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Cent. Eur. J. Chem. • 10(3) • 2012 • 802-835 DOI: 10.2478/s11532-012-0037-y

Central European Journal of Chemistry

Analysis of from different chemical groups by different liquid methods

Review Article

Anna Petruczynik

Department of Inorganic Chemistry, Medical University of Lublin, 20-093 Lublin, Poland

Received 29 October 2011; Accepted 27 January 2012

Abstract: Alkaloids are biologically active compounds widely used as pharmaceuticals and synthesised as secondary methabolites in . Many of these compounds are strongly toxic. Therefore, they are often subject of scientific interests and analysis. Since alkaloids - basic compounds appear in aqueous solutions as ionized and unionized forms, they are difficult for chromatographic separation for peak tailing, poor systems efficiency, poor separation and poor column-to-column reproducibility. For this reason it is necessity searching of more suitable chromatographic systems for analysis of the compounds. In this article we present an overview on the separation of selected alkaloids from different chemical groups by liquid chromatography thus indicating the range of useful methods now available for analysis. Different selectivity, system efficiency and peaks shape may be achieved in different LC methods separations by use of alternative stationary phases: silica, alumina, chemically bonded stationary phases, cation exchange phases, or by varying nonaqueous or aqueous mobile phase (containing different modifier, different buffers at different pH, ion-pairing or silanol blocker reagents). Developments in TLC (NP and RP systems), HPLC (NP, RP, HILIC, ion-exchange) are presented and the advantages of each method for alkaloids analysis are discussed. Keywords: Alkaloids • Thin layer chromatography • High performance liquid chromatography • Normal phase system • Reversed phase system © Versita Sp. z o.o.

1. Introduction they are often subject of scientific interests andanalysis. Since alkaloids are basic compounds that appear in Most of naturally occurring substances in their structure aqueous solutions as ionized and unionized forms, they have one or more acidic or basic groups. Such are difficult for chromatographic separation for peak compounds appear widely in different areas, such as the tailing, they have poor system efficiency, poor separation environment, food, extracts, biological fluids, and and poor column-to-column reproducibility. Reversed- [1,2]. Therefore, it is of crucial importance to develop phase (RP) chromatography continues to dominate efficient methods for the analysis of these compounds applications of high-performance liquid chromatography in several domains such as clinical analysis, quality (HPLC). The majority of silica based stationary phases control, therapeutic monitoring, toxicological analysis, are produced by reacting porous silica particles with an and studies. Among these compounds appropriate silane. Silanol groups (-Si-OH) on the silica are alkaloids, which are weak organic bases. Alkaloids gel surface are bonded in this reaction but steric effects are pharmacologically active compounds widely used prevent the reaction of only part of all the silanols. Further as pharmaceuticals and synthesised as secondary reaction with a short silane (endcapping) eliminates metabolites in plants. Many of these compounds are the most accessible silanol groups remaining from the strongly toxic. For diagnosis and prognosis of such initial bonding but typically does not substantially alter poisonings, analytical methods for detection and the total concentration of unreacted silanols. These quantification of the respective toxic alkaloids are residual silanols can then interact with basic compounds, required in clinical and forensic toxicology. Therefore, frequently leading to inferior separations, peak symmetry

* E-mail: [email protected] 802 A. Petruczynik Analysis of alkaloids from different chemical groups by different liquid chromatography methods

and systems efficiency [3]. Protonated basic compounds are regarded as rich sources of alkaloids. The can interact with residual silanol groups of the stationary principal alkaloids of medicinal interest in this group are phase, as shown in the equation: and which are agents in parasympathetic nervous system and they are XH+ + SiO-Na+ ↔ Na+ + SiO-XH+ (1) used as mydriatics and spasmolytics. alkaloid group is derived Because of that ionic analytes’ retention mechanism biosynthetically from the amino and is mixed and composed of ion-exchange mechanism with . Alkaloids belonging to phenethylamine the show kinetics and mass transfer and hydrophobic derivatives constitute an important class of natural interactions with much faster kinetics and mass transfer. products due to their structural similarity to many It gives rise to peak tailing [4]. For this reason, it is . Alkaloids from the group act as necessary to search for more suitable chromatographic () or (). systems for analysis of theses compounds. A great alkaloids are generally not used alone, but variety of analytical techniques have been applied rather as part of herbal formulas, and are known to the determination of different alkaloids, and liquid to induce pharmacological effects beyond their chromatography is the most frequently used method sympathomimetic activities such as anti-inflammatory, nowadays. The so-called silanol effect on silica based anti-anaphylactic, anti-microbial, anti-histaminic, and stationary phases has still been one of the major topics hypoglycemic effects. alkaloids are secondary of chromatographic studies even after the development metabolites produced by fungi of the species Claviceps. of advanced stationary phases that were supposed to Toxic effects after consumption of contaminated grains provide symmetrical peaks for basic compounds. have been described since mediaeval times. is currently being investigated for potential use as an anti-cancer . 2. Occurrence and medical alkaloids, biogenetically derived from significance of some alkaloids tyrosine, represent a manifold class of alkaloids within the plant kingdom. are spread is an aromatic heterocyclic . It mainly in the Papaverales, Rutales, Ranunculales, has a bicyclic structure, consisting of a six-membered Geraniales, Plumboginales, Myrtiflore and Rosales benzene ring fused to a five-membered - species. Among them alkaloids form containing pyrrole ring. Indole alkaloids are biogenetically an important group with different potent pharmacological derived from . These alkaloids contain two activity, including compounds of , nitrogen atoms, one of which is contained within the antitussives of and and anti-infective five-membered part of indole nucleus. agents of , palmatine, and magnoflorine. Indole alkaloids constitute an important class of serves as . Thebain is natural products which include a large number of worked up by pharmaceuthical industry to produce biologically important substances such as antitumor semi synthetic compounds such as the analgesic alkaloids (, , and and the antagonists and ), cardioarrithmic alkaloid () the . containing isoquinoline alkaloids: blood pressure lowering substances (reserpine), and morphine, codeine, noscapine, papaverine, tebaine, hallucinatory lysergic and its derivatives. Some laudanozine, retykuline has strong properties indole alkaloids are strongly toxic (), or are and demonstrates analgesic, spasmolytic, antitussive psychoactive substances (psylocibine, ). and obstructive properties. Tropane alkaloids are an important class of alkaloids are based on a bicyclic system in structurally related compounds having in common the with benzene and a ring are fused together. Most azabicyclo[3.2.1]octane-3-ol skeleton, with usually of them occur in the plant family Rutaceae, especially are estrified with various organic acids: (-)-S-tropic, rute. Quinoline alkaloids have also been identified in apotropic, cinnamic, tiglic, angelic, isovaleric and members of Malvaceae, Acanthaceae, Saxifragaceae α-truxillic. and Zygophyllaceae families. Cinchonidine and Tropane alkaloids mainly occurs in Solanacae, from the bark of the tree () Erythroxylacae and Convolvulacae plant families, but are well known for their antimalarial properties. they occur also sporadically in a number of other families The structures of pyridine alkaloids contain a e.g. , Rhizophoraceae plants. Genuses pyridine ring together with a ring (in ) Atropa, , Duboisia, Hyoscyamus and Scopolia or a unit (in ), the latter rings arising

803 804 A. Petruczynik Analysis of alkaloids from different chemical groups by different liquid chromatography methods

from and respectively. The alkaloids are salts. Samples containing a large number of naturally occurring in the solanaceous family. Nicotine, soluble compounds (e.g. phenols, tannines) should be the main alkaloid in the group, in small doses can act as extracted with organic immiscible with water a respiratory , though in larger doses it causes after addition of alkali to obtain the alkaloids in the respiratory depression. Nicotine is being used by former organic as free bases. smokers who wish to stop the habit. alkaloids of pilocarpine type are derived 3.1. Indole alkaloids biosynthetically from . Pilocarpine has the Various methods are used for extraction of indole muscarinic actions of . It is used as a miotic alkaloids from plant material. Generally in liquid-solid in the treatment of the open-angle glaucoma. systems acidic extractants are used. The following The alkaloids are a widely used group mixtures of solvents were used: MeOH with HCl (5%)

of alkaloids as they are constituents of popular daily [5], or 2% [6], MeOH with 1% CH3COOH [7], H2O with

beverages such as tea and coffee. These alkaloids TFA (0.1%) [8], n-hexane with 1% HCl [9], MeOH/H2O

stimulate the central nervous system, respiratory with HCl (1%) [10], 2-propanol/H2O with lactic acid (1%)

system, muscles and heart. They have also therapeutic [11], MeCN/H2O with 1% CH3COOH [12], water with properties such as acting as an analgesic, diuretic, or 10% [13], water with 2% sulfuric acid [14]. bronchodilator. The extraction of indole alkaloids was performed Pyrrolizidine alkaloids are derived biosynthetically also without addition of acids, for example extraction from the ornithine. They are spread in the with pure MeOH [15-17], EtOH [18-21], dichloromethane

Boraginaceae, and Composite plant families. [15], acetone [15] or MeOH/H2O [23] was reported. Their has drawn a lot of attention. Sometimes extraction was performed in extractant at

Diterpene alkaloids have a diterpene skeletal basic pH, e.g. MeOH/H2O with addition of ammonia structure. The group of alkaloids comprises highly toxic [24], EtOH with ammonia [25]. The MeOH extract compounds: , mesaconitine and hypaconitine. was acidified with 10% acetic acid to pH 2.8, and then The toxicology of these alkaloids derives from activation dissolved in water. The aqueous solution was extracted

of the of excitable cell membranes with CH2Cl2, while the aqueous solution was made leading to rapid paralysis of cardiac, muscular and alkaline with 25% ammonia to pH 8.5, and extracted

neural tissues. again with CH2Cl2 [26]. Ergot alkaloids from flour or grounded rye were extracted by adding 100 mL of dichloromethane/ethylacetate//ammonia 3. Sample preparation (25%), (50/25/5/1, v/v/v/v) [1]. The macrolactam-type indole alkaloids from Sample preparation is the crucial first part in a natural obscura were extracted with methanol. The solvent was product analysis because it is necessary to extract the evaporated under reduced pressure at 40°C, the residue desired chemical components from the material, dissolve re-dissolved in 2% aq. tartaric acid and extracted with the analyte in a suitable solvent and remove as many ethyl acetate. After evaporation of the organic solvent, interfering compounds as possible from the solution. the residue was dissolved with methanol [27]. Another Application of chromatographic techniques, especially procedure of extraction of indole alkaloids HPLC, requires preliminary sample preparation from pusillus was applied. The plant providing a sample free of components that may material was mixed with absolute , homogenised, deteriorate the column. Various methods of extraction centrifuged and the ethanolic extract was transferred are used for isolation of different alkaloids from plant into a round bottom flask. The pellet was re-extracted material, pharmaceutical formulations and biological in a similar fashion and the combined ethanolic phases samples. In the extraction and isolation of alkaloids one concentrated to dryness under reduced pressure. For the has to consider that alkaloids usually occur in plants as extraction of vindoline, the plant material was mixed with salts of organic or inorganic acids, sometimes exist as buffer (composed with , NaCl and NaOH) at pH complexes, and often together with non-alkaloidal 10 and dichloromethane. The mixture was homogenized compounds. The procedure of extraction depends on and centrifuged. The organic liquid phase was taken the class of alkaloids and ballast substances coexisting and transferred to a round bottom flask. The slurry was with the alkaloidal fraction. Natural samples with a extracted again by addition of dichloromethane [28]. high concentration of nonpolar compounds (e.g. lipids) SPE method involves selective extraction of the should preferably be extracted with water containing analytes from liquid samples onto solid support of acids to obtain the alkaloids in aqueous solution as different varieties and types (e.g. silica gel, alumina,

803 804 A. Petruczynik Analysis of alkaloids from different chemical groups by different liquid chromatography methods

florisil, kieselguhr, reversed-phase sorbents such as: obtained by conventional solid-liquid extraction but in a octyl, octadecyl, diol, cyano, amino, ion-exchange more efficient manner. sorbents). The sample is directly passed through The pressurized liquid extraction (PLE) was used previously conditioned cartridges filled with a given for and benzoylecgonine extraction from sorbent and analytes are directly collected, whereas leaves [37]. Mroczek et al. optimized PLE conditions for co-extractives are retained on the sorbent. It can be extraction of l-hyoscyamine and scopolamine from thorn performed another way in which a sample is applied in apple leaves [35]. solvent of low elution strength. The analytes adsorbed El-Shazly et al. [38] applied SELLP procedure for are eluted with solvent of higher elution power. Extraction isolation of pure alkaloidal fractions from Hyoscyamus of indole alkaloids from Rauwolfia serpentina tissue sp. The basified aqueous extract was applied into was performed on SCX cation-exchanger [29]. Tissues dry ExtrelutTM column, and the liquid was completely were extracted with methanol. After filtering, the extract absorbed by the kieselguhr. Tropane alkaloids free was evaporated to dryness. Extract was dissolved in bases which are exposed on the surface of the methanol and acidified with 0.1 M HCl. Acidified solution kieselguhr particles are eluted by organic solvents such was extracted with SCX cation –exchanger. as . In some cases liquid-liquid extraction were used for 3.2. Tropane alkaloids isolation of tropane alkaloids. At first the acidic extract Due to thermal instability and sensitivity to strong acidic is extracted with chloroform in a separation funnel and basis conditions of tropane alkaloids the method of to remove acidic co-extractives. Then the remaining solid-liquid extraction should be carefully selected. The extract is alkalized with ammonia solution to pH about first step of extraction should be with diluted acids like 5% 9-9.5 and extracted with non-polar organic solvents such

HCl, 5-10% acetic acid or 0.01% H2SO4 [30-32]. Jia et as chloroform, benzene, , or dichloromethane al. [33] found the extraction of Datura alkaloids the most [39,40]. efficient at pH 2-3. When alkaloid free bases are to be SPE of tropane alkaloids was usually performed on extracted, alkaline organic phases are used. Fliniaux et RP-18 columns. The procedure was applied for plant al. compared the efficiencies of both acidic and alkaline extracts, blood serum, urine and egg yolk samples solutions for the extraction of tropane alkaloids from plant [41-43]. material. They used: 0.2 M sulphuric acid, methanol-0.1 Keiner and Dräger [44] applied cation-exchange M HCl (24:1, v/v), methanol-27% ammonia (24:1, v/v), SPE for isolation of calystegines from plant samples, methanol-chloroform-27% ammonia (24:1, v/v) [34]. where they were retained by charge of the secondary Similar results were obtained by all procedures. amino group. Kintz et al. have extracted of scopolamine from children hairs. After liquid–liquid extraction with 5 mL of 3.3. Isoquinoline alkaloids a mixture of methylene chloride/isopropanol/n-heptane Various methods are used for extraction of isoquinoline (50/17/33, v/v/v) and evaporation of the organic phase alkaloids from different natural samples. Usually to dryness, the residue was reconstituted in 100 μL of simple techniques of extraction were applied. It was methanol. maceration or percolation at room temperature with Mroczek et al. [35] analysed the content of aqueous extractants or [45-48]. For liquid- l-hyoscyamine and scopolamine extracted from thorn solid extraction of isoquinoline alkaloids, aqueous acidic apple’s leaves. When 1% tartaric acid in methanol solutions were often applied [49,50]. Acidified methanol o was used at 90 ± 5 C on heating mantle for 15 min, or ethanol with HCl or H2SO4 was also used [51,52]. the highest amounts of scopolamine were measured Sometimes organic solvents are used for extraction also in comparison to more sophisticated methods such as methanol and dichloromethane, methanol and such as UAE (ultrasound assisted extraction) or PLE chloroform or dichloromethane [53-55]. The extraction (pressurised liquid extraction). However, the amounts was often assisted by shaking or ultrasonification of l-hyoscyamine were comparable to USE at 60oC and [56-58]. lower than PLE procedures. For cocaine and benzoylecgonine extraction from 3.4. Phenylethylamine alkaloids coca leaves, microwave-assisted extraction (FMAE) The following procedure was used for isolation of was optimised with respect to the nature of the extracting phenylethylamine alakloids from Colchicum crocifolium. solvent, the particle size distribution, the moisture of the Dried plant material was extracted with MeOH in a sample, the applied microwave power and radiation Soxhhlet apparatus for 3 h. The solvent was evaporated time [36]. FMAE generated extracts similar to those under reduced pressure to yield a MeOH-extract, which

805 806 A. Petruczynik Analysis of alkaloids from different chemical groups by different liquid chromatography methods

was fractionated [59]. Briefly, the MeOH-extract was 2-phenylimidizole, 5% antifoam/phenol red solution, dissolved in 5% acetic acid and extracted with light 30% ammonia, and dichloroethane and mixed by petroleum, after which the aqueous acid residue was gentle inversion for 1 min. The solution was centrifuged re-extracted three times with . The acidic 15 min using a Microfuge. The supernatant was discarded aqueous residues were made alkaline (pH 9) with 10% and the clear bottom layer was placed into a tube and

NH4OH followed by extraction three times with CH2Cl2. dried under N2 gas. The sample was reconstituted

The aqueous residues were then adjusted to pH 12 with with HPLC buffer (30 mM citric acid, 30 mM KH2PO4, 10% NaOH, and then extracted three times with diethyl 3.65 g L-1 triethylamine, 0.6 g L-1 1-heptanesulfonic acid, -1 ether and finally three times with CH2Cl2. 90 mL L acetonitrile, pH 4.8) was added. A sonification extraction was usually used for Hair samples containing alkaloids were separation of ephedrine alkaloids. The sonification washed three times with 3.0 mL of dichloromethane and microwave extraction were used for isolation of by vortex-mixing. After drying, the samples were ephedrine alkaloids from Ephedra natural products digested with 1.0 mol L-1 NaOH for 14 h at 50oC and [60]. Sonification was performed by use solvent then centrifuged. Afterwards, the clear supernatant was containing methanol or a mixture of hydrochloric acid diluted with an equal volume of a buffer of ammonium and methanol (0.8:99.2, v/v)) at different temperatures acetate + ammonia (pH 10.0) [67]. (room temperature, 40 or 50oC) for 15 min. Regarding In another procedure serum samples containing microwave extraction, a weighed amount of ground nicotine and its metabolites were prepared by SPE sample (0.25 g of E. vulgaris aerial parts) was extracted method [68]. Serum sample was added to internal with 5mL of solvent (methanol or a mixture of hydrochloric standard solution, water and 25% (w/v) trichloroacetic acid methanol (0.8:99.2, v/v)) by using a monomode acid to remove . The solutions were centrifuged microwave apparatus with a closed vessel system and at 10,000 g for 5 min after vortexing. The supernatant subjected to different temperatures for different times of was applied to SPE cartridges. An Oasis MCX cartridge irradiation (40oC for 15 min or 60oC for 4 min or 80oC () was conditioned with 1mL of methanol and for 1 min). The comparison between sonication and 1 mL of water. Serum samples were loaded and allowed microwave extractions indicated that sonication was the to flow by gravity. Cartridges were washed with 1mL most efficient procedure, allowing the highest yield of all water and 1 mL methanol, and dried for 5 min. Analytes considered analytes in a short time. were eluted with freshly prepared 1 mL methanol with 1% ammonia (v/v). Eluates were evaporated to dryness 3.5. Quinoline derivatives under a nitrogen stream at 50°C. Samples were The extraction of major alkaloids from Cinchona bark is reconstituted in acetonitrile with 0.1% formic acid (v/v). usually performed after preliminary pulverizing, grinding, The acidified plasma supernatant and urine sieving and drying of the bark at 110oC followed by containing nicotine and its metabolites were then treatment with alkali and Soxhlet extraction in hot subjected to solid-phase extraction (SPE) using a toluene [61], benzene or methanol [62]. combination of Oasis HLB and Oasis MCX mixed mode Quinine and are extracted from plasma or cartridges [69]. The SPE cartridges for both plasma urine also by making the sample basic with, for instance and urine were conditioned with methanol followed by sodium or aqueous ammonia, and extracting into an 10% aqueous trichloroacetic acid for plasma and 5 mM organic solvent such as dichloromethane or diethyl aqueous ammonium formate (pH 2.5) for urine. The ether [63,64]. samples were loaded onto the cartridges and the target For determination of quinine in plasma, samples analytes were subsequently eluted with 2 mL methanol were subjected to precipitation with acetonitrile. containing 5% concentrated aqueous ammonium The mixture was finally centrifuged at 4oC for 10 min. hydroxide (v/v). 1% concentrated aqueous hydrochloric The supernatant was transferred into a polypropylene acid in methanol (v/v) was added prior to evaporation tube and evaporated to dryness under nitrogen at room of the eluent. Extracts were evaporated to dryness temperature. The solid residue was reconstituted in and residues were reconstituted in initial mobile phase MeOH/ammonium formate 20 mM 1:1 adjusted to pH conditions. 4.0 with formic acid, vortex-mixed and centrifuged again SPE procedure was also used for extraction of [65]. tobacco alkaloids [70]. SPE column was pre-washed with methylene chloride, methanol, and finally with water. 3.6. Pyridine and piperidine alkaloids After the acidified sample was loaded onto the column, Nicotine and were extracted from rat plasma the column was washed with acetic acid and dried under

[66]. Plasma was added to a centrifuge tube containing positive pressure N2. The column was then washed with

805 806 A. Petruczynik Analysis of alkaloids from different chemical groups by different liquid chromatography methods

hexane and then of hexane-ethyl acetate (1:1), followed submitted to the following extraction step. Extractions by methanol. After washing, the analytes were slowly were carried out on a multi-frequency ultrasonic bath eluted with 2×3 mL of methylene chloride/isopropanol/ operating at 25 kHz at 100% intensity output. After ammonium hydroxide (78:20:2). The final extract was the last extraction, supernatants were combined and dried in the vacuum evaporator without heat, and the brought up with water and an aliquot was collected, residue was reconstituted in methanol with 0.1% formic which was filtered through 0.2-m nylon syringe filter acid for the analysis. before the HPLC analysis. analogues were extracted from plasma by SPE [71]. The various steps involved in the recovery 3.9. Pyrrolizidine alkaloids procedure were: (a) conditioning of SPE cartridge C18 Isolation of pyrrolizidine alkaloids was obtained by SPE with 1.0 mL methanol, followed by 1.0 mL water, (b) using C18 cartridges [76]. loading of diluted (1:4, v/v) plasma samples (1.0 mL) For extraction of pyrrolizidine alkaloids from onto cartridge and drying under positive pressure, and Boraginaceae species, the plant organs or cultured (c) samples were washed with 2 mL of water followed by roots were washed with tap water, dabbed dry, weighed, elution with 2 mL of methanol. and ground in a mortar with liquid nitrogen and sea sand before they were extracted twice for 30 min with 3.7. Imidazole alkaloids methanol containing 1% HCl (25%) and centrifuged [77]. alkaloids were prepared from cord serum, or The supernatant of the combined methanol extracts urine with 10 internal standards and NH4Cl saturated was evaporated. The resulting residue was dissolved in solution at pH 9.5 added, were transferred to a screw- methanol. capped glass tube with chloroform/isopropanol In another procedure, the sample preparation internal (95:5, v/v) [72]. The tubes were placed in a horizontal standard was added to serum and mixed well before shaker for 5 min. After centrifugation, the organic being deproteinized with 6% HClO4 (v/v) [78]. Then layer was transferred to another screw-capped glass KH2PO4–KOH (1 M; pH 8.1) was added. The mixture tube, and back-extracted with 0.5M HCl for 5 min. was vortex mixed and centrifuged. The supernatant was After centrifugation, the acidic layer was neutralized applied to a C18 solid phase extraction (SPE) cartridge with NaOH, or ammonia solution. Re-extraction with which had been conditioned with methanol followed by chloroform/isopropanol (95:5, v/v) was finally conducted water. Each cartridge was then washed with water and for 10 min. The organic phase was evaporated to 1% ammoniated methanol (v/v). They were evaporated dryness under a stream of nitrogen at 40°C. The dried to dryness under a flow of nitrogen in a heating block residue was dissolved in 10 mM ammonium acetate at 40°C. The residue was dissolved by initial mobile (pH 4.3) solution. phase. Alakloids from Pilocarpus sp. Were extracted with 10% ammonia; after 15 min extraction is carried out 3.10. Quinolizidine alkaloids Alkaloid extraction procedure was proposed by Wink 3 times with CHCl3; the pooled organic extracts are et al. [79]. According to this method, plant material re-extracted twice with 2% H2SO4; the pooled acid was homogenized in 0.5 M HCl. After 30 min at room extracts are adjusted to pH 12 with NH4OH and extracted temperature, the homogenate was centrifuged for twice with CHCl3 [73]. 10 min at 10 000 x g. The supernatant was made alkaline 3.8. Xanthine alkaloids by adding ammonia or 2 M NaOH and was applied to

Caffeine from traditional Chinese medicinal prescriptions Extrelut columns. Alkaloids were eluted with CH2Cl2 and which contain Theae folium was extracted by SPE the solvent evaporated in vacuo. [74]. In the procedure, the SPE C18 cartridges were Liu et al. used SPE for extraction of quinolizidine washed with water and then dichloromethane was alkaloids from dog plasma [80]. The sample was vortexed used to elute the compounds. The eluate was collected for 30 s and extracted with OASIS HLB Extraction and concentrated under reduced pressure to dryness. Cartridges under reduced pressure. Activation of the Finally, the residue was dissolved in 50% methanol. cartridges was achieved by sequential washing with The sequential extraction of samples containing methanol and distilled water. The cartridges loaded with was applied [75]. First samples with 50% the samples were firstly washed with 2% acetonitrile methanol, than with 75% methanol, and finally with 100% solution and then were eluted with acetonitrile. The methanol for 20 min at 60°C were extracted. After each eluate was reduced to dryness using a centrifugal extraction step, the sample was centrifuged at 10°C vacuum concentrator. The residue was reconstituted in for 10 min and the supernatant collected and the solid mobile phase.

807 808 A. Petruczynik Analysis of alkaloids from different chemical groups by different liquid chromatography methods

4. Thin layer chromatography (TLC) Centrifugal TLC (silica, MeOH/CHCl3 or AcOEt/hexane/ ammonia) was successfully applied for isolation of TLC is a chromatographic method widely used for alkaloids from Kopsia species [85]. Subramaniam qualitative rather than quantitative analysis of alkaloids, et al. have analyzed monoterpenoid indole alkaloids isolation of individual substances from multicomponent from Kopsia singapurensis by centrifugal TLC by use mixtures, and preparative-scale isolation. TLC provides different mobile systems [86]. Monoterpenoid indole a chromatographic plant extract and drug fingerprint. alkaloids in extract are separated Multiple samples can be analyzed at the same time on by TLC (on silica plates and eluent containing: ethanol

a single TLC plate, reducing the time of analysis and (EtOH), CHCl3, ammonia) and identified based on

solvent volume used per sample. TLC with densitometry their Rf values, as well as on their chromogenic is the method most frequently applied for quantitative reaction to ceric ammonium sulfate spray reagent analysis of biological samples, e.g. plant extracts. [87]. TLC was applied for micropreparative isolation of Advances in instrumental high-performance thin-layer indole alkaloids from yunnanensis [88]. The chromatography (HPTLC) have resulted in increasing authors used preparative silica plates and a mixture application of planar chromatography in quantitative of AcOEt, MeOH and diethylamine (DEA) as mobile analysis. The possibility of separation of components phase. Micropreparative TLC was used for isolation present in a mixture and the simultaneous handling of of ervatamine-type indole alkaloids from Ervatamia

a large number of samples has led to extensive use of officinalis in system: silica gel as adsorbent and CHCl3/ HPTLC for analysis of natural samples. TLC is the easiest MeOH as eluent. TLC method was applied to purification technique with which multidimensional separations can of isolated alkaloids on silica plates in eluent systems:

be performed. Particularly valuable separation results CHCl3/MeOH or petroleum ether/AcOEt/DEA [89]. On can be achieved when using various stationary and preparative silica gel plates with mixtures of MeOH and

mobile phase systems as an advantage over different dichloromethane or CHCl3 and AcOEt/hexane fractions separation mechanism. TLC coupled with densitometry of plant extract from Strychnos cathayensis were can be used for quantitative analysis of investigated purified [90]. For isolation of henricinols (indole alkaloids compounds. TLC also was often used for preparative obtained from Melodinus henryi) silica preparative

isolation of alkaloids, purification of multicomponent plates and mixture of AcOEt and CHCl3 was used [91]. samples and control of the separation efficiency of the The alkaloidal fraction obtained from Tabernaemontana different chromatographic methods. Nowadays, HPTLC catharinensis was analyzed on silica plates by use

is a routine analytical technique. mixture of MeOH and CHCl3 [92]. Isolated alkaloids Most TLC procedures for analysis of indole alkaloids were identified by UV-ViS or IR spectra. Silica gel plates use an adsorbent stationary phase such as silica gel, were applied for purification of indole alkaloidal fraction often with fluorescence agent added, since all indole obtained from leaf extracts of Rauvolfia bahiensis [93]. alkaloids adsorb UV light and mobile phase containing The alkaloidal extract of herbacea was purified by

strongly polar modifier (methanol, ethanol), medium preparative TLC on neutral Al2O3 in toluene/AcOEt/DEA or weakly polar diluent (toluene) and addition of basic solvent system [26]. Two curarizing quaternary indole compounds such as ammonia. Clavine and alkaloids from Strychnos quianensis were analyzed alkaloids were identified on silica gel as stationary on silica gel plates in eluent system containing MeOH,

phase and in chloroform (CHCl3), methanol (MeOH), Me2CO and MeCOONa [94]. Purification of extract from

ammonia mixture or ethyl acetate (AcOEt), MeOH, H2O, Strychnos moandaensis was performed on preparative

dimethyloformamide (DMF) as mobile phases [81]. TLC silica gel plates in mixtures of MeOH/CHCl3 as eluent separation of monoterpenoid oxindole alkaloids was [95]. For isolation of antileishmanial active indole

achieved on silica gel with CHCl3 and acetone mixture alkaloids from Aspidosperma ramiflorum preparative

as eluent [82]. Two indole alkaloids, 12-methoxykopsine silica gel plates and mixtures of MeOH/CHCl3 or CHCl3/ and danuphylline B, were obtained from the leaf extract AcOEt/MeOH/triethylamine were applied [96]. For of the Malayan Kopsia species, K. arborea, and isolated separation of indole alkaloids by TLC, gradient elution on silica plates by centrifugal TLC method with mobile was successfully used. Components of plant extract

phases containing: CHCl3/MeOH, AcOEt/Hexane, or from Cicer arietinum were separated on silica gel in

diethyl ether (Et2O)/hexane/ammonia [83]. Fractions eluent gradient system containing CHCl3/MeOH/H2O of Alstonia angustiloba plant extract, containing indole with increasing polarity [97]. Gradient TLC elution was alkaloids, were re-chromatographed by centrifugal used for separation of indole alkaloids TLC using different nonaqueous eluent systems [84]. from Psychotria stachyoides [98]. Mixtures containing

807 808 A. Petruczynik Analysis of alkaloids from different chemical groups by different liquid chromatography methods

CH2Cl2/MeOH/NH4OH as eluent with increasing polarity been employed for the analysis of alkaloid extracts from were applied. Aspidosperma and Hunteria type indole [113], Solanum tuberosum [114], alkaloids were separated on silica gel with AcOEt/ Ceropegia juncea [115] and Erythroxylum emarginatum hexane [99]. Lundurines, cytotoxic indole alkaloids [116]. Bringmann et al. have separated alkaloids from Kopsia tenuis, were isolated initially by column from Erythroxylum zeylanicum plant extract using chromatography, followed by re-chromatography using preparative silica gel plates and eluents containing, e.g. centrifugal TLC with mixtures: Et2O/hexane, Et2O/ MeOH/CH2Cl2, MeOH/AcOEt/ammonia, and MeOH/

MeOH, CHCl3/MeOH as eluents [100]. Centrifugal TLC CHCl3/water [117]. The procedure of separation and method was used for re-chromatography of partially quantification of tropane alkaloids from Datura species resolved fractions containing macroline indole alkaloids by TLC has been described by Mroczek et al. [118]. from Alstonia angustifolia [101]. Solvent systems used Alkaloids were separated on silica gel HPTLC plates for separation of the alkaloids were mixtures containing with two mobile phase systems: acetone/MeOH/water/

MeOH, EtO2, CHCl3, and hexane saturated NH3. ammonia and MeCN/MeOH/HCOOH. Fig. 1 presents Fractions of Ambelania occidentalis plant extract were densitograms of the plates obtained for separation of purified on preparative silica gel plates using AcOEt/ mixture of alkaloid standards and Datura fastuosa plant

CHCl3 or MeOH/CHCl3 [102]. Monoterpenoid indole extract. The authors compared quantitative results for alkaloids from Catharanthus roseus plant extract were investigated alkaloids obtained by HPLC and HPTLC separated on silica gel with various solvent systems and received good correlation between both methods. and the radioactivity was visualized and quantified by Singh et al. proposed a TLC densitometric method for exposure of the TLC to a storage phosphor screen the determination of scopolamine in chromatographic

[103]. Mixtures of CH2Cl2 with MeOH, acetone or hexane system: silica gel HPTLC plates and aqueous mobile were applied as eluents on silica gel for separation of phase - acetone/MeOH/water/ammonia [119]. indolo[2,3a]quinolizine alkaloids [104]. Indole alkaloids TLC of mescaline was carried out on silica gel plates from Kopsia arborea were initially isolated by column in the eluent system: CHCl3/BuOH/ammonia [120]. Fast chromatography followed by re-chromatography of determination of colchicine from pharmaceuticals and partially resolved fractions using centrifugal TLC in vegetal extracts was performed by TLC-densitometry solvent systems containing Et2O/hexane or AcOEt/ [121]. The extract was separated on silica gel layers hexane saturated NH3 [105]. Indole alkaloids from with a mixture of acetone/CHCl3/DEA as a mobile phase. different cyanobacteria were isolated on preparative HPTLC determination of colchicine in a pharmaceutical silica gel plates with mobile phase containing AcOEt/ formulation has been reported [122]. Analysis was

MeOH/H2O [106]. The quantification of yochimbine in performed on silica gel plates with MeCN/AcOEt/water Pausinystalia yochimbe was performed on silica HPTLC /HCOOH. Ephedra alkaloids from Sida species were plates with mixture of toluene/AcOEt/DEA [107]. Thoden determined on nano silica HPTLC plates with toluene/ et al. proposed the use of centrifugal TLC for fractionation AcOEt/DEA as eluent [123]. of Crotalaria species plant extracts containing pirolizidine Opium alkaloids were chromatographed on silica alkaloids [108]. As eluent mixtures of MeOH and CH2Cl2 gel plates with nonaqueous eluent containing toluene/ and silica gel plates were used. acetone/EtOH/ammonia [124]. Samples containing Silica gel plates and nonaqueous eluents were alkaloids were analyzed by TLC, subsequently by HPLC commonly used for analysis of tropane alkaloids. and additionally identified by MS (Fig. 2). Dimeric tropane alkaloids were analyzed in normal Piperine was determined on silica plates by use of phase system containing silica gel as stationary phase petroleum ether, CH2Cl2 and HCOOH [125]. The plates and mixture of MeOH/CHCl3 as mobile phase [109]. were saturated in eluent vapors for 20 minutes before On silica gel plates tropane alkaloids from Merremia developing of chromatograms. genus plant extracts in eluent containing CHCl3/ A simple spectrophotometric-TLC method was used MeOH/ammonia were isolated [110]. TLC separation for determination of colchicine in Colchicum species of alkaloids from Schizanthus litoralis was performed [126]. Analysis was performed on silica gel plates with on silica gel with mixtures of CHCl3/Me2CO or CHCl3/ eluent consisting with acetone/CH2Cl2/DEA. Ellington et

Me2CO/ammonia and on aluminum oxide with Et2O/ al. have separated colchicine from genus Androcybium

EtOH [111]. Brachet et al. described separation of on preparative silica gel plates with MeOH/CH2Cl2 in an tropane alkaloids from Erythroxylum lucidum on silica ammonium-saturated atmosphere [127]. gel with use of Me2CO/ammonia and on aluminum In TLC especially on plates with polar bonded oxide using Et2O/EtOH [112]. TLC on silica gel plates phases (CN-, Diol-, and NH2-silica), two-dimensional and eluents containing MeOH/CHCl3/ammonia have separations can be realized using NP adsorption

809 810 A. Petruczynik Analysis of alkaloids from different chemical groups by different liquid chromatography methods

(a) systems with non-aqueous eluents and RP partition systems with aqueous eluents, which allows for better or full separation of complex mixtures. The method can be applied during the analysis of complex plant mixtures and allows selected compounds to be identified by retention coefficients in two directions. The application of the most selective systems in 2D-TLC separation of standards’ mixture of isoquinoline alkaloids and alkaloid extracts from herb of Fumaria officinalis is presented in Fig. 3 [128]. Special modes of developing a TLC chromatogram (b) such as automated multiple development (AMD) were also used for analysis of alkaloids. AMD is an instrumental technique of planar chromatography which uses an eluent gradient starting from the most polar to the least polar. The migration is performed by successive steps and at each new development the proportions of the eluent constituents change; so the polarity is decreasing when the distance increases. Gradient development with a linear eluotropic profile leads to a band re-concentration improving the separation. A successful separation depends mainly on the choice of the solvent components and optimization of the Figure 1. HPTLC-densitometric assay of the mixture of (1a) scopolamine–N-oxide (Sk–NO), scopolamine–N-methyl shape of the gradient. The stepwise movement of the bromide (Sk-Me), l-hyoscyamine (H) and scopolamine elution front and the repeated developments increase (Sk). Stationary phase: silica gel 60 F254 HPTLC plates, 20×10 cm, 0.25 mm thickness; mobile phase: the resolution. AMD has proved to be an efficient planar (a) acetone–methanol–water–25% ammonia chromatographic technique that provides increased (82:5:5:8, v/v), then after evaporation of the solvents; (b) acetonitrile–methanol–85% formic acid (120:5:5, separation for compounds with neighboring structures. v/v). Scan was recorded at 205 nm [35]; and (1b) of Pothier and Galand applied AMD for separation of l-hyoscyamine (H) and scopolamine (Sk) in the extract opium alkaloids by using different solvents as eluents from of Datura fastuosa purified by SPE procedure. Details as in Fig. 1a [35]. (Fig. 4) [129].

Table 1. Methods used for TLC of alkaloids.

Alkaloids Source Stationary Eluent Detaection Ref. phase

Fluorinated synthesis SiO MeOH/CH Cl UV [139] quinine alkaloids 2 2 2 Quinoline UV and Dragendorff synthesis SiO Toluene/AcOEt [140] alkaloids 2 reagent The plates were sprayed with 10%

Quinine Khaya anthotheca SiO2 Me2CO/hexane H2SO4 reagent (in [141] EtOH) and heated for detection.

Et2O/hxane/ Piperine standards SiO2 UV [142] CH3COOH Curcuma longa,

Piperine Capsicum annuum, SiO2 Benzene/EtOH/water/CH3COOH UV [143] Piper nigrum

Piperine Polyherbal formulation SiO2 Toluene/AcOEt UV [144] CHCl /CH Cl / Methylxanthines Different types of tea SiO 3 2 2 UV [145] 2 iPrOH Caffeine, n-buthanol/water/ Coffea arabica cellulose Radioactivity [146] CH3COOH

CHCl3/EtOH/acetone/water/ Caffeine Energy drinks SiO2 UV-Wis [147] CH3COOH

809 810 A. Petruczynik Analysis of alkaloids from different chemical groups by different liquid chromatography methods

Over Pressured Layer Chromatography (OPLC) is 5. High Performance Liquid nearly equivalent to HPLC. It is a planar chromatographic Chromatography (HPLC) method using a pressured chamber in which the vapor phase above the sorbent is practically eliminated. In recent years there has been a remarkable The eluent is pushed through the sorbent layer and development in the format of the chromatographic continuous development can be performed by a pump. columns and stationary phases, as well as highly The method was applied by Mincsovics for separation specialized organization of HPLC components. Currently of xanthine alkaloids in black tea [130]. The separation HPLC is the most versatile and most widely applied was performed on silica layer with eluent containing technique in the analysis of natural products including toluene and CH3COOH. Fig. 5 presents schematic alkaloids. In most cases, compounds are detected with diagram of bilateral band compression – the method ultraviolet (UV) detectors or photodiode array detectors used in this study. Bilateral band compression has been (DAD). Sophisticated coupled techniques like HPLC- used to increase the sensitivity of detection of minor mass spectrometry (LC-MS) and HPLC-molecular components of a mixture. magnetic resonance (HPLC-NMR) are increasingly Recently, TLC method was conducted with mass used in the analysis of alkaloids. HPLC is also useful for spectrometry (MS). The TLC/MS combination has the quantification of alkaloids for pharmacokinetic studies. potential to provide low-level, highly specific detection of targeted compounds and molecular mass and structure 5.1. Normal-phase LC determinations for unknowns. TLC/MS has been NP-HPLC is rarely applied in separation of alkaloids. employed for the analysis of xanthine alkaloids. Caffeine However, the use of these systems for the analysis of was analyzed in Ilex vomitoria extract on C18 plates with alkaloids is sometimes reported. Usually silica column mixture of MeOH and water as mobile phase by TLC/MS and strongly polar modifier (acetonitrile) and medium [131]. Aranda and Morlock described determination of polar diluent (dichloromethane) are used for these caffeine in pharmaceuticals and energy drink samples purposes. For the analysis of indole alkaloids from by TLC coupled with MS [132]. Schematic diagram Haraldiophyllum species, a chromatographic system of the interface components is presented in Fig. 6. containing cyanopropyl stationary phase and mixture of Analysis was performed on silica gel plates with eluents 2-propanol/hexane as mobile phase was used [148]. containing MeOH/formate buffer at pH 4.0. The TLC/MS Rarely, for analysis of alkaloids, silica stationary was also applied for determination of caffeine, codeine phase coated with metal ions was used. Piperine isomers and ephedrine on C18 plates and eluent consisting with were separated on a silver-modified cation-exchange MeOH and water [133]. ligand-covered silica material as stationary phase with Shariatgorj et al. have applied thin-layer eluent system containing MeCN/iPrOH/hexane [149]. chromatography/laser desorption ionization mass spectrometry for facile separation and identification 5.2. Reversed-phase LC of quaternary protoberberine alkaloids from Berberis The optimization of alkaloids’ analysis in RP systems barandana [134]. The silica gel TLC plates were consists in reducing of ion-exchange interactions developed with buthanol, water and CH3COOH. between basic analytes and residual surface silanols. A further field of application of TLC is the purification There are several methods to achieve the reduction of of chromatographic fractions. Tood et al. described the ionic interactions such as: using a mobile phase separation of tropane alkaloids from Convolvulus at low pH (suppression of silanols’ ionization); using arvensis on preparative silica gel plates using eluent a mobile phase at high pH (suppression of alkaloids’ consisting with MeOH/CHCl3/water/ammonia [135]. The ionisation); addition of ion-pairing reagent to mobile extract from was chromatographed phase (formation of non-polar, non-charged ion-pairs on preparative TLC silica gel plates [136]. Alkaloids with analyte); addition of relative strong bases to eluent were eluted with mixture of CHCl3/DEA. The alkaloidal playing the role of silanol blockers and/or alkaloids fractions from Erythroxylum species were separated on ionization suppressants; selecting a stationary phase. preparative silica gel plates in eluent system containing Some alkaloids were separated in RP system with

MeOH/CHCl3 [137]. Mixture of MeOH/CHCl3/ammonia eluents containing only organic modifier and water. was applied on silica gel preparative plates for purification The ergot alkaloids, fungal secondary metabolites of of officinarum plant extract [138]. significant toxicological and pharmacological activities, Table 1 covers some selected TLC methods for were successfully analysed on C18 stationary phase analysis of alkaloids from different classes. with a gradient of 20-70% MeCN in water [150]. The

811 812 A. Petruczynik Analysis of alkaloids from different chemical groups by different liquid chromatography methods

Figure 2. Thin layer chromatography (TLC), high-performance liquid chromatography (HPLC) and liquid chromatography/mass spectrometry (LC/MS) analyses of : (a) TLC of three families indicates the presence of the novel salutardine band not seen in the control. The track labeled standards includes , codamine and laudanine in order of increasing distance from the origin; (b) HPLC confirmed the novel accumulating alkaloid as salutaridine, the salutaridine peak is clearly present at just under 7.6 min retention time, and is absent in the control sample [124]. analyses of alkaloids from Psychotria leiocarpa were Rarely, indole alkaloids were chromatographed in carried out using C18 column and mixture of MeOH / eluent system containing buffer at neutral pH. Mixture water as eluent [151]. The eluent containing only MeOH of MeOH and phosphate buffer at pH 7.0 was applied and water was applied for separation of indole and for separation of oxindole alkaloids from leaves of carbazole alkaloids in Glycosmis montana plant extract Mitragyna inermis [161]. Tang et al. used for analysis of [152] and from Alstonia scholaris [153]. catharanthine and vindoline from Catharanthus roseus Xanthine alkaloids were often successfully analysed an eluent system containing MeCN and phosphate on RP columns in simple eluent systems containing only buffer at slightly acidic pH (6.0) [162]. Eluent system organic modifier and water. Caffeine, and containing buffer at pH 6.5 has been employed for the were determined in food samples by HPLC analysis of colchicine in a human specimen [163]. Mobile in eluent system containing MeCN and water [154-156] phase containing mixture of MeOH and water was or MeOH and water [157-159]. The use of a monolithic applied for determination of nicotine [164]. The addition column provided excellent and rapid separation of of ammonium carbonate to the mobile phases was caffeine from the endogenous sample components often used for analysis of ergot alkaloids. Separation of and from structurally similar compounds in less than a these compounds from was carried minute [160]. on C18 column with eluent containing MeCN/water/

811 812 A. Petruczynik Analysis of alkaloids from different chemical groups by different liquid chromatography methods

Figure 3. Videoscans of chromatogram (a) for standards of alkaloids scanned at 366 nm and 254 nm. Eluent systems: I direction: 60% MeOH in water + 2% ammonia; II direction: 10% MeOH in diisopropyl ether + 2% ammonia; and (b) of Fumaria officinalis herb extract scanned at 366 and 254 nm. Eluent systems: I direction: 60% MeOH in water + 2% ammonia; II direction: 10% MeOH in diisopropyl ether + 2% ammonia [128].

(NH4)2CO3 [165-168]. Wang et al. have used for analysis phosphate buffer and (NH4)2SO4 [173]. Nicotine, cotinine of ergot alkaloids the mobile phase consisting of MeCN, and related alkaloids were determined in human urine water and CH3COONH4 [169]. Ergot alkaloids were also and saliva by LC-MS on C18 column with MeOH and analysed with mobile phase containing MeCN/water/ aqueous solution of HCOONH4 [174]. tartaric acid/ammonium chloride [170]. Zhang et al. described LC/ESI-MS method for Quinine was determined on C18 column with eluent determination of isoquinoline alkaloids from Tinospora containing MeOH, MeCN, water and ammonium formate sagittata and Tinospora capillipes [175]. Separation of [171]. Ammonium acetate was added to mixture of alkaloids was performed on C18 column with eluent MeOH and water for separation of nicotine and related consisting of MeOH, MeCN, water and ammonium alkaloids in human plasma [172]. The simple method acetate. should be useful for monitoring tobacco exposure, for The mobile phase containing MeCN/water/ nicotine pharmacokinetic studies, and for determining CH3COONH4 has been employed for determination the usefulness of nicotine biomarkers, including of hepatotoxic pyrrolizidine alkaloids in a medicinal metabolite ratios. Nicotine was also analyzed on C18 plant - Symphytum officinale on C18 column by LC-MS column with multicomponent eluent containing MeOH, [176]. The eluent containing addition of HCOONH4 to a

813 814 A. Petruczynik Analysis of alkaloids from different chemical groups by different liquid chromatography methods

Figure 4. HPTLC chromatogram by AMD of opium extract and standard alkaloids of opium: (1) morphine; (2) codeine; (3) ; (4) papaverine; (5) noscapine; (6) opium extract; eluent used was universal gradient: methanol 100, methanol–dichloromethane 50/50, dichloromethane 100, dichloromethane 100, hexane100; derivatization by Dragendorff reagent [129].

mixture of MeOH and water was used for determination of quinolizidine alkaloids and oxymatrine in rat blood and derma by LC-MS [177]. , a polyhydroxy alkaloid, was chromatographed by LC-MS/MS on C18 column with the

mobile phase containing MeOH, water and CH3COONH4 [178]. Often, particularly in toxicological investigation, there is a need for simultaneous determination of alkaloids belonging to different chemical groups in complex matrices. Plant alkaloids from different classes in serum and urine were separated on C18 column in eluent system MeCN/phosphate buffer at pH 6.5 (Fig. 7) [179].

5.2.1. Mobile phases at acidic pH The use of mobile phase at acidic pH by addition of appropriate buffers or acids suppresses the ionization of free silanol groups on silica surface, which reduces ion exchange mechanism of retention. In the acidic mobile phases, decreases of retention of alkaloids, more symmetrical peaks and increases in system efficiency were often obtained. Indole alkaloids were often analysed on C18 column in eluent systems containing addition of acid or buffer at Figure 5. Schematic diagram of bilateral band compression. acidic pH. In these conditions, alkaloids are in ionic form, A, initial period of the process; B, final stage of the but dissociation of free silanol groups is suppressed. C8 compression. 1, foam to transfer the mobile phase for band compression; 2, mobile phase fronts. Arrows stationary phase and mobile phase containing MeOH, represent eluent movement for band compression; the water and trifluoroacetic acid (TFA) was used for arrow with curly brackets at the end indicates the effect on minor component [130]. separation of alkaloid brachycerine from callus cultures

813 814 A. Petruczynik Analysis of alkaloids from different chemical groups by different liquid chromatography methods

Table 2. Methods used for HPLC of alkaloids with mobile phase at acidic pH.

Alkaloids Source Stationary Eluent Detection Ref. phase

Cinchonine, MeOH/water/CH COONH / synthesis C18 3 4 ESI-MS [211] cinchonidine CH3COOH MeCN/MeOH/water/CH COONH / Fluorescence Quinine tablets C18 3 4 [212] perchloric acid (pH3.0) detection MeCN/MeOH/water/sodium Fluorescence Quinine gels C18 [213] perchlorate/perchloric acid (pH 2.5) detection MeCN/MeOH/water/CH COONH / Fluorescence Quinine pellets C18 3 4 [214] perchloric acid (pH3.0) detection Quinine alkaloids

and their Human plasma C18 MeCN/water/HCOONH4/HCOOH ESI-MS [65] metabolites MeCN/MeOH/water/CH COONa/ Cotinine Serum, urine C8 3 UV [215] CH3COOH/citric acid/TEA (pH4.4)

Nicotine Mushroom C18 MeOH/water/CH3COONH4 (pH 3) UV [216] Nicotine and Human plasma and PFP MeOH/water/ CH COONH /HCOOH MS [69] metabolites urine 3 4 Nicotine, cotinine, trans- 3’hydroxycotinine and norcotinine Human plasma C18 MeCN/water/ CH3COONH4/HCOOH MS/MS [217]

Piperine and its Urine CN MeOH/water/CH COOH MS/MS [218] metabolites 3 Piperine Rat plasma C18 MeCN/water/HCOOH MS/MS [219] Neonantal biological Arecoline C18 MeCN/water/CH COONH (pH 4.3) MS [72] matrices 3 4 Pilocarpine Ocular hydrogels C18 MeCN/water/TFA UV [220] MeCN/water/HCOONH / HCOOH Pilocarpine Pilocarpine species C 2 4 MS/MS [73] (pH 4) Steroidal alkaloids Sarcococca coriacea C18 MeCN/water/HCOOH MS/