HPLC method development for fampridine using Analytical Quality by Design approach.

Offering a systematic and multivariate analysis of the analytical procedure, development and validation of HPLC methods using Quality by Design approach are in the limelight of current research trends. A new, experimental design-aided HPLC method for fampridine was developed and preliminarily validated according to current in-force international guidelines for linearity, accuracy, robustness and precision. The method offers a high throughput sample analysis, with an elution time of 2.9 minutes, and signal detection without excipient interference performed at 262 nm. The method proved to be linear between 1-15 µg mL-1 (R2= 0.9996). The mean recovery was found to be 98.7 ± 1.9 % in the tested range of 2.5-7.5 µg mL-1. Low RSD values (< 1 %) were obtained for both model, intra- and inter-day precision. The limit of detection and limit of quantification were 0.24 and 0.78 µg mL-1, resp. The method proved to be applicable for active substance assay in a pharmaceutical dosage form.

Novel High-Throughput Quantitation of Potent hERG Blocker Dofetilide in Human Plasma by Liquid Chromatography Tandem Mass Spectrometry: Application in a Phase 1 ECG Biomarker Validation Study.

The authors developed a novel, sensitive high-throughput ultra-performance liquid chromatography-tandem mass spectrometric method for the determination of dofetilide in human plasma. To compensate for the matrix effect, a deuterated internal standard was used. The method employed a very low sample volume (50 µL) of plasma for sample processing by using simple protein precipitation extraction in a 96-well plate. The extracted samples were chromatographed on an Acquity BEH C18 column (2.1 × 100 mm, 1.7 µm) and eluted in a gradient manner at a flow rate of 0.5 mL/min for 2 min using 5 mM ammonium formate (0.1% formic acid) and methanol. The calibration curve was linear from 25 to 2,500 pg/mL with a correlation coefficient (r2) ≥ 0.99 (0.9969-0.9980; n = 3). The developed method was validated as per the current United States Food and Drug Administration's guidance for industry on 'Bioanalytical Method Validation'. The multiple reaction-monitoring mode was employed for quantitation of dofetilide with m/z 442.2/198.2 and dofetilide d4 with m/z 446.2/198.2. The validated method was used for evaluation of dofetilide concentration in the Comprehensive in vitro Proarrhythmia Assay phase 1 electrocardiogramic biomarker validation study.

Thermostable potassium channel-inhibiting neurotoxins in processed scorpion medicinal material revealed by proteomic analysis: Implications of its pharmaceutical basis in traditional Chinese medicine.

The neurotoxins of venomous scorpion act on ion channels. Whether these neurotoxins are retained in processed Buthus martensii Karsch scorpions used in traditional Chinese medicine materials is unknown. Comprehensive mass spectrometry-based proteomic characterization of functionally active toxins in the processed medicinal scorpion material revealed 22 full-length and 44 truncated thermostable potassium channel-modulatory toxins that preserved six conserved cysteine residues capable of forming the three disulfide bonds necessary for toxicity. Additionally, a broad spectrum of degraded toxin fragments was found, indicating their relative thermal instability which enabled toxicity reduction. Furthermore, the suppression of interleukin-2 (IL-2) production in Jurkat cells and the reduced delayed-type hypersensitivity (DTH) response demonstrated that the extracts have immunoregulatory activity both in vitro and in vivo. Our work describes the first "map" of functionally active scorpion toxins in processed scorpion medicinal material, which is helpful to unveil the pharmaceutical basis of the processed scorpion medicinal material in traditional Chinese medicine. BIOLOGICAL SIGNIFICANCE: Scorpions have been used as medicinal materials in China for more than one thousand years. This is an example of the well-known "Combat poison with poison" strategy common to traditional Chinese medicine. In the past 30 years, extensive investigations of Chinese scorpions have indicated that the neurotoxins in the scorpion venom are the main toxic components and they target various ion channels in cell membranes. However, whether these neurotoxins are retained in processed Buthus martensii Karsch scorpions used for traditional Chinese medicine remains unknown. Our study described the thermal stability and instability of potassium channel-modulatory neurotoxins in processed scorpions and helps to understand the pharmaceutical basis underling the strategy of "combat poison with poison to cure diseases".

Transcriptomic and Proteomic Analyses Reveal the Diversity of Venom Components from the Vaejovid Scorpion Serradigitus gertschi.

To understand the diversity of scorpion venom, RNA from venomous glands from a sawfinger scorpion, Serradigitus gertschi, of the family Vaejovidae, was extracted and used for transcriptomic analysis. A total of 84,835 transcripts were assembled after Illumina sequencing. From those, 119 transcripts were annotated and found to putatively code for peptides or proteins that share sequence similarities with the previously reported venom components of other species. In accordance with sequence similarity, the transcripts were classified as potentially coding for 37 ion channel toxins; 17 host defense peptides; 28 enzymes, including phospholipases, hyaluronidases, metalloproteases, and serine proteases; nine protease inhibitor-like peptides; 10 peptides of the cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 protein superfamily; seven La1-like peptides; and 11 sequences classified as "other venom components". A mass fingerprint performed by mass spectrometry identified 204 components with molecular masses varying from 444.26 Da to 12,432.80 Da, plus several higher molecular weight proteins whose precise masses were not determined. The LC-MS/MS analysis of a tryptic digestion of the soluble venom resulted in the de novo determination of 16,840 peptide sequences, 24 of which matched sequences predicted from the translated transcriptome. The database presented here increases our general knowledge of the biodiversity of venom components from neglected non-buthid scorpions.

Graphene nanoribbon/FePt bimetallic nanoparticles/uric acid as a novel magnetic sensing layer of screen printed electrode for sensitive determination of ampyra.

A novel electrochemical sensor for sensitive determination of ampyra (Am) based on graphene nanoribbons modified by iron-platinum bimetallic nanoparticles and uric acid (SPCE/FePtGNR/UA) dropped on the screen-printed carbon electrode (SPCE) surface and magnetically captured onto an SPCE working electrode surface is reported in the present work. The modified nanocomposite and sensing layer was characterized by different techniques, including cyclic voltammetry (CV), linear sweep voltammetry (LSV), electrochemical impedance spectroscopy (EIS), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR) and X-ray powdered diffraction (XRD). Am determination by conventional electrochemical methods is not possible, because of its high redox overpotential. Therefore, the differential pulse voltammetry (DPV) signals of UA were used as a redox probe for indirect electrochemical determination of Am. The limit of detection (LOD) and linear concentration range were obtained as 0.028 and 0.08-9.0µmolL-1 (3Sb/m = 3), respectively. The feasibility of the proposed method was examined by the detection of Am in biological and pharmaceutical samples with satisfactory results. The constructed electrochemical sensor was applied for fast, simple and sensitive detection of Am in real environments.

Characterization of the venom from the Australian scorpion Urodacus yaschenkoi: Molecular mass analysis of components, cDNA sequences and peptides with antimicrobial activity.

The Urodacidae scorpions are the most widely distributed of the four families in Australia and represent half of the species in the continent, yet their venoms remain largely unstudied. This communication reports the first results of a proteome analysis of the venom of the scorpion Urodacus yaschenkoi performed by mass fingerprinting, after high performance liquid chromatography (HPLC) separation. A total of 74 fractions were obtained by HPLC separation allowing the identification of approximately 274 different molecular masses with molecular weights varying from 287 to 43,437 Da. The most abundant peptides were those from 1 K Da and 4-5 K Da representing antimicrobial peptides and putative potassium channel toxins, respectively. Three such peptides were chemically synthesized and tested against Gram-positive and Gram-negative bacteria showing minimum inhibitory concentration in the low micromolar range, but with moderate hemolytic activity. It also reports a transcriptome analysis of the venom glands of the same scorpion species, undertaken by constructing a cDNA library and conducting random sequencing screening of the transcripts. From the resultant cDNA library 172 expressed sequence tags (ESTs) were analyzed. These transcripts were further clustered into 120 unique sequences (23 contigs and 97 singlets). The identified putative proteins can be assorted in several groups, such as those implicated in common cellular processes, putative neurotoxins and antimicrobial peptides. The scorpion U. yaschenkoi is not known to be dangerous to humans and its venom contains peptides similar to those of Opisthacanthus cayaporum (antibacterial), Scorpio maurus palmatus (maurocalcin), Opistophthalmus carinatus (opistoporines) and Hadrurus gerstchi (scorpine-like molecules), amongst others.

Short-chain peptides identification of scorpion Buthus martensi Karsch venom by employing high orthogonal 2D-HPLC system and tandem mass spectrometry.

Scorpion venom contains a considerable variety of neurotoxic peptides that can act on ionic channels. Here, we describe an orthogonal 2D-reversed phase/hydrophilic interaction chromatography system (RPLC/HILIC) and use it to separate short-chain peptides from Asian scorpion Buthus martensi Karsch (BmK) venom in a high throughput format. Due to its high orthogonality and efficiency, 18 homogenous peptides were purified and sequence identified by MS/MS with collision-induced dissociation. Among them, four peptides were discovered, which only have evidence at transcript-level, were first purified from crude venom in this study. Two peptides named BmKK2-b and Martentoxin-b were found the new cleaved chains of known BmKK2 and Martentoxin. In addition, two novel peptides named BmKK12 and BmKK16 in this paper were sequenced by de novo MS/MS, which we predict, are members of potassium channel toxin α-KTx 17 subfamily by homology to other known peptides found in the Swiss-Prot protein database.

Identification of novel Kv1.3 blockers using a fluorescent cell-based ion channel assay.

A functional cell-based assay was developed using a generic proprietary assay protocol, based on a membrane-potential sensitive dye, for the identification of small-molecule antagonists against the Kv1.3 potassium ion channel. A high-throughput screen (HTS) was subsequently performed with 20,000 compounds from the Evotec library, preselected using known small molecule antagonists for both sodium and potassium ion channels. Following data analysis, the hit rate was measured at 1.72%, and subsequent dose-response analysis of selected hits showed a high hit confirmation rate yielding approximately 50 compounds with an apparent IC50 value lower than 10 microM. Subsequent electrophysiological characterization of selected hits confirmed the initial activity and potency of the identified hits on the Kv1.3 target and also selectivity toward Kv1.3 through measurements on HERG as well as Kv1.3-expressing cell lines. Follow-up structure-activity relationship analysis revealed a variety of different clusters distributed throughout the library as well as several singlicates. In comparison to known Kv1.3 blockers, new chemical entities and scaffolds showing potency and selectivity against the Kv1.3 ion channel were detected. In addition, a screening strategy for ion channel drug discovery HTS, medicinal chemistry, and electrophysiology is presented.

A subfamily of acidic alpha-K(+) toxins.

Three homologous acidic peptides have been isolated from the venom of three different Parabuthus scorpion species, P. transvaalicus, P. villosus, and P. granulatus. Analysis of the primary sequences reveals that they structurally belong to subfamily 11 of short chain alpha-K(+)-blocking peptides (Tytgat, J., Chandy, K. G., Garcia, M. L., Gutman, G. A., Martin-Eauclaire, M. F., van der Walt, J. J., and Possani, L. D. (1999) Trends Pharmacol. Sci. 20, 444-447). These toxins are 36-37 amino acids in length and have six aligned cysteine residues, but they differ substantially from the other alpha-K(+) toxins because of the absence of the critical Lys(27) and their total overall negative charge. Parabutoxin 1 (PBTx1), which has been expressed by recombinant methods, has been submitted to functional characterization. Despite the lack of the Lys(27), this toxin blocks several Kv1-type channels heterologously expressed in Xenopus oocytes but with low affinities (micromolar range). Because a relationship between the biological activity and the acidic residue substitutions may exist, we set out to elucidate the relative impact of the acidic character of the toxin and the lack of the critical Lys(27) on the weak activity of PBTx1 toward Kv1 channels. To achieve this, a specific mutant named rPBTx1 T24F/V26K was made recombinantly and fully characterized on Kv1-type channels heterologously expressed in Xenopus oocytes. Analysis of rPBTx1 T24F/V26K displaying an affinity toward Kv1.2 and Kv1.3 channels in the nanomolar range shows the importance of the functional dyad above the acidic character of this toxin.