A General Description of Apocynaceae Iridoids Chromatography

Iridoids are considered atypical monoterpenoid compounds, based on a methylcyclopentan[C]-pyran skeleton, often fused to a six-membered oxygen ring consisting of ten, nine or in rare cases, eight carbon atoms (Figure 1a) [1, 2]. More than 2500 iridoid compounds have been described in nature, with structural differences related mainly to the degree and type of substitution in the cyclopentane ring skeleton [3]. Iridoids can be found in nature as secoiri‐ doids (Figure 1b), a large group characterized by cleavage of the 7,8-bond on the cyclopentane ring, glycosides, mainly 1-O-glucosides, and nor-iridoids, originating from oxidative decar‐ boxylation on C10 or C11 (Figure 1) [3, 4]. A General Description of Apocynaceae Iridoids Chromatography


Iridoids
Iridoids are considered atypical monoterpenoid compounds, based on a methylcyclopentan-[C]-pyran skeleton, often fused to a six-membered oxygen ring consisting of ten, nine or in rare cases, eight carbon atoms ( Figure 1a) [1,2]. More than 2500 iridoid compounds have been described in nature, with structural differences related mainly to the degree and type of substitution in the cyclopentane ring skeleton [3]. Iridoids can be found in nature as secoiridoids ( Figure 1b), a large group characterized by cleavage of the 7,8-bond on the cyclopentane ring, glycosides, mainly 1-O-glucosides, and nor-iridoids, originating from oxidative decarboxylation on C 10 or C 11 ( Figure 1) [3,4].

Iridoids
Iridoids are considered atypical monoterpenoid comp six-membered oxygen ring consisting of ten, nine or compounds have been described in nature, with struc cyclopentane ring skeleton [3]. Iridoids can be found i of the 7,8-bond on the cyclopentane ring, glycosid decarboxylation on C10 or C11 ( Figure 1) [3,4].  Iridoids are derived from isoprene units¸ which are considered the universal building blocks of all terpenoids, formed through intermediates of the mevalonic acid (MVA) pathway in the citosol, and the novel 2-methyl-D-erythritol 4-phosphate (MEP) pathway in the plastids of plant cells [2,5,6]. The participation of two pathways in iridoid biosynthesis has not yet been clarified, but recent analyses have described the major role of the MEP in the yield of the source for the iridoid isoprene units when compared with the MVA pathway [7,8,9]. Iridoid biosynthesis shows two pathways, called route I and II, in which secoiridoids and carboxylated or decarboxylated iridoids are formed, respectively. Route I, considered the main pathway, is responsible for yielding the precursor of the carboxylic iridoids, from iridodial which is oxidized a iridotrial and subsequently converted to a series the iridoids, as occurs in loganin, secologanin, derived secoiridoids, and complex indole alkaloids. In route II, the presence of 8epi-iridodial, 8-epi-iridotrial and 8-epi-deoxyloganic acid have been reported, forming a source of decarboxylated carbocyclic iridoids such as aucubin and catalpol [10,3,11,12].
The distribution of iridoids in the Eudicotyledoneae has potential usefulness in the taxonomy of the families, related to their presence in a restricted number of families. Iridoid are considered good chemotaxonomic markers of different taxononomic groups, and can be used, in combination with order, tribe and family, to establish the phylogenetic relationship [22,23,10,3,24,25].
According to an update of the Angiosperm Phylogeny Group (APGIII) [28,29], the presence of iridoids has been reported in approximately fifty plant families, and can be considered as one of the synapomorphies of the Asterid clade. It is divided into Lamiids, which presents iridoids of the Gentianales, Garryales and Lamiales orders, and Campanulids, which presents secoiridoids of the Asterales and Dipsacales orders. The Gentianales order comprises five families: Apocynaceae, Gelsemiaceae, Gentianaceae, Loganiaceae and Rubiaceae (APG III, 2009), highlighted for the diversity of their iridoids [11,29,30].

Thin layer chromatography
Preparative chromatography was performed with thin layer chromatography (TLC) aluminum sheets and 8:2 chloroform/ methanol as mobile phase of a fraction from the 95% EtOH extract of stems of Alstonia macrophylla. This procedure led to the isolation of the compounds sweroside (2 mg) and naresuanoside (3 mg) [128] Another interesting application of preparative chromatography is described in [47] with ethanol extract of ground stems of A. schottii. This extract was fractionated by bioassay monitoring and after successive liquid-liquid partition and flash column chromatography; the authors obtained the iridoids allamandin; allamcin and a mixture of plumericin and isoplumericin. This separation was performed with a Chromatotron rotor (silica gel (Si gel), 2-mm) with 1 % methanol in chloroform as solvent system.
The bioassay-guided fractionation of the extracts of Plumeria rubra barks also proved to be a successful strategy, leading to the isolation of eleven substances, of which eight were iridoids. In this context, the aqueous extract of Plumeria rubra bark has iridoids such as the epimers, αallamcidin and β-allamcidin, which were resolved by preparative TLC on Si-gel G plates (20 × 20 cm, 250 µm, Merck ® ), using chloroform/ ethyl acetate/ methanol (3:3:1) as solvent system [44].
Ferreira and coworkers [95] describe the HPTLC analysis of the bark, latex and leaf extracts and substances of Himatanthus sucuuba. The solutions of the extracts in methanol (10 mg mL -1 ) and the isolated iridoids (3 mg mL -1 -plumieride; 1 mg mL -1 -isoplumieride) were applied (5 µL) using Linomat IV, a Camag semi-automatic spotter. The analysis was carried out on a precoated silica gel 60 F254 (Merck) HPTLC plate (0.2 mm of layer thickness and 10 × 10 cm size) using chloroform/ methanol (8:2) as developing system. The resulting chromatogram was dried and the spots were visualized by spraying with vanillin -sulfuric acid solution, followed by heating at 100 °C. Table 3 shows other studies using TLC analysis of Apocynaceae iridoids.

Open column chromatography
The methanol extract of the leaves of Cerbera manghas and its fruit contain the iridoids theveside and theviridoside, as described in [124]. The methanol extract of the leaves, after the addition of water, was sequentially partitioned with chloroform, acetic acid and butanol. This extract and the aqueous phase were submitted to column chromatography with charcoal and water/ methanol as eluent. Theveside was isolated from the aqueous phase. Fractions of the butanol extract, which turn blue after heating with mineral acid, were chromatographed over a silica gel column with a gradient of increasing polarity of chloroform/methanol to afford theviridoside.
Cerberidol, epoxycerberidol, cyclocerberidol, cerberidol-3-O-β-D-allopyranoside, cerberidol-3,10-bis-O-β-D-allopyranoside, epoxycerberidol-3-O-β-D-allopyranoside and cyclocerberidol-3-O-β-D-allopyranoside are present in the leaves of C. manghas [55]. The methanol extract from 1.45 kg of dried leaves was concentrated, re-suspended in water, and sequentially partitioned with benzene and chloroform. The aqueous phase was chromatographed using MCI-gel column (elution with gradients of water/methanol) and the fraction eluted with 20% methanol was chromatographed in two steps: using RQ-1 (Fuji-gel phase) column (elution with water/acetonitrile) and using a silica gel column (elution with gradient of chloroform/ methanol/water 7:3:1 to 7:3:1. The iridoids cerbinal, cerberic acid and cerberinic acid are found in the methanol extract of the bark of C. manghas [67]. The crude extract obtained by percolation from 4 kg of stem bark and 1.9 kg of root bark were diluted with water to 50% water:methanol. The mixture was washed with hexane and partitioned with benzene. Benzene fractions were re-suspended in methanol and cerbinal (120 mg from stem bark extract and 300 mg from root bark extract) precipitated. The supernatant was chromatographed on a silica gel column and cerbinal, cerberic acid and cerberinic acid were eluted with benzene/acetone.
The iridoids isoplumericin, plumericin, plumieride, plumieride coumarate and plumieride coumarate glucoside can be detected and quantified in several species of Plumeria and Allamanda by TLC using silica gel 60 and the following mobile phases: benzene/ethyl acetate 4:1, chloroform/methanol 4:1, chloroform/methanol 7:3, propanol/ethyl acetate/water 7:2:1 [84]. Visualization of the chromatograms is achieved by spraying with 50% sulfuric acid/ ethanol solution and heating. For the analyses, it is necessary to use iridoids as standards that can be isolated from the roots of Allamanda cathartica. For the isolation of iridoids during the development of the method, chloroform and methanol extracts, sequentially obtained in a Soxhlet apparatus from the root bark and inner roots of Allamanda cathartica (15.0 g for each extract) were successively fractionated on column chromatography. In the first chromatography step, the chloroform extract from the bark (1.7 g) was applied to a silica gel column (80 g) and eluted with gradient of petrol, ethyl ether, chloroform and methanol. This procedure yielded a mixture containing isoplumericin and plumericin (160 mg). Fractions eluted with chloroform/ methanol (3:2) were mixed with the methanol extract of the bark (total mass: 2.8 g) and chromatographed on a silica gel column (150 g deactivated with water) with gradient of chloroform and methanol as mobile phase to furnish plumieride coumarate (150 mg), plumieride coumarate glucoside (480 mg) and two mixtures: one containing plumieride and plumieride coumarate, and the other containing plumieride and plumieride coumarate glucoside. The first mixture was further resolved on partition between water and ethyl acetate and afforded 180 mg of plumieride and 150 mg of plumieride coumarate. The other mixture was rechromatographed to afford plumieride (300 mg) and plumieride coumarate glucoside (410 mg). Neither the chloroform (810 mg) nor the methanol (920 mg) extracts from the inner part of the roots contained isoplumericin and plumericin. These extracts were purified on silica gel column (75 g) deactivated with water and eluted with chloroform and methanol gradient, to give plumieride (70 mg), plumieride coumarate (80 mg) and plumieride coumarate glucoside (200 mg).
The fresh leaves of Cerbera manghas contain the iridoids 10-O-benzoyltheveside, 10-dehydrogeniposide, loganin, theviridoside and theveside [124]. For the isolation, the methanol extract obtained by percolation using 2.6 kg of fresh leaves was extracted with butanol, and this extract was partitioned with benzene. After partition, the remaining butanol fraction (22.8 g) was subjected to column chromatography using MCI-gel (CHP-20) as stationary phase and a gradient of methanol/water as eluent. The fraction eluted with 20% methanol was subjected to C-18 column (elution with acetonitrile/water) to afford 20 mg of 10-dehidrogeniposide and 20 mg of loganin. The fraction eluted with pure water (2.6g) in the first chromatographic step was also subjected to C-18 column (elution with acetonitrile/ water) to furnish 23 mg of 10-Obenzoyltheveside and 270 mg of theveside.
The iridoids, plumericin, isoplumericin, plumieride and fulvoplumierin, were present in the extracts of Plumeria rubra bark. After maceration of the powdered bark (3.5 kg) with dichloromethane/methanol (1:1) and pure methanol, the combined extracts were partitioned between water and ethyl acetate. To isolate the four iridoids, the organic layer was chromatographed twice in a column using silica gel and gradient of increasing polarity with hexane and ethyl acetate, ethyl acetate and methanol, and then pure methanol. The amounts of the compounds isolated were not reported [75]. The flowers of Plumeria rubra L. cv. acutifolia can provide plumericidine, as described by [97]. The ethanol (95%) extract, obtained from 2.9 kg of flowers, was successively partitioned with petroleum ether, ethyl acetate and butanol. The ethyl acetate fraction was sequentially submitted twice to column chromatography using silica gel and gradient of chloroform/ methanol as mobile phase. Chromatography on a Sephadex LH20 column yielded 20 mg of plumericidine.
According to [72], several iridoids can be isolated from the aerial parts of Plumeria obtusa: obtusadoid A, obtusadoid B, plumieridin A, 1α-plumieride, 15-demethylplumieride and plumieridine. The methanol extract (400 g) was obtained from 10 kg of the plant material and sequentially partitioned with hexane and ethyl acetate. The ethyl acetate extract was chromatographed using a silica gel column and gradients of hexane, ethyl acetate and methanol. The less polar fractions were rechromatographed in the same stationary phase, and eluted with hexane/dichloromethane (1:1) to afford obtusadoid A (6 mg), obtusadoid B (11.5 mg), plumieridin A (8 mg) and plumieridine (12 mg). The more polar fraction obtained in the first chromatography was filtered on a Sephadex LH20 column using methanol, and further submitted to RP-8 flash column chromatography. Elution with 50% methanol afforded 1αplumieride (22 mg) and 15-demethylplumieride (13 mg).
Plumieride also can be isolated from the bark of Plumeria bicolor [114]. Powdered bark (4 kg) was extracted in methanol, and the crude extract was washed with acetonitrile. The material was re-extracted with chloroform, and this extract was fractionated in column chromatography using silica gel (900 g) and different solvents of increasing polarity. Plumieride was eluted with chloroform/ethyl acetate (1:1) and recrystallized from methanol.
The bark of Plumeria bicolor also contains plumericin and isoplumericin, as described in [86]. The methanol extract (100 g), after washing with acetonitrile, was extracted with chloroform and chromatographed on a column containing 800 g of silica gel G (60-120 mesh). Elution was carried out using gradients of increasing polarities with benzene, chloroform and methanol. Plumericin and isoplumericin was recrystallized from methanol.
Isoplumericin and plumericin are present in the bark of Himatanthus sucuuba [88]. For the isolation of these iridoids, 95% ethanol extract (2 g), obtained from 50 g of plant material was submitted to column chromatography using silica gel and gradients of increasing polarities with hexane, ethyl acetate and methanol. After recrystallization, isoplumericin (18 mg) was obtained from ethyl acetate and plumericin (70 mg) from methanol.
The stem bark of Winchia calophylla contains loganin (1.25 g) [129]. The 95% ethanol extract (600 g) from the dried stem bark (10.5 kg) was partitioned between petroleum ether and water. The petroleum ether extract was submitted to acid-base extraction and after adjustment to pH 9-10 with ammonium hydroxide; the aqueous layer was extracted with petroleum ether, chloroform and butanol. The chromatography of the butanol fraction using silica gel H column led to the isolation of loganin.
The iridoids, scholarein A, B, C and D, can be obtained by the fractionation of the ethanol extract from bark of Alstonia scholaris [60]. The crude extract, obtained from 15 kg of the plant material, was partitioned between ethyl acetate and water. The organic layer (190 g) was sequentially chromatographed on a column. The first chromatography, over silica gel (2.1 kg) and using gradient of chloroform and ethyl ether, furnished five fractions (1)(2)(3)(4)(5). From fraction 2, 8 mg of scholarein B and 7 mg of scholarein D were isolated after silica gel chromatography and elution with petroleum ether/ethyl ether (3:1). From fraction 3, 25 mg of scholarein A and 60 mg of scholarein C were obtained after successive columns using silica gel and chloroform/ ethyl ether as stationary and mobile phases, respectively.

Gas Chromatography (GC)
In the study on iridoids, the technique of gas chromatography is generally used for analytical purposes. Gas chromatography represents an advantage over thin layer chromatography, particularly for detecting substances in small amounts, and mass spectrometry can be used to distinguish most iridoid and secoiridoid glucosides by fragmentation patterns [112].
For the gas chromatography-mass spectrometry studies, a Hitachi K-53 gas chromatograph and a Hitachi RMU-6 E mass spectrometer were used. The glass columns, 0.5 m x 3 mm in I.D., were packed with 1.5% OV-17 on 80-100-mesh Shimalite W AW/DMCS and 1.5 % OV-1 on 80-100-mesh Shimalite W AW/DMCS, and were used to the oleuropein-type glucosides detection. The authors considered the GC-MS identification of some iridoids was not satisfactory: any important peak different from the sugar moiety was detected in asperuloside and paederoside TMS-derivatives of amaroswerin, amarogaentin asperuloside and paederoside exhibited different retention times, but the same fragmentation pattern; separation using this technique was unsuccessful [112].
Aqueous extracts of different plant species with known presence of iridoid and secoridoid glucosides were analyzed by GC-FID and GC-MS [112]. One of these species was Allamanda cathartica var. schottii (Pohl) Rafill (Apocynaceae) cultivated in a greenhouse. Aqueous extracts obtained from 3-5 g of fresh plant material and hot water were treated in a column of charcoal (active carbon for column chromatography) for removal of sugars by elution with water. The sample was eluted with methanol and concentrated under reduced pressure. TMS-derivatives were prepared. For the analyses, a 1.5% OV-17 column with 1.8 m in length at 280 ºC was used. GC-FID and GC-MS (70 eV) showed the presence of one iridoid glycoside. The fragmentation pattern indicated that the original glucoside was plumieride, and that relative retention time was the same as that of asperuloside.
Isoplumericin, plumericin, plumieride, plumieride coumarate and plumieride coumarate glucoside can be detected by GC-FID [116]. For the development of the method, it was necessary to isolate these iridoids for use as standard. The methanol extract of Allamanda cathartica L. roots, obtained with 500 g of the dried plant material and boiling methanol, was submitted to silica gel 60 column (2.5 kg) with the eluents: petrol, petrol/ethyl ether, ethyl ether, ethyl ether/chloroform, chloroform/ methanol and pure methanol [116]. Fractionation was monitored by TLC [84]. The fractions were eluted with petrol/ethyl ether until chloroform/ methanol contained isoplumericin and plumericin (4.2 g). This mixture (1.0 g) was suspended in ethyl ether and rechromatographed on silica gel (40 g). Elution with petrol and a gradient of increasing polarity with petrol/ethyl ether furnished isoplumericin (250 mg), plumericin (140 mg) and a mixture of both (290 mg). A second fraction eluted with chloroform/methanol in the first chromatographic step (8.0 g) was resubmitted to column chromatography on silica gel (300 g). Elution with a gradient of chloroform and methanol led to the isolation of plumieride coumarate (5.1 g). The third fraction of the first chromatograph step (10.0 g), eluted with chloroform/methanol, was partitioned between water and ethyl acetate to give plumieride coumarate in the organic phase (3.8 g) and plumieride in the aqueous phase (1.8 g). Finally, 30.0 g of the forth fraction of the first chromatograph step, eluted with chloroform/methanol to methanol, was submitted to column chromatography on silica gel (1.0 kg) deactivated with water. The elution was carried out with a gradient of chloroform and methanol and furnished plumieride coumarate glucoside (6.2 g). GC-FID analyses were used to evaluate the pure grade of fractions and isolated substances. For these analyses, trimethylsilylation of iridoids using HMDS-TMCS and pyridine, and acetylations with acetic anidride and pyridine, were necessary. Plumieride coumarate (isomer mixture) and plumieride coumarate glucoside were acetylated and their products were purified on chromatography with silica gel column and ethyl ether as eluents, to afford pure penta-acetylplumieride coumarate and octa-acetylplu-mieride coumarate glucoside, respectively. Furthermore, plumieride, plumieride coumarate and plumieride glucoside were hydrolyzed under heating and acid conditions (sulfuric acid, 1 N for 2-3 h), extracted with ethyl acetate and both organic phase and aqueous phase (after neutralization with Amberlite) were analyzed by TLC and GC. Analyses were performed on a 1.5% OV-17 glass column with 0.4 m length and 4 mm I.D. Other analytical conditions were: nitrogen as carrier gas at 50 mL/min; detector temperature, 320 ºC; column temperatures: 190 ºC for isoplumericin and plumericin, 240 ºC for TMS-plumieride, 300 ºC TMS-plumieride coumarate. Glucose was detected in aqueous phases from acid hydrolyses of plumieride, plumieride coumarate and plumieride coumarate glucoside, while p-coumaric acid was detected in the organic phases of plumieride coumarate and plumieride coumarate glucoside. GC analyses also showed that plumieride coumarate was isolated as an isomer mixture (20% cis and 80% trans isomers) [116].

High, Medium and Low Performance Liquid Chromatography (HPLC, MPLC and LPLC)
Studies on HPLC with iridoids of Apocynaceae focus mainly on the separation of components from extracts or fraction. The chromatography profile, the identification and quantification of these terpenes in the extracts are described.

Counterflow
Protoplumericin and plumieride can be extracted from the methanol extract of Allamanda neriifolina stems [117] by droplet counter-current chromatography. Crude extract, obtained from 1.6 kg of plant material, was successively partitioned with benzene, chloro-form and butanol. Plumericin (420 mg) was directly obtained from the benzene fraction (0.02% yield). The butanol fraction was subjected to sequential chromatographic steps, using XAD-2 column and gradient of water/methanol (mobile phase), silica gel column and solvent system containing chloroform/methanol/water or chloroform/methanol, followed by droplet current chromatography with chloroform/methanol/ water. Plumieride (1.3 g) and protoplumericin (13.2 g) were obtained with yields of 0.08% and 0.83%, respectively.
The iridoids allamcidin B β-D-glucoside, plumiepoxide and protoplumericin B were isolated from Allamanda neriifolia extract obtained by percolation with methanol, also by droplet counter-current chromatography [39]. The crude methanol extracts from 2.6 kg of stem and 6.7 kg of leaves were sequentially fractionated with benzene, chloroform and butanol. Previous chromatographic treatment with MCI gel (elution with water/methanol), silica gel column (mobile phases: chloroform/methanol/water; benzene/acetone; ethyl acetate/methanol/ water and ethyl acetate/hexane) and Sephadex LH20 column (mobile phase: chloroform/methanol) led to the isolation of isoallamandicin (10 mg from the stem), allamcin (230 mg from the leaves), 3-O-methylallamcin (30 mg from the leaves), allamancin (102 mg from the stem), 3-Omethylallamancin (41 mg from the leaves), allamcidin (125 mg from the leaves), plumieride 13-O-acetate (760 mg from the stem and ca. 2 g from the leaves). Fractions of butanol extracts from the stem and leaves were subjected to droplet counter-current chromatography using chloroform/methanol/water (5:6:4, ascending mode) to obtain allamcidin B β-D-glucoside (17 mg from the stem), plumiepoxide (7 mg from the stem and 374 mg from the leaves) and protoplumericin B (70 mg from the leaves).

Capillary electrophoresis
For analytical purposes, iridoids can be analyzed by capillary electrophoresis. A method to separate nine iridoids described in [131] uses a Hewlett-Packard (HP 3D CE) capillary electrophoresis system coupled to a photodiode array detector (210 nm and 230 nm) and equipped with a fused-silica capillary tube (90 cm × 75µm I.D.). The distance to the detector was 81.5 cm.
Other conditions: sample injection at 50 mbar for 3 s and further deionized water injection at 50 mbar for 3 s; constant voltage, 16 kV (positive to negative); cartridge temperature, 30 °C; electrolyte (buffer), 50 mM sodium borate and 30 mg/mL 2,6-di-O-methyl-β-cyclodextrin (DMβ-CD); run time, 32 min. Before the analyses, the capillary column was sequentially purged with 0.5 M NaOH, 0.1 M NaOH, deionized water and buffer solution. The iridoids studied eluted in the following order: geniposide, loganin, shanzhiside, aucubin, catalpol, harpago- side, gardenoside, geniposidic acid and loganic acid. All were commercially purchased and only loganin, loganic acid and gardenoside were described for the Apocynaceae family. Several conditions of analyses were studied, including different pH, surfactants, concentrations of sodium borate and the addition of cyclodextrins (CD) to the buffer, and it was concluded that the less polar DM-β-CD added to 50 mM borate solution was the most suitable running buffer. In this condition, only aucubin and catalpol could not be separated, even with the addition of organic solvents and/or valine, urea and barium ion. The greatest advantage of capillary electrophoresis compared to HPLC analyses (the most commonly used technique) is its speed.
According to [132], capillary electrophoresis can be used to analyze a mixture of eleven iridoid glycosides: unedoside, harpagide, methyl catalpol, morroniside, asperuloside, griselinoside, catalpol, ketologanin, verbenalin, loganin and 10-cinnamoyl catalpol. Only loganin was found in the Apocynaceae family. For the analyses, iridoids were diluted in purified water. A Hewlett-Packard 3D CE system coupled to a diode array detector and equipped with an aircooling device was used. The fused-silica capillary tube measured 80 cm in length, 50 µm in I.D. and 375 µm in O.D. Distance to the detector was 71.5 cm only for UV detection (197 nm, 235 nm, 239 nm and 283 nm). When coupled to a mass spectrometer system (Bruker ESQUIRE) with an electrospray ionization source, the drying gas was nitrogen at 200 ºC and flow-rate 100 L/h. In this case, the distance between injector and UV detector was 20 cm. Other conditions: sample injection at 50 mbar for 5 s (only UV detection) or 25 s (with MS system); voltage, +20 kV; cartridge temperature, 25 °C; electrolyte solution, 20 mM ammonium acetate with 100 mM sodium dodecyl sulfate (SDS), pH 9.5; sheath liquid, 1 mM lithium acetate mixture to water/methanol (1:1 v/v) at a flow rate of 200 µL/h. When the MS system was used: scan range, 100-550 m/z; cut-off, 80 m/z; glass capillary exit, 95 V; skimmer, 32 V; electrospray voltage for the capillary, -4.0 kV; for the cylinder, -1,8 kV; for the end plate, -3.5 kV. In the comparison among the counterions sodium dodecyl sulfate (SDS), ammonium dodecyl sulfate and lithium dodecyl sulfate, diluted in water and running buffer, the best resolution for separating iridoid glucosides, lower noise in the MS system, and better repeatability and sensitivity were found with SDS in the running buffer. The volatility of ammonium acetate in buffer enables MS analyses, and concentrations higher than 20 mM did not represent better resolution. Quite the contrary, higher SDS concentrations furnished better results. In the study of the influence of pH, the best one was 9.5, although its influence in the range of 8.7-10.0 was lower than the SDS effect. Good linearity was observed for all the iridoids glucosides analyzed, but in different ranges.