Cyclodextrin based polymer sorbents for micro-solid phase extraction followed by liquid chromatography tandem mass spectrometry in determination of endogenous steroids.

Sorbents were prepared by cross-linking ß-cyclodextrin (ß-CD) using two different types of cross-linker units at variable reactant mole ratios. The resulting polymers containing ß-CD were evaluated as sorbents in micro-solid phase extraction (µ-SPE) format for the extraction of the endogenous steroids testosterone (T), epitestosterone (E), androsterone (A), etiocholanolone (Etio), 5α-androstane-3α,17ß-diol (5αAdiol) and 5ß-androstane-3α,17ß-diol (5ßAdiol). The best sorbent (C1; cyclodextrin polymer) showed superior extraction characteristics compared with commercial sorbents (C18 and Bond Elut Plexa). Parameters influencing the extraction efficiency of the C1 sorbent such as extraction and desorption times, desorption solvent and volume of sample were investigated. The extracts were separated using a Hypersil Gold column (50 × 2.1 mm, 1.9 µm) under gradient elution coupled to a LC-MS/MS. The compounds were successfully separated within 8 min. The method offers good repeatability (RSD < 10%) and linearity (r2 > 0.995) were within the range of 1-200 ng mL-1 for T and E, 250-4000 ng mL-1 for A and Etio and 25-500 ng mL-1 for 5αAdiol and 5ßAdiol, respectively. The method was applied for the determination of steroid profile of urine from volunteers.

Biochemical and radical-scavenging properties of sea cucumber (Stichopus vastus) collagen hydrolysates.

The molecular mass distribution, amino acid composition and radical-scavenging activity of collagen hydrolysates prepared from collagen isolated from the sea cucumber Stichopus vastus were investigated. ß and α1 chains of the collagen were successfully hydrolysed by trypsin. The molecular mass distribution of the hydrolysates ranged from 5 to 25 kDa, and they were rich in glycine, alanine, glutamate, proline and hydroxyproline residues. The hydrolysates exhibited excellent radical-scavenging activity. These results indicate that collagen hydrolysates from S. vastus can be used as a functional ingredient in food and nutraceutical products.

Micro-solid phase extraction with liquid chromatography-tandem mass spectrometry for the determination of aflatoxins in coffee and malt beverage.

A single step extraction-cleanup procedure using porous membrane-protected micro-solid phase extraction (µ-SPE) in conjunction with liquid chromatography-tandem mass spectrometry for the extraction and determination of aflatoxins (AFs) B1, B2, G1 and G2 from food was successfully developed. After the extraction, AFs were desorbed from the µ-SPE device by ultrasonication using acetonitrile. The optimum extraction conditions were: sorbent material, C8; sorbent mass, 20mg; extraction time, 90 min; stirring speed, 1,000 rpm; sample volume, 10 mL; desorption solvent, acetonitrile; solvent volume, 350 µL and ultrasonication period, 25 min without salt addition. Under the optimum conditions, enrichment factor of 11, 9, 9 and 10 for AFG2, AFG1, AFB2 and AFB1, respectively were achieved. Good linearity and correlation coefficient was obtained over the concentration range of 0.4-50 ng g(-1) (r(2) 0.9988-0.9999). Good recoveries for AFs ranging from 86.0-109% were obtained. The method was applied to 40 samples involving malt beverage (19) and canned coffee (21). No AFs were detected in the selected samples.

Isolation and characterization of pepsin-solubilized collagen from the integument of sea cucumber (Stichopus vastus).

BACKGROUND: Sea cucumber (Stichopus vastus) is considered an underutilized resource, since only its stomach and intestines are eaten raw as salad in a few countries and the remaining parts, especially the integument rich in collagen, is discarded. Hence a valuable by-product having potential nutraceutical and pharmaceutical applications is wasted. In the present investigation, pepsin-solubilized collagen (PSC) from the integument of S. vastus was isolated, purified and characterized. RESULTS: Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis showed that the purified collagen was of type I, consisting of three α1 chains of approximately 122 kDa each. The peptide map of PSC digested by V8 protease was different from that of calf skin type I collagen. Fourier transform infrared spectroscopy revealed that the triple helical structure was well preserved in isolated collagen. The denaturation temperature of PSC was 21.23 °C and showed good gel-forming capability at pH 6.5 and 300 mmol L⁻¹ NaCl. CONCLUSION: It is inferred that the collagen isolated from S. vastus integument has potential for use as an alternative to land-based mammalian collagen in food, nutraceuticals and pharmaceutical industries.

Sulfated glycosaminoglycans from crown-of-thorns Acanthaster planci - extraction and quantification analysis.

In this article, the novel inventive steps for the extraction and quantification of sulfated glycosaminoglycan (GAG) from Acanthaster planci starfish, generally known as crown-of-thorns (COT), are reported. Starfish have been implicated with collagenous distributions within their body anatomy, thus making it a prima facie fact searching for the possibility that GAGs can be isolated from COT. In this study, total-, N-, and O-sulfated GAGs were extracted from three anatomical regions of the COT (integument, internal tissue, and coelomic fluid) and comparison was made. The result showed that body region of COT seemed to contain higher amount of sulfated GAGs as opposed to the arm region (55.79 ± 0.65 µg/mg was the highest amount in the body extracted from its coelomic fluid and 32.28 ± 3.14 µg/mg was the highest amount in the arm extracted from its internal tissue). COT's integument and coelomic fluid from its body region possessed the highest total of sulfated GAGs content with no significant difference (P < 0.05) between the two. All GAGs from COT comprised a higher percentage of N-sulfated GAGs than its counterpart, the O-sulfated GAGs. When compared with a similar previous study that used sea cucumbers as the sulfated GAGs source, COT possessed more total sulfated GAGs content per milligram as compared with the sea cucumber generally. This result seems to unveil this marine species' advantage per se pertaining to GAGs extraction biomass applicability. Thus, COT could now be the better alternative source for production technology of total-, N-, and O-sulfated GAGs.

Associations of serum aflatoxin B1-lysine adduct level with socio-demographic factors and aflatoxins intake from nuts and related nut products in Malaysia.

Aflatoxins are one of the major risk factors in the multi-factorial etiology of human hepatocellular carcinoma. Therefore, the information on aflatoxins exposure is very important in the intervention planning in order to reduce the dietary intake of aflatoxins, especially among the children. This study investigated the relationship between aflatoxin B(1) (AFB(1)) lysine adduct levers in serum and socio-demographic factors and dietary intake of aflatoxins from nuts and nut products in Penang, Malaysia. A cross-sectional field study was conducted in five districts of Penang. A survey on socio-demographic characteristics was administered to 364 healthy adults from the three main ethnic groups (Malay, Chinese and Indian). A total of 170 blood samples were successfully collected and tested for the level of AFB(1)-lysine adduct. 97% of the samples contained AFB(1)-lysine adduct above the detection limit of 0.4 pg/mg albumin and ranged from 0.20 to 23.16 pg/mg albumin (mean±standard deviation=7.67±4.54 pg/mg albumin; median=7.12 pg/mg albumin). There was no significant association between AFB(1)-lysine adduct levels with gender, district, education level, household number and occupation when these socio-demographic characteristics were examined according to high or low levels of AFB(1)-lysine. However, participants in the age group of 31-50 years were 3.08 times more likely to have high AFB(1) levels compared to those aged between 18 and 30 years (P=0.026). Significant difference (P=0.000) was found among different ethnic groups. Chinese and Indian participants were 3.05 and 2.35 times more likely to have high AFB(1) levels than Malay. The result of AFB(1)-lysine adduct suggested that Penang adult population is likely to be exposed to AFB(1) but at a level of less than that needed to cause direct acute illness or death.

Exposure measurement of aflatoxins and aflatoxin metabolites in human body fluids. A short review.

Aflatoxins are highly toxic secondary fungal metabolites mainly produced by Aspergillus flavus and A. parasiticus. Human exposure to aflatoxins may result directly from ingestion of contaminated foods, or indirectly from consumption of foods from animals previously exposed to aflatoxins in feeds. This paper focuses on exposure measurement of aflatoxins and aflatoxin metabolites in various human body fluids. Research on different metabolites present in blood, urine, breast milk, and other human fluids or tissues including their detection techniques is reviewed. The association between dietary intake of aflatoxins and biomarker measurement is also highlighted. Finally, aspects related to the differences between aflatoxin determination in food versus the biomarker approach are discussed.

Exposure assessment and risk characterization of aflatoxin B1 in Malaysia.

Aflatoxin B1 (AFB1) was detected in 57% of the nuts and nut products marketed in Penang, Malaysia using the liquid chromatography tandem mass spectrometry. The contamination levels ranged from 0.40 to 222 µg/kg and 17 out of 128 samples (13.3%) contained AFB1 above the European Commission permitted level (2 µg/kg). Estimated dietary exposure of AFB1 in nuts and nut products were 0.36 ng per kg body weight and day and 8.89 ng per kg body weight and day, representing the low and high-level of exposure, respectively. Dose-response modelling resulted in benchmark dose lower confidence limit (BMDL10) values of 0.305 ng per kg body weight and day, with the best fitted from the log-logistic model. The derived margin of exposure (MoE) values ranged from 34 to 847 suggested that AFB1 would be of public health concern and might reasonably be considered as a high priority for risk management actions.

Crystallization of Ranasmurfin, a blue-coloured protein from Polypedates leucomystax.

Ranasmurfin, a previously uncharacterized approximately 13 kDa blue protein found in the nests of the frog Polypedates leucomystax, has been purified and crystallized. The crystals are an intense blue colour and diffract to 1.51 A with P2(1) symmetry and unit-cell parameters a = 40.9, b = 59.9, c = 45.0 A, beta = 93.3 degrees . Self-rotation function analysis indicates the presence of a dimer in the asymmetric unit. Biochemical data suggest that the blue colour of the protein is related to dimer formation. Sequence data for the protein are incomplete, but thus far have identified no model for molecular replacement. A fluorescence scan shows a peak at 9.676 keV, indicating that the protein binds zinc and suggesting a route for structure solution.