Comparison of porcine urine and bile as matrices to screen for the residues of two sulfonamides using a semi-automated enzyme immunoassay.

Porcine urine enzyme immunoassays for sulfamethazine and sulfadiazine have previously been employed as screening tests to predict the concentrations of the drugs in the corresponding tissues (kidneys), If a urine was found positive (> 800 ng ml(-1)) the corresponding kidney was then analysed by an enzyme immunoassay and, if found positive, a confirmatory analysis by HPLC was performed. Urine was chosen as the screening matrix since sulfonamides are mainly eliminated through this body fluid, However, after obtaining a number of false positive predictions, an investigation was carried out to assess the possibility of using an alternative body fluid which would act as a superior indicator of the presence of sulfonamides in porcine kidney, An initial study indicated that serum, plasma and bile could all be used as screening matrices. From these, bile was chosen as the preferred sample matrix and an extensive study followed to compare the efficiencies of sulfonamide positive bile and urine at predicting sulphonamide positive kidneys, Bile was found to be 17 times more efficient than urine at predicting a sulfamethazine positive kidney and 11 times more efficient at predicting a sulfadiazine positive kidney, With this enhanced performance of the initial screening test, the need for the costly and time consuming kidney enzyme immunoassay, prior to HPLC analysis, was eliminated

Comparison of ELISA and SPR biosensor technology for the detection of paralytic shellfish poisoning toxins

An enzyme labeled immunosorbent assay (ELISA) and surface plasmon resonance (SPR) biosensor assay for the detection of paralytic shellfish poisoning (PSP) toxins were developed and a comparative evaluation was performed. A polyclonal antibody (BC67) used in both assay formats was raised to saxitoxin–jeffamine–BSA in New Zealand white rabbits. Each assay format was designed as an inhibition assay. Shellfish samples (n = 54) were evaluated by each method using two simple rapid extraction procedures and compared to the AOAC high performance liquid chromatography (HPLC) and the mouse bioassay (MBA). The results of each assay format were comparable with the HPLC and MBA methods and demonstrate that an antibody with high sensitivity and broad specificity to PSP toxins can be applied to different immunological techniques. The method of choice will depend on the end-users needs. The reduced manual labor and simplicity of operation of the SPR biosensor compared to ELISA, ease of sample extraction and superior real time semi-quantitative analysis are key features that could make this technology applicable in a high-throughput monitoring unit.

Helix pomatia agglutinin lectin-binding oligosaccharides of aggressive breast cancer

Predicting long-term outcome after breast-cancer diagnosis remains problematic, particularly for patients with clinically small, axillary lymph node- negative tumours, Evidence suggests that the lectin Helix pomatia agglutinin (HPA) identifies oligosaccharides associated with poor-prognosis cancer. Our aim was to identify oligosaccharides that bind HPA in aggressive breast cancers. Breast-cancer cell lines (MCF-7, BT-549 and BT-20) and a cell line From human milk (HBL-100), which showed a range of HPA-binding intensities, were used to extract HPA-binding glycoproteins, Oligosaccharides were released using anhydrous hydrazine and separated on a range of HPLC matrices. We investigated whether HPA-binding oligosaccharides from cell lines were present in human breast-cancer tissues, using 69 breast-cancer specimens from patients with between 5 and 10 years' follow-up. A monosialylated oligosaccharide was over-expressed in the cell line that bound HPA strongly. Further analysis by normal-phase HPLC showed that the 2-aminobenzamide-conjugated oligosaccharide had a hydrodynamic volume of 4.58 glucose units (HPAgly 1), Increased expression of HPAgly 1 was associated with HPA staining of breast-cancer specimens (Student's t-test p = 0.025). Analysis of oligosaccharide levels and disease-free survival after treatment for breast cancer indicated a shorter disease-free interval for patients with elevated levels of HPAgly 1, This is the first time that histochemical lectin staining has been correlated with biochemical mapping of oligosaccharides, Using this approach, we have identified a monosialylated HPA lectin-binding oligosaccharide present in breast-cancer cells grown in vitro which is elevated in breast-cancer specimens that bind the lectin, (C) 2001 Wiley-Liss, Inc.

Peptide Tyrosine Arginine, a potent immunomodulatory peptide isolated and structurally characterized from the skin secretions of the dusky gopher frog, Rana sevosa

An octadecapeptide was isolated from the skin secretions of the dusky gopher frog (Rana sevosa) on the basis of histamine release from rat peritoneal mast cells. This peptide was purified to homogeneity by HPLC and found to have the following primary structure, YLKGCWTKSYPPKPCFSR, using both Edman degradation chemistry and peptide sequencing using high-resolution mass spectrometry (Q-TOF MS). The peptide, named peptide Tyrosine Arginine (pYR) shares 77.8% homology with peptide Leucine Arginine (pLR). The effects of the natural amidated peptide, non-amidated peptide and C-loop region of pYR on granulopoiesis and neutrophil apoptosis were investigated. All three analogues inhibited the early development of granulocyte macrophage colonies from bone marrow stem cells but did not induce apoptosis of the end stage granulocytes, the mature neutrophil. Thus, pYR is a novel member of an important and emerging new class of amphibian peptides with hemopoietic actions. (c) 2004 Elsevier Inc. All rights reserved.

Stir bar sorptive extraction of diclofenac from liquid formulations: A proof of concept study

A new stir bar sorptive extraction (SBSE) technique coupled with HPLC-UV method for quantification of diclofenac in pharmaceutical formulations has been developed and validated as a proof of concept study. Commercially available polydimethylsiloxane stir bars (Twister (TM)) were used for method development and SBSE extraction (pH, phase ratio, stirring speed, temperature, ionic strength and time) and liquid desorption (solvents, desorption method, stirring time etc) procedures were optimised. The method was validated as per ICH guidelines and was successfully applied for the estimation of diclofenac from three liquid formulations viz. Voltarol (R) Optha single dose eye drops, Voltarol (R) Ophtha multidose eye drops and Voltarol (R) ampoules. The developed method was found to be linear (r=0.9999) over 100-2000 ng/ml concentration range with acceptable accuracy and precision (tested over three QC concentrations). The SBSE extraction recovery of the diclofenac was found to be 70% and the LOD and LOQ of the validated method were found to be 16.06 and 48.68 ng/ml, respectively. Furthermore, a forced degradation study of a diclofenac formulation leading to the formation of structurally similar cyclic impurity (indolinone) was carried out. The developed extraction method showed comparable results to that of the reference method, i.e. method was capable of selectively extracting the indolinone and diclofenac from the liquid matrix. Data on inter and intra stir bar accuracy and precision further confirmed robustness of the method, supporting the multiple re-use of the stir bars. (C) 2010 Elsevier B.V. All rights reserved.

Peptide IC-20, encoded by skin kininogen-1 of the European yellow-bellied toad, Bombina variegata, antagonizes bradykinin-induced arterial smooth muscle relaxation

The objectives were to determine if the skin secretion of the European yellow-bellied toad (Bombina variegata), in common with other related species, contains a bradykinin inhibitor peptide and to isolate and structurally characterize this peptide. Materials and Methods: Lyophilized skin secretion obtained from this toad was subjected to reverse phase HPLC fractionation with subsequent bioassay of fractions for antagonism of the bradykinin activity using an isolated rat tail artery smooth muscle preparation. Subsequently, the primary structure of the peptide was established by a combination of microsequencing, mass spectroscopy, and molecular cloning, following which a synthetic replicate was chemically synthesised for bioassay. Results: A single peptide of molecular mass 2300.92 Da was resolved in HPLC fractions of skin secretion and its primary structure determined as IYNAIWP-KH-NK-KPGLL-. Database interrogation with this sequence indicated that this peptide was encoded by skin kininogen-1 previously cloned from B. variegata. The blank cycles were occupied by cysteinyl (C) residues and the peptide was located toward the C-terminus of the skin kininogen, and flanked N-terminally by a classical -KR- propeptide convertase processing site. The peptide was named IC-20 in accordance (I = N-terminal isoleucine, C = C-terminal cysteine, 20 = number of residues). Like the natural peptide, its synthetic replicate displayed an antagonism of bradykinin-induced arterial smooth muscle relaxation. Conclusion: IC-20 represents a novel bradykinin antagonizing peptide from amphibian skin secretions and is the third such peptide found to be co-encoded with bradykinins within skin kininogens.

Development and validation of a fast monoclonal based disequilibrium enzyme-linked immunosorbent assay for the detection of triphenylmethane dyes and their metabolites in fish

Malachite Green (MG), Crystal Violet (CV) and Brilliant Green (BC) are antibacterial, antifungal and antiparasitic agents that have been used for treatment and prevention of diseases in fish. These dyes are metabolized into reduced leuco forms (LMG, LCV, LBG) that can be present in fish muscles for a long period. Due to the carcinogenic properties they are banned for use in fish for human consumption in many countries including the European Union and the United States. HPLC and LC-MS techniques are generally used for the detection of these compounds and their metabolites in fish. This study presents the development of a fast enzyme-linked immunosorbent assay (ELISA) method as an alternative for screening purposes. A first monoclonal cell line producing antibodies to MG was generated using a hybridoma technique. The antibody had good cross-reactivates with related chromatic forms of triphenylmethane dyes such as CV, BC, Methyl Green, Methyl Violet and Victoria Blue R. The monoclonal antibody (mAb) was used to develop a fast (20 min) disequilibrium ELISA screening method for the detection of triphenylmethanes in fish. By introducing an oxidation step with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) during sample extraction the assay was also used to detect the presence of the reduced metabolites of triphenylmethanes. The detection capability of the assay was 1 ng g(-1) for MG, LMG, CV, LCV and BC which was below the minimum required performance limit (MRPL) for the detection method of total MG (sum of MG and LMG) set by the Commission Decision 2004/25/EC (2 ng g(-1)). The mean recoveries for fish samples spiked at 0.5 MRPL and MRPL levels with MG and LMG were between 74.9 and 117.0% and inter- and intra-assay coefficients of variation between 4.7 and 25.7%. The validated method allows the analysis of a batch of 20 samples in two to three hours. Additionally, this procedure is substantially faster than other ELISA methods developed for MG/LMG thus far. The stable and efficient monoclonal cell line obtained is an unlimited source of sensitive and specific antibody to MG and other triphenylmethanes. (C) 2011 Elsevier B.V. All rights reserved.

The association between macular pigment optical density and CFH, ARMS2, C2/BF, and C3 genotype.

Age-related macular degeneration (AMD) is the most common cause of blindness in older people in developed countries, and risk for this condition may be classified as genetic or environmental, with an interaction between such factors predisposing to this disease. This study investigated the relationship between AMD risk genes, macular pigment optical density (MPOD), which may protect against AMD, and serum concentrations of the macular carotenoids, lutein (L) and zeaxanthin (Z). This was a cross-sectional study of 302 healthy adult subjects. Dietary intake of L and Z was assessed by food frequency questionnaire, and MPOD was measured by customized heterochromatic flicker photometry. We also calculated MPOD Area as the area of MP under the spatial profile curve, to reflect MP across the macula. Serum L and Z were measured by HPLC. Genotyping of tag SNPs in the genes CFH, ARMS2, C3, C2 and BF was undertaken with multiplex polymerase chain reaction (PCR) and primer extension methodology (ABI Snapshot, ABI Warrington UK) on DNA extracted from peripheral blood. The mean ± SD (range) age of the subjects in this study was 48 ± 11 (21-66) years. There was a statistically significant association between CFH genotype and family history of AMD, with subjects having two non-risk CFH haplotypes (n =35), or one non-risk and one protective CFH haplotype (n = 33), being significantly more likely to have a negative family history of AMD (Pearson Chi square: p = 0.001). There was no significant association between the AMD risk genes investigated and either MPOD (One way ANOVA: p > 0.05) or serum concentrations of L or Z (One way ANOVA: p > 0.05, for both). Subjects who were homozygous for risk alleles of both CFH and ARMS2 (n = 4) had significantly lower MPOD at 0.5_ and 1_ retinal eccentricity (Independent samples t test: p

The natriuretic peptide/helokinestatin precursor from Mexican beaded lizard (Heloderma horridum) venom: Amino acid sequence deduced from cloned cDNA and identification of two novel encoded helokinestatins

Natriuretic peptides are common components of reptile venoms and molecular cloning of their biosynthetic precursors has revealed that in snakes, they co-encode bradykinin-potentiating peptides and in venomous lizards, some co-encode bradykinin inhibitory peptides such as the helokinestatins. The common natriuretic peptide/helokinestatin precursor of the Gila Monster, Heloderma suspectum, encodes five helokinestatins of differing primary structures. Here we report the molecular cloning of a natriuretic peptide/helokinestatin precursor cDNA from a venom-derived cDNA library of the Mexican beaded lizard (Heloderma horridum). Deduction of the primary structure of the encoded precursor protein from this cloned cDNA template revealed that it consisted of 196 amino acid residues encoding a single natriuretic peptide and five helokinestatins. While the natriuretic peptide was of identical primary structure to its Gila Monster (H. suspectum) homolog, the encoded helokinestatins were not, with this region of the common precursor displaying some significant differences to its H. suspectum homolog. The helokinestatin-encoding region contained a single copy of helokinestatin-1, 2 copies of helokinestatin-3 and single copies of 2 novel peptides, (Phe)(5)-helokinestatin-2 (VPPAFVPLVPR) and helokinestatin-6 (GPPFNPPPFVDYEPR). All predicted peptides were found in reverse phase HPLC fractions of the same venom. Synthetic replicates of both novel helokinestatins were found to antagonize the relaxing effect of bradykinin on rat tail artery smooth muscle. Thus lizard venom continues to provide a source of novel biologically active peptides. (C) 2011 Published by Elsevier Inc.

Identification of MRP-8 (calgranulin A) as a major responsive protein in chronic periodontitis

The purpose of the study was to analyse how the protein composition of the inflammatory exudate associated with chronic periodontitis differed from the exudate in periodontal health. Gingival crevicular fluid (GCF) was collected from sites with chronic periodontal inflammation and from non-diseased sites in healthy control subjects. Microbore HPLC analysis revealed one major difference in GCF protein profiles between healthy controls and periodontitis patients. The protein enhanced in periodontitis patients was identified as migration inhibitory factor-related protein-8 (MRP-8) by a combination of N-terminal amino acid sequencing, mass spectrometry, and SDS-PAGE. Together, these data demonstrate, for the first time, the presence of monomeric MRP-8 in an inflammatory exudate. Whether monomeric MRP-8 is a unique feature of chronic periodontal inflammation is not yet clear, but the chemotactic properties of this peptide support a functional role for MRP-8 in periodontal inflammation. Copyright (C) 2000 John Wiley & Sons, Ltd.

Rapid synthesis of nucleotide pyrophosphate linkages in a ball mill

Using a ball mill, rapid, atom-economic coupling between adenosine-5'-phosphoromorpholidate and phosphorylated ribose derivatives as their sodium or barium salts was achieved. Facile purification by reversed-phase HPLC enabled product isolation within hours.

Development and validation of a dried blood spot LC-MS/MS assay to quantify ranitidine in paediatric samples.

A novel approach has been developed to determine ranitidine in paediatric samples using dried blood spots (DBS) on Guthrie cards (Whatman 903). A selective and sensitive HPLC-MS/MS assay has been developed and validated using small volumes of blood (30µl). A 6mm disc was punched from each DBS and extracted with methanolic solution of the internal standard (IS) nizatidine. This was further subjected to solid phase extraction (SPE), followed by reversed phase HPLC separation, using a XBridge™ C18 column and mobile phase 10mM ammonium acetate/methanol (98:2 v/v) with a flow rate of 0.3mL/min. This was combined with multiple reaction monitoring (MRM) mass detection using electrospray ionisation (ESI). The calibration curve for ranitidine was found linear over the range 10-500ng/mL (r=0.996). The limit of quantification (LOQ) of the method was validated at 10ng/mL. Accuracy and precision values for within and between days were

Enantioselective formal total synthesis of the Dendrobatidae frog toxin, (+)-pumiliotoxin B, via O-directed alkyne free radical hydrostannation

Herein we describe our application of the O-directed free radical hydrostannation of disubstituted alkyl-acetylenes (with Ph3SnH and Et3B) to the (+)-pumiliotoxin B total synthesis problem. Specifically, we report on the use of this method in the synthesis of the Overman alkyne 8, and thereby demonstrate the great utility of this process in a complex natural product total synthesis setting for the very first time. We also report here on a new, stereocontrolled, and highly practical enantioselective pathway to Overman's pyrrolidine epoxide partner 9 for 8, which overcomes the previous requirement for use of preparative HPLC to separate the 1:1 mixture of diastereomeric epoxides that was obtained in the original synthesis of 9.

Comparison of biosensor platforms for surface plasmon resonance based detection of paralytic shellfish toxins

Paralytic shellfish poisoning (PSP) toxins are produced by certain marine dinoflagellates and may accumulate in bivalve molluscs through filter feeding. The Mouse Bioassay (MBA) is the internationally recognised reference method of analysis, but it is prone to technical difficulties and regarded with increasing disapproval due to ethical reasons. As such, alternative methods are required. A rapid surface plasmon resonance (SPR) biosensor inhibition assay was developed to detect PSP toxins in shellfish by employing a saxitoxin polyclonal antibody (R895). Using an assay developed for and validated on the Biacore Q biosensor system, this project focused on transferring the assay to a high-throughput, Biacore T100 biosensor in another laboratory. This was achieved using a prototype PSP toxin kit and recommended assay parameters based on the Biacore Q method. A monoclonal antibody (GT13A) was also assessed. Even though these two instruments are based on SPR principles, they vary widely in their mode of operation including differences in the integrated mu-fluidic cartridges, autosampler system, and sensor chip compatibilities. Shellfish samples (n = 60), extracted using a simple, rapid procedure, were analysed using each platform, and results were compared to AOAC high performance liquid chromatography (HPLC) and MBA methods. The overall agreement, based on statistical 2 x 2 comparison tables, between each method ranged from 85% to 94.4% using R895 and 77.8% to 100% using GT13A. The results demonstrated that the antibody based assays with high sensitivity and broad specificity to PSP toxins can be applied to different biosensor platforms. (C) 2011 Elsevier B.V. All rights reserved.

Physical and immunoaffinity extraction of paralytic shellfish poisoning toxins from cultures of the dinoflagellate Alexandrium tamarense

In the present study the extraction of paralytic shellfish poisoning (PSP) toxins from a toxic strain of the marine dinoflagellate Alexandrium tamarense CCMP-1493 using various mechanical and/or physical procedures was investigated. PBS buffer was investigated as the extraction solvent in order for these procedures to be used directly with immuno-magnetic Ferrospheres-N. The extraction was performed following the determination of when toxin content by the algae was at its highest during batch culture. The methods used for cell lysis and toxin extraction included freeze-thawing, freeze-boiling, steel ball bearing beating, glass bead beating, and sonication. The steel ball bearing beating was determined to release a similar amount of toxin when compared to a modified standard extraction method which was reported to release 100% of toxins from the algal cells and was therefore used in the next phase of the study. This next phase was to determine the feasibility of utilising an antibody coupled to novel magnetic microspheres (Ferrospheres-N) as a simple, rapid immune-capture procedure for PSP toxins extracted from the algae. The effects of increasing mass of Ferrospheres-N on the immuno-capture of the PSP toxins from the toxic algal strain extracts were investigated. Toxin recovery was found to increase when an increasing mass of Ferrospheres-N was used until 96.2% (+/- 1.3 SD) of the toxin extracted from the cells was captured and eluted. Toxin recovery was determined by comparison to an appropriate PSP toxin standard curve following analysis by the AOAC HPLC method. (C) 2011 Elsevier B.V. All rights reserved.