv-ing plants (leaves of Eucalyptus citriodora). By use ofcoated SPME fibers they were able to identify 33 com-pounds emitted by the living plant and, using diffusion-based SPME quantitation, it was possible to quantify sub-parts-per-billion amounts of isoprene after a very shortextraction time (15 s).

The effect of the fiber coating in HS-SPME–GC analy-sis of aromatic and medicinal plants has been investigatedin depth by Bicchi and co-workers [19]. Interestingly, itwas found that fibers consisting of two components, a liq-uid polymeric coating for the less polar analytes and asolid polymeric coating for the more polar analytes, weremore effective for HS-SPME analysis of the volatile frac-tion of the four aromatic and medicinal plants investigated(rosemary, sage, thyme, and valerian). More recently, re-placement of the fiber needle with a stir-bar, a new sam-pling technique known as stir-bar sorptive extraction(SBSE), has been described by Bicchi and co-workers[20] for the headspace sampling of the same four aro-matic/medicinal plants. Because of the larger amounts oftrapping material (e.g. polydimethylsiloxane) coated onthe stir-bar, the concentration capability is significantlygreater than that of SPME.

Supercritical- and subcritical-fluid extraction

Supercritical fluid extraction (SFE) has been used formany years for the extraction of volatile components, e.g.

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essential oils and aroma compounds from plant materials,on an industrial scale [21]. Recently, the application ofthis technique on an analytical scale has started to attractwide interest for sample preparation before chromato-graphic analysis [22]. The potential advantages includethe ability to perform rapid (often less than 30 min) ex-tractions, to reduce the use of hazardous solvents (i.e. car-bon dioxide is commonly used as the extraction solvent),and to couple the extraction step with gas, liquid, or su-percritical-fluid chromatography.

An important advantage of applying SFE to the extrac-tion of active compounds from medicinal plants is thatdegradation as a result of lengthy exposure to elevatedtemperatures and atmospheric oxygen are avoided. Forexample:

1. Smith and Burford [23] showed that the active com-pound of feverfew, i.e. the sesquiterpene lactoneparthenolide (well known to be unstable and to de-grade during storage), can be efficiently and selec-tively extracted from dried feverfew by SFE withoutthermal degradation;

2. Bartley and Foley [24] demonstrated that althoughvolatile compounds (essential oils) in ginger were ex-pected to be strongly influenced by heat treatment,possibly as a result of hydrolysis, oxidation, and re-arrangement, a very low concentration of gingeroldegradation products were, however, found after SFEof Australian-grown ginger, which attested to the mildnature of the extraction procedure; and

3. in the determination of the enantiomeric purity of at-ropine (a tropane alkaloid with significant medical in-terest found in plants of the Solanaceae family), Ma-teus et al. [25] found that SFE induced less racemiza-tion than classical liquid–solid extraction procedures.

In SFE several experimental conditions can be adjusted tooptimize the recovery, kinetics, and selectivity of the ex-traction. For example:

1. during optimization of SFE conditions for extractionof active ingredients from Curcuma zedoaria, Ma andco-workers [26] found that the density of the CO2 andthe fluid volume passing through the plant matrix arethe most important factors affecting extraction effi-ciency, whereas increasing the temperature has littleeffect;

2. when using SFE to extract indirubin (the active ingre-dient in the herbs Strobilanthes cusia, Isatis tinctoria,and Polygonum tinctorium) Li et al. [27] found thatfaster extraction kinetics were achieved by employingfactorial design to optimize experimental variablessuch as temperature, pressure, modifier concentration,static extracting time, and CO2 dynamic extracting vol-ume; and

3. it is interesting to note from another study byHawthorne and co-workers [28] that the rate of extrac-tion of volatile active components (essential oils) fromplant matrices by SFE seemed to be governed by ana-lyte–matrix interaction rather than the bulk solubility

of the analyte in pure CO2, because the extraction rateswere found to increase greatly due to an addition of or-ganic modifiers.

In another fundamental study Fahmy and co-workers [29]clearly demonstrated that swelling of the plant matrix, asa result of interaction between modifiers and matrix, wasan important factor in enhancing extraction recovery andkinetics in SFE. Also, by use of different conditions ofpressure and temperature, Reglero and co-workers [30]found that SFE conditions can be fined tuned for selectiveextraction of an antioxidant fraction with almost no resid-ual aroma from rosemary plants. It has also been shown,by operating under sub-critical temperature and pressureconditions, that CO2 fluid can be used as solvent for theselective extraction of essential oils [31] and diterpeneglycosides [32] from plants of medicinal interest. Morerecent examples of the application of SFE for the opti-mum extraction of analytes from medicinal plants underdifferent extraction conditions are listed in Table 1.

The on-line coupling of SFE with supercritical-fluidchromatography (SFC) has recently been shown to affordenhanced speed and sensitivity, as a result of the ability touse this technique to perform consecutive extraction, con-centration, and separation of the constituents of herbalmaterials. For example, using an amino column for trap-ping and separation, Suto and co-workers [44] demon-strated that the on-line SFE–SFC analysis of the activecompounds (magnolol and honokiol) in Magnoloae cortexcan be completed within 5 min. Also, by using silica gelas the trapping and separation column and sodium sulfo-succinate as the counter-ion for ion-pair formation, thesame research group recently showed that the on-line cou-pling of ion-pair SFE–SFC enabled the rapid analysis(within 10 min) of berberine and palmatine (positivelycharged active species) in Phellodendri cortex [45].

Another interesting recent development is the on-linecoupling of SFE to a uterotonic bioassay by Sewram andco-workers [46]. In South Africa, the use of traditionalmedicine is popular and the ingestion of plant extractsduring pregnancy to provide health supplements or to in-duce labor is common. In the on-line SFE uterotonicbioassay system [46] SFE extracts from four local medic-inal plants were transferred directly to a uterus musclechamber to identify the active fractions (i.e. the fractionscapable of inducing muscle contraction can be determinedrapidly, safely, and sensitively). This novel on-line SFEbioassay method could also be adapted for screeningplants with other therapeutic properties, e.g. those usedfor treatment of diabetes mellitus and hypertension. An-other novel approach involving the off-line coupling ofSFE to GC in such a way that the glass liner of a pro-grammed temperature vaporizer is placed after the separa-tion vessels of the SFE extraction module has been re-cently demonstrated by Blanch et al. [47] to be effectivefor the sensitive and selective analysis of complex plantmatrices.

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Microwave-assisted extraction

In contrast with conventional liquid–solid extractionmethods (e.g. Soxhlet extraction) in which a relativelylong extraction time (typically 3–48 h) is required, the useof microwave energy for solution heating (the techniqueknown as microwave-assisted extraction, MAE) results insignificant reduction of the extraction time (usually lessthan 30 min), because the microwaves heat the solvent orsolvent mixture directly, thus accelerating the speed ofheating. Besides having the advantage of high extractionspeed, MAE also enables a significant reduction in theconsumption of organic solvent (typically less than 40 mL,compared with the 100–500 mL required in Soxhlet ex-traction) [48].

Among the conditions commonly studied for optimiza-tion of MAE process, the effects of solvent composition,solvent volume, extraction temperature, and matrix char-acteristics seem to be most important for plant materials.For example, Chen and Spiro [49] recently performed akinetic study of the effects of these conditions on theMAE extraction of plant materials with medicinal signifi-cance, i.e. leaves from rosemary and peppermint. Theirresults indicated that for a sample matrix such as a plant,which usually contains water as a component with dielec-tric loss (a measure of the efficiency of converting mi-crowave energy into heat), the use of pure, microwave-

transparent solvents such as hexane could result in therapid extraction of essential oil components. This is prob-ably because of the direct interaction of microwaves withthe free water molecules present in the glands and vascu-lar systems, which results in the subsequent rupture of theplant tissue and the release of the essential oil into the or-ganic solvent (hexane). More effective microwave heatingof this particular system (hexane+leaves) could thereforebe achieved by increasing the weight of leaves relative tothe volume of hexane. For a system involving use of anorganic solvent which absorbs microwaves more strongly(ethanol+leaves), more effective heating of sample mix-ture could, on the other hand, be achieved by increasingthe microwave power output, because in this system theethanol (rather than water molecules in the leaves) ab-sorbs the bulk of the microwave energy.

In traditional medicine the preparation of “herbaldrinks” for oral intake usually involves cooking the herbalmaterials with water for 30–60 min. In modern herbalmedicines the herbs are cooked with water or ethanol andthen processed into tablets, pills, liquid, or injection solu-tions. The use of conventional extraction methods, e.g. ul-trasonic processing and heating under reflux, to leach theactive compounds from herbs is very time consuming. Toresolve this problem, Gou et al. [50] recently demon-strated that with the aid of MAE the extraction of puerarinfrom Radix puerariae took 1 min only when water was

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Table 1 Recent applications of SFE in medicinal plant analysis

Analyte Medicinal plant Extraction conditions/remarks References

Taxanes English yew tree (Taxus baccata) Methanol-modified CO2; 50°C; 400 atm; 100 min; [33](Taxol) extraction efficiency comparable with that of liquid solvent

extractionVolatile Frankincense, myrrh and Evodia CO2 only: 20 MPa; 50°C; 40 min; good extraction efficiency [34]compounds rutaecarpa (traditional for high molecular-weight and oxygenated components

Chinese medicine)Diosgenin Tubers of Dioscorea nipponica CO2; range of temperature and pressure studied: 3100 psig; [35](a steroid 44°C; 70 min gave the highest recoveryintermediate)Taxol and Needles of Taxus cuspidata Ethanol-modified CO2; 40°C; 300 bar; using a continuous [36]baccatin III flow-through cell for extractionEssential oils Feverfew, tansy, and German CO2: 250 bar and 450°C; GC chromatograms can be used [37]

chamomile to discriminate between the different plant materialsOxindole Uncaria tormentosa (one of the most CO2 only and methanol-modified CO2; 250 atm; 60°C; [38]alkaloids important botanicals in the rain forest 30 to 60 min

of Peru)Kava lactones Piper methysticum Forst CO2 alone and ethanol-modified CO2; 250 to 450 atm; [39]

60°C; 60 min; to maximize extraction efficiency, use CO2alone for another 4 h and 3 min

Volatile Roots from different species of CO2: 15 MPa; 60°C; 30 min; GC patterns can be used for [40]compounds Echinacea (herbal extracts marketed rapid identification and to verify the authenticity of different

widely in Europe) speciesTanshinone II A Root and rhizome of Salvia miltiorrhiza Methanol–CO2 gave the highest recovery; 350 kg cm–2; [41]

bunge (popular traditional Chinese 60°C; 30 minmedicines)

Flavanones and Root bark of osage orange tree CO2 alone and methanol-modified CO2; 40.5 MPa; [42]xanthones (Maclura ponifera) 40 to 100°C; 45 min; addition of methanol to CO2 essential

for achieving high yieldsFlavonoids Roots of Scutellaria baicalensis A range of conditions were studied; optimum conditions: [43]

(traditional Chinese medicine) CO2 + methanol + H2O (20:2.1:0.9), 50°C and 200 bar

used as the extraction solvent, because water not only ab-sorbed the microwaves efficiently but also readily dis-solved the polar active constituents (iso-flavone com-pounds) of this important Chinese herb.

For the analysis of Chinese herbs Liu and co-workers[51, 52, 53] have developed new MAE methodologies forextraction of glycyrrhizic acid from licorice root, whichhas been used in traditional medicines for the treatment ofstomach ulcers for over 2000 years [51, 52]. The root con-tains approximately 1–10% (w/w) of glycosides includingtwo glucuronic acid units and glycyrrhetinic acid andavloids. Their results indicated that the use of water and amixture of water and ethanol were suitable for rapid andefficient extraction of glycyrrhizic acid. The recoverycould be enhanced by addition of appropriate amounts ofammonia or salt to form more water soluble species, e.g.glycyrrhizic ammoniate and the potassium salt of glyc-yrrhizic acid. New MAE methodologies were also devel-oped for the optimum extraction of tanshinones from thedried roots of Salvia miltiorrhiza; this was complete in 2 min only [53]. Other recent applications of MAE to theextraction of medicinal plants include the leaching of:

• alpha-hederin and hedera-saponin from Hedera helixleaves [54];

• saponin and sapoganin from Paris polyphylla [55];• sallidroside and tyrosol from Rhodioda sachalinensis

[56];• essential oils from the leaves of Lippoa sidoides [57];• Lupin alkaloid (sparteine) from seeds [58]; and• Taxanes from Taxus biomass [59].

The effectiveness of using microwave and aqueous min-eral acids for the digestion of solid materials in metalanalysis is well known. In herbal medicine the signifi-cance of measuring the concentration of metals is relatedto their clinical efficacy, safety, and toxicity. For example,raw and processed herbs frequently contain heavy metalsthat exceed the safety regulation limits set by many coun-tries. Clinical studies, on the other hand, show that someheavy metals, despite their reported toxicity, actual playan important role in the therapeutic effects of the herbscontaining these metals [60, 61, 62]. It is, therefore, ofcritical importance to develop reliable sample treatmentand analytical techniques for metal analysis and specia-tion in herbal products [5, 6].

In a recent study Wang and co-workers [63] comparedthe effectiveness of three different digestion methods (twoconventional wet acid digestion and one microwave aciddigestion) for the determination of metals in traditionalChinese medicine (TCM). Although all three methods ledto comparable results using inductively-coupled plasma(ICP)–MS for analysis, microwave acid digestion wasconcluded to be the method of choice because of itshigher speed and lower reagent consumption.

Similarly Ong and co-workers [64] have recently re-ported a simple and rapid method for determination ofmercury in TCM. Two different types of vessel for closed-vessel microwave were compared; the results indicatedthat, in addition to improved speed, closed-vessel mi-

crowave digestion also minimizes loss of volatile analytessuch as mercury. To enable higher sample throughput andprecision, lower washout time and memory effect, andsmaller sample volume capability, the same research grouprecently coupled closed-vessel microwave digestion andflow injection methods with ICP–MS for the determina-tion of arsenic and lead [65, 66].

Pressurized liquid extraction

For rapid and efficient extraction of analytes from solidmatrices such as plant materials, extraction temperature isan important experimental factor, because elevated tem-peratures could lead to significant improvements in thecapacity of extraction solvents to dissolve the analytes, inthe rates of mass transport, and in the effectiveness ofsample wetting and matrix penetration, all of which leadto overall improvement in the extraction and desorption ofanalytes from the surface and active sites of solid samplematrices. To achieve all these advantages, however, ele-vated pressure is needed to maintain the extraction sol-vents as liquids at high temperatures (usually above theirboiling points); this can be accomplished by use of amodern extraction and sampling technique known as pres-surized-liquid extraction (PLE) or, more commonly, by itstrade name (accelerated solvent extraction) [67].

PLE emerged in the mid-nineteen-nineties, but it issurprising to find it has rarely been applied to the extrac-tion/analysis of plant materials. Benthin et al. [68] wereamong the first to conduct a comprehensive study on thefeasibility/usefulness of applying PLE in medicinal herbanalysis. In their study PLE extracts from a selection ofrepresentative herbs were compared with extracts ob-tained according to pharmacopoeia monographs; their re-sults indicated that PLE is often superior to other extrac-tion methods currently used in crude herb analysis interms of recovery, extraction time, and solvent consump-tion (i.e. for all the herbs studied, a significant saving intime and solvents was realized and extraction recoveriesof the analytes were equivalent or higher). Similarly, Ongand co-workers [69] recently found that PLE is superior toconventional extraction methods (ultrasonic and Soxhletextraction) for the extraction of berberine and aristolochicacids in medicinal plants.

In addition to extraction temperature, the choice of ex-traction solvent is another important factor in PLE. MostPLE applications reported in the literature employed theorganic solvents commonly used in conventional tech-niques, e.g. methanol, in which many organic compoundsare very soluble. A recent application of PLE reported byKawamura and co-workers [70] for the extraction of anactive compound with significant medicinal interest, pac-litaxel (commonly known as taxol, which has anticanceractivity), from the bark of Taxus cuspidata indicated,however, that use of water alone as the extraction solventis a viable alternative. An interesting result from thisstudy was that although in conventional extraction meth-ods the taxol content of the water extract was very low,

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this was dramatically improved by use of the elevatedtemperature and pressure conditions of PLE. Sumni andco-workers [71] compared conventional and modern ex-traction methods for the rapid and efficient extraction ofmedicinal iridoid glycosides from a plant matrix (Veronicalongifoloa leaves) and reported that use of hot water as theextraction solvent under atmospheric or higher pressureconditions was the most efficient.

Ultrasonic extraction

Although the use of ultrasonic energy to aid the extractionof medicinal compounds from plant materials can befound in the literature as early as the nineteen-fifties,mechanistic aspects of the usefulness of ultrasonically as-sisted extraction are worth noting. Fundamentally, the ef-fects of ultrasound on the cell walls of plants can be de-scribed as follows [72]:

1. Some plant cells occur in the form of glands (externalor internal) filled with essential oil. A characteristic ofexternal glands is that their skin is very thin and can beeasily destroyed by sonication, thus facilitating releaseof essential oil contents into the extraction solvent; and

2. Ultrasound can also facilitate the swelling and hydra-tion of plant materials to cause enlargement of thepores of the cell wall. Better swelling will improve therate of mass transfer and, occasionally, break the cellwalls, thus resulting in increased extraction efficiencyand/or reduced extraction time.

As a novel approach to extraction and sample preparationfor medicinal herbs, Huie and co-workers [73] recentlyemployed ultrasound to assist the surfactant-mediated ex-traction of ginsenosides from American ginseng (a verypopular herb consumed worldwide). In this approach thesurfactant-mediated extraction process can be divided intotwo parts:

1. solubilization of active ingredients from the solidherbal material into the extraction solvent (aqueoussurfactant solution); and

2. the cloud-point phase separation of the aqueous surfac-tant solution containing the active ingredients into abulk aqueous phase and a smaller volume surfactant-rich phase (analyte concentration).

In ultrasonically assisted extraction the use of aqueoussurfactant solution containing 10% Triton X-100 as theextraction solvent was found to result in faster extractionkinetics and higher recovery compared to methanol andwater.

Solid-phase extraction

A common drawback of classical and modern extractionmethods in sample preparation for complex matrices isthat additional clean-up procedures are often required be-fore gas or liquid chromatographic analysis. For medici-

nal plants the use of sampling techniques such as Soxhletextraction, MAE, or PLE often results in non-selective co-extraction of relatively large amounts of undesirable com-ponents (e.g. lipids, sterols, chlorophylls, etc.), which canseverely affect the separation and detection performanceof subsequent GC or HPLC analysis [74].

Solid-phase extraction (SPE) is a simple preparationtechnique based on the principles used in liquid chro-matography, in which the solubility and functional groupinteractions of sample, solvent, and adsorbent are opti-mized to effect sample fractionation and/or concentration.A wide range of chemically modified adsorbent materials(silica gel or synthetic resins) enable precise group sepa-ration on the basis of different types of physicochemicalinteraction, i.e. reversed-phase (C2, C8, C18), cation- andanion-exchange, etc. It should, in particular, be noted thatSPE is well suited to the treatment of sample matriceswith high water content, e.g. extracts of herbal materials[75].

Most traditional medicines, e.g. TCM, involve use ofboiling water to extract the herbal materials for prepara-tion of the medicinal prescriptions. For example, theleaves of a tea plant known as Theaw folium, which con-tain caffeine as a major constituent, are commonly used asa Chinese herb [76]. Without pretreatment HPLC analysisof aqueous extracts of this tea plant was rather difficult,because of the presence of many highly polar complexconstituents. Ku and co-workers [76] recently resolvedthese difficulties by combining SPE (C18 reversed-phaseadsorbent) and HPLC for the sensitive and reproducibledetermination of caffeine in six different TCM prescrip-tions (complex herb mixtures) that contained Theae foliumas one of the herbs.

Similarly, Hurlbut and co-workers [77] from the USFood and Drug Administration (FDA) demonstrated theimportance and effectiveness of using SPE and HPLC forthe determination of ephedrine alkaloids extracted fromplants of the genus Ephedra. Large amounts of these ex-tracts are imported into the US annually for sale to con-sumers as dietary supplements that promote weight loss,body building, and energy increase. Ephedrine alkaloidscan, however, affect the cardiovascular and nervous sys-tem and are known to cause illnesses and injuries to con-sumers. Using a propylsulfonic acid SPE column (cation-exchange clean-up) the FDA developed a simple and reli-able method for HPLC analysis of ephedrine alkaloids inherbal products. Lino and co-workers [78] have recentlyshown that, in addition to biologically active compounds,SPE with C18 or Florisil adsorbents was also very usefulfor extraction and clean-up of herbal materials beforeanalysis of the organochlorine pesticide residues presentin medicinal plants.

In addition to conventional SPE columns, new poly-meric adsorbents have recently been developed for theimproved retention of polar organic compounds, which isa major limitation of the C18 adsorbent. Klejdus et al. [79]recently compared classical adsorbents and new poly-meric adsorbents (e.g. Waters Oasis HLB extraction car-tridges, which contain a unique copolymeric adsorbent

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designed to have a hydrophilic–lipophilic balance) for de-termination of isoflavones in plants; these compounds areknown to be important in cancer prevention and to haveother health benefits. The results indicated that the use ofpolymeric adsorbents for sample pretreatment enabledhigher recoveries, higher reproducibility, and lower con-sumption of plant materials for the HPLC analysis ofthese isoflavones. As an alternative to a single SPE col-umn, the use of combined or mixed-mode SPE columnswas shown to be effective for the purification and isola-tion of active components from medicinal plants. For ex-ample, Glowniak et al. [80] demonstrated the usefulnessof combining C18 and quaternary amine adsorbents for thefractionation of free phenolic acids (naturally occurringcompounds with a broad spectrum of pharmaceutical ac-tivity) in Echinacea species. Stobiecki et al. [81] com-bined C18 and an adsorbent containing benzene sulfonicgroups as an effective sample-preparation method for pro-filing quinolizidine alkaloids and phenolic compounds inLupinus albus.

Concluding remarks

Among the modern sampling techniques described in thisreview, SFE seems to offer unique advantages in the ex-traction of medicinal plants – high selectivity, minimumdegradation of thermally labile analytes, and eliminationof the use of hazardous organic solvents (e.g. use of pureCO2 as the extractant). The main drawbacks of SFE, onthe other hand, such as difficulties in extracting polarcompounds and high susceptibility to matrix effects, areproblematic in the extraction of herbal materials. As plantmatrices are highly complex, factors such as the watercontent and particle size of the matrix and strong ana-lyte–matrix interactions, etc., can severely limit the ca-pacity of SFE to effect high extraction efficiency andrapid kinetics, especially for polar analytes.

The basic principles of MAE and PLE are very similarto those of classic extraction techniques, i.e. use of liquidsolvents is still needed; however, partly because thesenewer technologies are automated and the solvents are un-der “superheated” conditions (the effect of microwaves inMAE or elevated temperature and/or pressure in PLE),they are more user-friendly, much quicker, and requiresignificantly less organic solvent. Compared with SFE,MAE and PLE are more easily optimized and should bepreferred when less selectivity is acceptable, because, forexample, the use of “hot” liquid solvents in these tech-niques can partly overcome strong analyte–matrix interac-tions, even for more polar compounds. For some medici-nal plants, however, thermal degradation of the analytescan be a problem under typical MAE and PLE conditionsand co-extraction of large amounts of matrix materials,e.g. lipids and chlorophylls, would require extensive addi-tional sample clean-up and concentration before chro-matographic analysis.

In the coming years, it seems that the use of PLE withsubcritical water as the extractant for the extraction and

analysis of analytes from medicinal plants might have in-teresting potential [70, 71], because water is inexpensive,non-toxic, and environmentally friendly. Also, by simplyincreasing the temperature at constant pressure, the rela-tive permittivity of water can be reduced, so that analyteswith a wide range of polarity can be extracted. The use ofSPME, especially head-space SPME, also shows goodpromise as a convenient and effective analytical tool forthe sampling of volatile compounds, e.g. essential oilsfrom medicinal herbs, from plant materials before GCanalysis [15, 17]. Although SPE and ultrasonic extractionhave been around for a relatively long time, it is likelythese methods will remain popular and effective tools forthe extraction, clean-up, and/or concentration of analytesfrom a variety of herbal materials.

Acknowledgements Financial support provided by a Competi-tive Earmarked Research Grant from the Research Grant Council(HKBU 2056/98P) is gratefully acknowledged.

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