Investigation of extraction and analysis techniques for Lyngbya wollei derived Paralytic Shellfish Toxins

Paralytic Shellfish Toxins (PSTs) are highly toxic metabolic by-products of cyanobacteria and dinoflagellates. The filamentous cyanobacterium Lyngbya wollei produces a unique set of PSTs, including L. wollei toxins (LWT) 1-6. The accurate identification and quantification of PSTs from Lyngbya filaments is challenging, but critical for understanding toxin production and associated risk, as well as for providing baseline information regarding the potential for trophic transfer. This study evaluated several approaches for the extraction and analysis of PSTs from field-collected L. wollei dominated algal mats. Extraction of PSTs from lyophilized Lyngbya biomass was assessed utilizing hydrochloric acid and acetic acid at concentrations of 0.001-0.1 M. Toxin profiles were then compared utilizing two analysis techniques: pre-column oxidation (peroxide and periodate) High Performance Liquid Chromatography (HPLC) with Fluorescence (FL) detection and LC coupled with Mass Spectrometry (MS). While both acid approaches efficiently extracted PSTs, hydrochloric acid was found to convert the less toxic LWT into the more toxic decarbamoylgonyautoxins 2&3 (dcGTX2&3) and decarbamoylsaxitoxin (dcSTX). In comparison, extraction with 0.1 M acetic acid preserved the original toxin profile and limited the presence of interfering co-extractants. Although pre-chromatographic oxidation with HPLC/FL was relatively easy to setup and utilize, the method did not resolve the individual constituents of the L. wollei derived PST profile. The LC/MS method allowed characterization of the PSTs derived from L. wollei, but without commercially available LWT 1-6 standards, quantitation was not possible for the LWT. In future work, evaluation of the risk associated with L. wollei derived PSTs will require commercially available standards of LWT 1-6 for accurate determinations of total PST content and potency.     

Paralytic shellfish poisoning toxin profile of mussels Perna perna from southern Atlantic coasts of Morocco

During the monitoring programme of harmful algal blooms established along the south Atlantic coast of Morocco, a bimonthly determination of harmful algae and phycotoxins analysis in Perna perna was carried out from May 2003 to December 2004. Results of mouse bioassay (in organs and whole flesh) showed a seasonal evolution of paralytic shellfish poisoning (PSP) toxin. The mussel's contamination was associated with the occurrence in water of Alexandrium minutum.The PSP toxin profile obtained with high-performance liquid chromatography (HPLC/FD) revealed the dominance of gonyautoxins GTX2 and GTX3 and a minority of GTX1, GTX4 and saxitoxin (STX). This profile explains that the toxicity was mainly associated with A. minutum.     

Accumulation and depuration rates of paralytic shellfish poisoning toxins in the shore crab Telmessus acutidens by feeding toxic mussels under laboratory controlled conditions.

Accumulation and depuration rates of paralytic shellfish poisoning toxins (PSP) in the crab Telmessus acutidens were investigated by feeding toxic and non-toxic mussels under laboratory controlled conditions. The crab accumulated toxins in the hepatopancreas in proportion to the amount of toxic mussels they ingested, and the toxicity in the crab hepatopancreas became 3.2 fold of that in the prey mussels after 20 days of feeding. During depuration, a fast reduction of the total toxicity was observed in the crab, and the retention rate of the toxicity after 5 days depuration with feeding of non-toxic mussels was 45.8+/-18.7%. The reduction of the toxicity was moderated in the later period of depuration, and the retention rates of the total toxicity after 10 and 20 days were 54.1+/-29.8% and 14.5+/-9.0%, respectively. The toxin profiles in the crab and mussel were investigated by high performance liquid chromatography, and reductive conversions of the toxins were observed when the toxins were transferred from the mussel to the crab. Consequently, high concentrations of GTX2 and GTX3, and STX that were not detected in the prey mussels, were found in the crab.     

Paralytic shellfish toxin profiles and toxin variability of the genus Alexandrium (Dinophyceae) isolated from the Southeast China Sea.

Paralytic shellfish toxin (PST) profiles of 16 Alexandrium isolates from the Southeast China Sea were analyzed by high-pressure liquid chromatography. Toxin content and composition of three A. tamarense isolates, ATDH01, ATGX02 and ATMJ02, were also investigated at different growth phases and under various culture conditions. Our results showed that six strains of A. affine were non-toxic, while 10 strains of A. tamarense and A. catenella were toxic. These toxic isolates grown in the same culture conditions consistently produced an unusually high proportion of the N-sulfocarbamoyl toxin C1/2 (around 60-80% of total toxins) and medium amounts of gonyautoxin GTX5 (around 15-30% of total) with only trace quantities (<5% of total) of other saxitoxin derivatives (i.e. GTX1, GTX3, GTX4 and neoSTX). The toxin composition of three A. tamarense isolates did not vary with the growth phases, although higher toxin contents (Qt, fmolcell(-1)) were found in the exponential phase. Variations in temperature, salinity and nutrient levels affected toxin content of three A. tamarense isolates but they did not have pronounced effects on the toxin composition (mole %). These results indicate that toxin composition remained relatively constant under various culture conditions, suggesting that toxin composition could be used as a stable biomarker for the Alexandrium species in this region. However, comparison of toxin profiles between isolates from different localities require special caution since isolates even from the same region can have distinct toxin profiles.     

An in vitro comparative study upon the toxic properties of the venoms from Hemiscorpius lepturus, Androctonus crassicauda and Mesobuthus eupeus scorpions

The aim of the present study was to compare the toxic effects of the venoms from Hemiscorpius lepturus (H. lepturus), Androctonus crassicauda (A. crassicauda) and Mesobuthus eupeus (M. eupeus). For this purpose, three in vitro models were employed to compare the toxic effects of various concentrations of the venoms from these three scorpions, namely: hemolytic potential using human RBCs, phospholipase activity using Saubouraud's dextrose agar (SDA) supplemented with 2% egg yolk and lactate dehydrogenase (LDH) enzyme releasing effect using K562 leukemia cell line. In addition, the neutralizing effectiveness of the antivenom against these toxic properties was assessed. The results showed that, unlike the venoms from A. crassicauda and M. eupeus, the venom from H. lepturus produced dose-dependent lysis of human RBCs and showed phospholipase activity. However, all the tested venoms showed variable degrees of LDH releasing properties. The venom from H. lepturus had highest and the venom from M. eupeus had the lowest LDH releasing effect. The antivenom effectively inhibited all the tested toxicities. In conclusion, these results suggest that the venoms from the studied scorpions have variable toxic properties, which may explain the underlying reason for the differences in their clinical manifestations. In addition, the antivenom was effective in neutralizing all the tested toxic effects.     

Analysis of the bioactive constituents of ChanSu in rat plasma by high performance liquid chromatography with mass spectrometric detection

A plasma pharmacochemistry analysis of the bioactive constituents in rat plasma after oral administration of ChanSu was performed. High performance liquid chromatography coupled with the electrospray ionization mass spectrometry techniques of HPLC/ESI-IT-MS/MS and RRLC/ESI-Q-TOF-MS were used for the chemical profiling of samples of dosed plasma, control plasma and ChanSu extract. Comparative analysis of the resulting chemical profiles revealed 20 prototype compounds and 4 metabolites derived from ChanSu as potential bioactive components. By MS/MS analysis and accurate molecular weight assessments, the majority of these bioactive components (19 prototype compounds and 2 metabolites) were structurally identified. Moreover, seven were confirmed by comparing their retention behaviors using MS and MS/MS analysis. This study proposes a series of potential bioactive components of ChanSu, which we hope will lead to new drug discovery based on ChanSu and other traditional Chinese medicines.     

HPLC法测定藏药螃蟹甲中苯乙醇苷类化合物的含量

目的建立RP-HPLC法同时测定藏药螃蟹甲中3个苯乙醇苷的含量。方法色谱柱为Symmetry C18(4.6 mm×150 mm,5μm),流动相为甲醇-水(36∶64),冰醋酸调pH至4.5;流速1.0 ml/min;柱温20℃,检测波长330 nm。结果毛蕊花糖苷、alyssonoside、异毛蕊花糖苷分别在20～500(r=0.999 6),10～130(r=0.999 5),10～130(r=0.999 7)μg/ml范围间与峰面积线性关系良好;平均加样回收率均在96%～104%之间,RSD均小于5%(n=6)。结论该方法专属性强、灵敏度高、精确度与重现性好,可作为螃蟹甲药材的质量控制方法。     

Differential accumulation of paralytic shellfish toxins from Alexandrium minutum in the pearl oyster, Pinctada imbricata

To investigate the potential for differential accumulation of paralytic shellfish toxins (PSTs) in various tissues of the akoya pearl oyster, Pinctada imbricata, two feeding trials were carried out using the PST-producing dinoflagellate, Alexandrium minutum. When fed with A. minutum at concentrations between 100 and 1300 cells ml⁻¹, the maximum clearance by P. imbricata was shown to occur at a density of 300 cells ml⁻¹. When fed twice daily at this rate for up 12 days, P. imbricata accumulated analogues of gonyautoxins (GTXs): GTXs 1,4 and 2,3. The levels of GTXs in the viscera increased progressively on days 4, 8 and 12 to peak at 17.9 ± 4.47 μg STX-equivalent 100 g⁻¹ biomass. Following 12 days of depuration, in the absence of A. minutum, GTX levels fell by approximately 65% to 6.0 ± 2.20 μg STX-equivalent 100 g⁻¹ biomass. No GTX was found in the oysters at the start of the trial or in untreated controls. The accumulation of GTX was found to be tissue specific. No GTX was detected in the muscle tissue of P. imbricata during the feeding trial.     

Antioxidant activity and ultra-performance LC-electrospray ionization-quadrupole time-of-flight mass spectrometry for phenolics-based fingerprinting of Rose species: Rosa damascena, Rosa bourboniana and Rosa brunonii

Roses are one of the most important groups of ornamental plants and their fruits and flowers are used in a wide variety of food, nutritional products and different traditional medicines. The antioxidant activity of methanolic extracts from fresh flowers of three rose species (Rosa damascena, Rosa bourboniana and Rosa brunonii) was evaluated by 1,1-diphenyl-2-picryl hydrazyl (DPPH) free-radical method. The ability to scavenge DPPH radical was measured by the discoloration of the solution. The methanolic extract from R. brunonii exhibited maximum free-radical-scavenging activity (64.5 ± 0.38%) followed by R. bourboniana (51.8 ± 0.46%) and R. damascena (43.6 ± 0.25%) at 100 μg/ml. Simultaneously, ultra-performance liquid chromatography coupled with electrospray ionization-quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOF-MS) was used to study phenolic composition in the methanolic extracts from the fresh flowers of rose species. The phenolic constituents were further investigated by direct infusion-ESI-QTOF-MS/MS in negative ion mode. Characteristic Electrospray ionization tandem mass spectrometry (ESI-MS/MS) spectra with other diagnostic fragment ions generated by retro Diels–Alder (RDA) fragmentation pathways were recorded for the flavonoids. Distinct similarities were observed in the relative distribution of polyphenolic compounds among the three species. The dominance of quercetin, kaempferol and their glycosides was observed in all the three species.     

Immunome and venome of Bothrops jararacussu: A proteomic approach to study the molecular immunology of snake toxins

A combination of anti-bothropic and anti-crotalic sera has been reported to be more effective in neutralizing the effects of Bothrops jararacussu venom than anti-bothropic serum alone. The role of proteins from B. jararacussu venom in the horse immune response was evaluated via the analysis of cross-reactivity with homologous and heterologous sera. Many of the proteins in B. jararacussu venom were identified via 2D gel electrophoresis. Western blots revealed that anti-jararacussu showed higher reactivity to l-aminoxidase (LAOs) and snake venom metalloproteinase, (SVMPs) and weaker reactivity towards Snake venom serine proteases (SVSPs), PLA₂, C-type lectin and cysteine-rich proteins. Anti-jararaca preferentially recognized LAOs, SVMPs and SVSPs. Both of these sera failed to recognize low-molecular weight proteins. Anti-crotalic serum clearly recognized LAOs, C-type lectin, SVSP, cysteine-rich proteins, SVMP and Asp49-PLA₂. The cross-reactivity with anti-PLA₂ revealed the immunoreactivity of these antibodies to proteins with molecular masses in a range that is poorly recognized by other studied anti-sera. Our results suggest that the contribution of anti-crotalic serum to the neutralization of B. jararacussu by may be due to its cross-reactivity with proteins such as C-type lectins, SVSPs, Asp49-PLA₂. These results also reinforce the importance of neutralizing the highly toxic proteins inclusive those with low immunogenicity in commercial antivenom production to obtain a highly protective serum against snake venoms.     

Contents of major bioactive flavones in proprietary traditional Chinese medicine products and reference herb of Radix Scutellariae

A simple and efficient HPLC/UV method for the simultaneous determination of six bioactive flavones, namely baicalein, baicalin, wogonin, wogonoside, oroxylin A and oroxylin A-7-O-glucuronide, has been developed and applied for their content determination in reference herb and proprietary traditional Chinese medicine (PTCM) products of Radix Scutellariae. The chromatographic separation was carried out on a Thermo C₁₈ column and linear gradient elution was employed with a mobile phase containing acetonitrile and 20 mM sodium dihydrogen phosphate buffer (pH 4.6). All the analytes were detected by PDA detector at a wavelength of 270 nm. Contents of the analytes in Radix Scutellariae containing PTCM products in forms of capsule, soft capsule, tablet and dripping pill and the reference herb of Radix Scutellariae were analyzed by sonicator extraction with methanol and water mixture (80:20) containing 1 mM HCl for 30 min followed by HPLC analysis. Separation of the six analytes was achieved within 25 min with good linearity (R² > 0.99). The R.S.D. of both the intra-day and inter-day precision for all the six analytes was below 10.14%. The accuracy at different concentrations was within the range of −7.83 to 4.06%. The extraction recovery was within the range of 89.22–107.33% for all the analytes. Contents of the six flavones were found to vary significantly among different products with glycosides, such as baicalin, wogonoside and oroxylin A-7-O-glucuronide, in much greater quantity than their corresponding aglycones. In addition to baicalin (18.54 ± 0.71%, w/w), the commonly used marker compound for Radix Scutellariae, wogonoside (3.54 ± 0.18%, w/w) and oroxylin A-7-O-glucuronide (2.84 ± 0.14%, w/w) also existed in abundant amount in the reference herb. Our findings suggested that wogonoside and oroxylin A-7-O-glucuronide should also be served as the chemical markers together with baicalin for the quality control of herbs and PTCM products of Radix Scutellariae.     

应用高效液相色谱法测定猫人参中积雪草酸的含量

目的 建立猫人参药材中积雪草酸的含量测定方法。 方法 采用高效液相色谱法,色谱柱为Agilent HC-C₁₈柱(4.6 mm×250 mm,5.0 μm),流动相为乙腈(30 mmol/L)-醋酸铵水溶液(35:65),流速1.0 ml/min,柱温25 ℃,紫外检测波长210 nm,进样量25 μl,运行时间35 min。 结果 积雪草酸与周围干扰峰达到基线分离,线性范围25.3~506.0 μg/ml, r=0.999 6;日内及日间精密度均<3% (n=5),平均回收率为99.4%(RSD=1.9%, n=6),猫人参中积雪草酸的含量为0.751 mg/g。 结论 该法简便快捷、测定结果准确、实用性强,可用于猫人参药材中积雪草酸的含量测定。     

HPLC法测定葛花中鸢尾苷和鸢尾苷元的含量

目的建立测定葛花中鸢尾苷和鸢尾苷元含量的高效液相色谱方法。方法采用美国Agilent-C18柱(4.6 mm×250 mm,5μm),以甲醇-乙腈-水(2∶1∶2)为流动相,流速为1.0 ml/min;检测波长为264 nm,柱温为室温。结果鸢尾苷在2.0～120.0μg/ml呈线性关系,r=0.999 8;平均加样回收率为99.7%,RSD为1.9%;鸢尾苷元在0.8～80.0μg/ml呈线性关系,r=0.998 6;平均加样回收率为99.4%,RSD为1.7%。结论该方法简便易行,结果准确、可靠,重复性好,可用于葛花中鸢尾苷和鸢尾苷元的含量测定。     

Comparative analysis of the antioxidant and DNA protection capacities of Anadenanthera colubrina, Libidibia ferrea and Pityrocarpa moniliformis fruits

This study aimed to explore the antioxidant and DNA protection abilities of hydroalcoholic extracts from fruits of Anadenanthera colubrina (ACHE), Libidibia ferrea (LFHE) and Pityrocarpa moniliformis (PMHE). These extracts were tested by five antioxidant methods (phosphomolibdenium and reducing power assays; superoxide, hydrogen peroxide and nitric oxide scavenging) and DNA protection capacity. Total phenolic content was measured by Folin-Ciocalteu method. ACHE exhibited the highest phenolic content (578 mg/g GAE), followed by LFHE (460 mg/g GAE) and PMHE (448 mg/g GAE). In phosphomolibdenium assay, ACHE showed 24.81% of activity in relation to ascorbic acid, whereas LFHE and PMHE had 21.08% and 18.05%, respectively. These plants showed high ability to inhibit reactive species tested with IC50 values ranged from 10.66 to 14.37 μg/mL for superoxide radical; 26.05 to 45.43 μg/mL for hydrogen peroxide; 178.42 to 182.98 μg/mL for reducing power; and 199.2 to 283 μg/mL for nitric oxide. Furthermore, these extracts had capacity to break the DNA damage induced by hydroxyl radicals. The antioxidant activity of these plants is related with their higher phenolic content and show that they may be used as source of bioactive compounds, relevant to the maintenance of oxidative stability of the food matrix, cosmetics and/or pharmaceutical preparations.     

Bites by the colubrid snake Philodryas patagoniensis: A clinical and epidemiological study of 297 cases

We retrospectively analyzed 297 proven cases of Philodryas patagoniensis bites admitted to Hospital Vital Brazil (HVB), Butantan Institute, São Paulo, Brazil, between 1959 and 2008. Only cases in which the causative animal was brought and identified were included. Part of the snakes brought by the patients was still preserved in the collection maintained by the Laboratory of Herpetology. Of the 297 cases, in 199 it was possible to describe the gender of the snake, and seventy three (61.3%) of them were female. The length of snakes (snout-vent length) ranged from 160 to 1080 mm. In 117 snakes their state of preservation enabled the dissection and examination of their stomach contents. The stomach was empty in 106 snakes (89.1%). Most bites occurred in the seasons of spring and summer (n = 196, 66.0%) and during warmer periods of the day. The mean age of the victims was 24.1 ± 15.1 years old and 206 (69.4%) patients were men. Around 92% of the patients sought medical care within 6 h after the bite. Both lower (n = 188, 63.3%) and upper limbs (n = 102, 34.3%) were most frequently bitten, especially the feet and hands (n = 205, 69.0%). The local clinical manifestations were pain (n = 151, 50.8%), transitory bleeding (n = 106, 35.7%), erythema (n = 47, 15.8%) and edema (n = 39, 13.1%). Ecchymosis was not observed. Only 7 (2.4%) patients reported systemic symptoms characterized by mild dizziness and 88 patients (29.6%) showed no evidence of envenoming. The whole blood clotting time was performed in 76 (25.6%) patients on admission and all of them had coagulable blood. Supportive treatment was offered to only 13.4% of patients, namely administration of antihistamines (n = 19, 6.4%) and analgesics (n = 12, 4.1%). Eight patients (2.7%) were mistreated with Bothrops antivenom before their admission to HVB. No sequels or relevant complications were observed in patients, and the prognostic was benign. Therefore, although P. patagoniensis accidents can cause mild local symptomatology, it is very important that health professionals know how to make the correct diagnosis to avoid unnecessary use of antivenom.     

A novel TTX-producing Aeromonas isolated from the ovary of Takifugu obscurus

Puffer fish (Takifugu obscurus) from the Yangtze River of China were screened for tetrodotoxin-producing bacteria. An Aeromonas strain was isolated from the ovary of the puffer fish and was shown to produce tetrodotoxin; this strain was denoted Ne-1. The identity of tetrodotoxin produced by strain Ne-1 was confirmed by mouse bioassay, high performance liquid chromatography coupled with mass chromatography (LC-MS), and ELISA. Strain Ne-1 was characterized morphologically, biochemically, and by 16S rDNA phylogenetic analysis; these analyses suggested that strain Ne-1 is closely related to Aeromonas molluscorum. Given that strain Ne-1 was isolated from the ovary of T. obscurus, we propose that the TTX-producing Aeromonas sp. is a parasite or symbiotic bacterium rather than a sample contaminant. Collectively, our studies suggest that Aeromonas sp. strain Ne-1 produces tetrodotoxin in T. obscurus.     

Global distribution of ciguatera causing dinoflagellates in the genus Gambierdiscus

Dinoflagellates in the genus Gambierdiscus produce toxins that bioaccumulate in tropical and sub-tropical fishes causing ciguatera fish poisoning (CFP). Little is known about the diversity and distribution of Gambierdiscus species, the degree to which individual species vary in toxicity, and the role each plays in causing CFP. This paper presents the first global distribution of Gambierdiscus species. Phylogenetic analyses of the existing isolates indicate that five species are endemic to the Atlantic (including the Caribbean/West Indies and Gulf of Mexico), five are endemic to the tropical Pacific, and that two species, Gambierdiscus carpenteri and Gambierdiscus caribaeus are globally distributed. The differences in Gambierdiscus species composition in the Atlantic and Pacific correlated with structural differences in the ciguatoxins reported from Atlantic and Pacific fish. This correlation supports the hypothesis that Gambierdiscus species in each region produce different toxin suites. A literature survey indicated a >100-fold variation in toxicity among species compared with a 2 to 9-fold within species variation due to changing growth conditions. These observations suggest that CFP events are driven more by inherent differences in species toxicity than by environmental modulation. How variations in species toxicity may affect the development of an early warning system for CFP is discussed.     

First evidence of azaspiracids (AZAs): A family of lipophilic polyether marine toxins in scallops (Argopecten purpuratus) and mussels (Mytilus chilensis) collected in two regions of Chile

Azaspiracids are a family of lipophilic polyether marine biotoxins that have caused a number of human intoxication incidents in Europe since 1995 following the consumption by consumers of intoxicated shellfish (Mytilus edulis). These azaspiracids have now been identified in mussels (Mytilus chilensis) and scallops (Argopecten purpuratus) from two Chilean locations. This is the first report of the occurrence of azaspiracid toxins in these species (Mytilus chilensis and Argopecten purpuratus) from Chile. The areas studied were Bahía Inglesa (III Region, 27° SL) and Chiloé Archipelago, both important scallop and mussels farming areas. Separation of azaspiracid (AZA1), azaspiracid isomer (AZA6) and its analogues, 8-methylazaspiracid (AZA2) and 22-demethylazaspiracid (AZA3), was achieved using reversed-phase LC and toxins were identified using a turbo electrospray ionisation (ESI) source, to a triple quadrupole mass spectrometer.In mussels, AZA1 was the predominant toxin in mussel hepatopancreas with AZA2, AZA3 and AZA6 present in approximate equivalent amounts in the remaining tissues, 20-30% of the AZA1 level. AZA2 predominated in the scallop samples with the toxin almost entirely present in the hepatopancreas (digestive gland). AZA1 was only observed in some of the scallop samples and was present at 12-15% of the AZA2 levels.Whilst the levels of AZAs in Chilean samples are below the EU regulatory limit of 160 μg/kg, it is significant that this toxin is present in Pacific Ocean species. Consequently measures should be taken by regulatory authorities to implement regular seafood monitoring to ensure safety of harvested product.     

A study of anti-cancer effects of Funalia trogii in vitro and in vivo

Extracts of natural products have been used for many years for health benefits. We report on an in vitro and in vivo study into the anti-tumour efficacy of an aqueous extract of the mycelial form of basidiomycete, Funalia trogii. A variety of biological assays were used to show that a 4 h exposure of HT29, LNCaP, PC3, MCF-7 and MDA-MB-231 tumour cells to extract (0.5-5.0 mg/ml) resulted in significant cytotoxicity. In a clonogenic assay, IC₅₀ values were found to range from 0.4-0.72 mg/ml; exposing fibroblast cells to the extract resulted in no cell kill. The extract resulted in significant cell kill in proliferating endothelial cells but had no toxicity to quiescent cells, this is useful in targeting tumour tissue since endothelial cells in tumours proliferate more rapidly that those found in other parts of the body. When tumours grown in immune compromised mice were injected intratumourally with extract (5 mg/ml twice a week for two weeks), a 9 day tumour growth delay was observed. The results indicate that the mycelial extract of F. trogii has a promising anti-tumour property.     

Qualitative and quantitative analysis of steroidal saponins in crude extracts from Paris polyphylla var. yunnanensis and P. polyphylla var. chinensis by high performance liquid chromatography coupled with mass spectrometry

High performance liquid chromatography coupled with electrospray ionization multi-stage tandem mass spectrometry (HPLC–ESI-MSⁿ) and triple quadrupole mass spectrometric detection (HPLC–ESI-MS/MS), respectively, had been employed for the simultaneous identification and quantification of steroidal saponins in the rhizomes of Parispolyphylla var. yunnanensis and P. polyphylla var. chinensis, which are the qualified plants of “Chonglou” in Chinese. The HPLC experiments were performed by means of a reversed-phase C-18 column and a binary mobile phase system consisting of 0.1% aqueous formic acid and acetonitrile under gradient elution conditions. The characteristic fragmentation patterns of diosgenin- and pennogenin-type steroidal saponins were investigated using ESI-MSⁿ in negative ion mode. The MSⁿ data of the [M−H]⁻ ions provided structural information on the sugar sequence of the oligosaccharide chains and the aglycones of steroidal saponins. As a result, ten and seven saponins were determined in P. polyphylla var. yunnanensis and P. polyphylla var. chinensis, respectively, including four unknown compounds. One unknown compound was tentatively identified as diosgenin-3-O-α-l-rhamnopyranosyl(1 → 4) [α-l-rhamnopyranosyl(1 → 2)]-β-d-glucopyranoside and the aglycones of the other three new compounds were reported from Chonglou for the first time. The developed HPLC–ESI-MS/MS method was validated and found to be satisfactorily linear, selective and robust. The limits of detection (LODs) and quantitation (LOQs) ranged, respectively, from 0.5 to 10 ng/mL and 2 to 34 ng/mL depending on six various compounds. The intra- and inter-day precisions of the method were evaluated and were less than 5.0%. Recoveries ranged from 92% to 104% for all compounds. The established quality evaluation method was successfully used for simultaneous quantification of six predominant steroidal saponins in the rhizomes of these two Paris species.