Dried blood spot liquid chromatography assay for therapeutic drug monitoring of metformin

The use of blood spot collection cards is a simple way to obtain specimens for analysis of drugs for the purpose of therapeutic drug monitoring, assessing adherence to medications and preventing toxicity in routine clinical setting. We describe the development and validation of a microanalytical technique for the determination of metformin from dried blood spots. The method is based on reversed phase high-performance liquid chromatography with ultraviolet detection. Drug recovery in the developed method was found to be more than 84%. The limits of detection and quantification were calculated to be to be 90 and 150 ng/ml, respectively. The intraday and interday precision (measured by CV%) was always less than 9%. The accuracy (measured by relative error, %) was always less than 12%. Stability analysis showed that metformin is stable for at least 2 months when stored at -70 degrees C. The small volume of blood required (10 mu L), combined with the simplicity of the analytical technique makes this a useful procedure for monitoring metformin concentrations in routine clinical settings. The method is currently being applied to the analysis of blood spots taken from diabetic patients to assess adherence to medications and relationship between metformin level and metabolic control of diabetes. (c) 2006 Elsevier B.V. All rights reserved.

Components of the peptidome and transcriptome persist in lin wa pi: the dried skin of the Heilongjiang brown frog Rana amurensis as used in Traditional Chinese Medicine

Although the ancient practice of traditional Chinese medicine (TCM) utilizes predominantly herbal ingredients, many of which are now the subject of intense scientific scrutiny, significant quantities of animal tissue-derived materials are also employed. Here we have used contemporary molecular techniques to study the material known as lin wa pi, the dried skin of the Heilongjiang brown frog, Rana amurensis, that is used commonly as an ingredient of many medicines, as a general tonic and as a topical antimicrobial/wound dressing. Using a simple technology that has been developed and validated over several years, we have demonstrated that components of both the skin granular gland peptidome and transcriptome persist in this material. Interrogation of the cDNA library constructed from the dried skin by entrapment and amplification of polyadenylated mRNA, using a "shotgun" primer approach and 3'-RACE, resulted in the cloning of cDNAs encoding the precursors of five putative antimicrobial peptides. Two (ranatuerin-2AMa and ranatuerin-2AMb) were obvious homologs of a previously described frog skin peptide family, whereas the remaining three were of sufficient structural novelty to be named amurins 1-3. Mature peptides were each identified in reverse phase HPLC fractions of boiling water extracts of skin and their structures confirmed by MS/MS fragmentation sequencing. Components of traditional Chinese medicines of animal tissue origin may thus contain biologically active peptides that survive the preparation procedures and that may contribute to therapeutic efficacy.

Elements of the granular gland peptidome and transcriptome persist in air-dried skin of the South American orange-legged leaf frog, Phyllomedusa hypocondrialis.

The defensive strategy of amphibians against predator attack relies heavily on the secretion of noxious/toxic chemical cocktails from specialized skin granular glands. Bioactive peptides constitute a major component of secretions in many species and the most complex are produced by neotropical leaf frogs of the sub-family Phyllomedusinae. We recently reported that these skin secretions contain elements of both the granular gland peptidome and transcriptome and that polyadenylated mRNAs constituting the latter are protected from degradation by interactions with endogenous amphipathic peptides. This thus permits parallel amino acid sequencing of peptides and nucleic acid sequencing of cloned precursor transcripts from single lyophilized samples of secretion. Here we report that the protection afforded is sufficiently robust to permit transcriptome studies by cloning of full-length polyadenylated peptide precursor encoding mRNAs from libraries constructed using ambient temperature air-dried skin from recently deceased specimens as source material. The technique was sufficiently sensitive to permit the identification of cDNAs encoding antimicrobial peptides constituted by six different isoforms of phylloseptin and two dermaseptins. Also, for the first time, establishment of the nucleic acid and amino acid sequence of the precursor encoding the phyllomedusine frog skin bradykinin-related peptide, phyllokinin, from cloned cDNA, was achieved. These data unequivocally demonstrate that the granular gland transcriptome persists in air-dried amphibian skin—a finding that may have fundamental implications in the study of archived materials but also in the wider field of molecular biology.

Volatile compounds in Bacillus-fermented soybeans

Thua nao, a traditional, proteolytic, fermented soybean condiment of northern Thailand, was prepared from cooked whole soybeans by natural flora fermentation. The microbial flora during the fermentation was dominated by Bacillus species. The formation of volatile compounds during the fermentation was studied. In addition, the volatile compounds of two samples of commercial dried thua nao and two samples of commercial Japanese natto were analysed. Fermentation led to a large increase in the concentration of total volatile compounds, from 35 mug kg(-1) wet weight in cooked soybeans to 3500 mug kg(-1) wet weight in 72h fermented material. The major volatile compounds in fermented beans were 3-hydroxybutanone (acetoin), 2-methlybutanoic acid, pyrazines, dimethyl disulphide and 2-pentylfuran. Sun drying of 72 h fermented material resulted in the loss of 65% of total volatiles, including important aroma compounds. The commercial dried thua nao samples had low concentrations of total volatile compounds (380 mug kg(-1) wet weight). It is suggested that improved drying/preservation methods are needed to retain aroma compounds in the traditional products. The natto samples were devoid of aldehydes, aliphatic acids and esters, and sulphur compounds, whereas the thua nao samples contained a diversity of these compounds. Previous investigators have reported these compounds in natto and it is not possible to suggest the existence of systematic differences between the volatile compounds in traditional thua nao prepared with an undefined, mixed microbial flora and those in natto fermented with Bacillus subtilis. (C) 2001 Society of Chemical Industry.

Development and validation of a dried blood spot-LC-APCI-MS assay for estimation of canrenone in paediatric samples

A selective and sensitive liquid chromatography (LC)-atmospheric pressure chemical ionisation (APCI)-mass spectroscopic (MS) assay of canrenone has been developed and validated employing Dried Blood Spots (DBS) as the sample collection medium. DBS samples were prepared by applying 30 mu l of spiked whole blood onto Guthrie cards. A 6 mm disc was punched from the each DBS and extracted with 2 ml of methanolic solution of 17 alpha-methyltestosterone (Internal Standard). The methanolic extract was evaporated to dryness and reconstituted in acetonitrile:water (1:9, v/v). The reconstituted solution was further subjected to solid phase extraction using HLB cartridges. Chromatographic separation was achieved using Waters Sunfire C18 reversed-phase column using isocratic elution, followed by a high organic wash to clear late eluting/highly retained components. The mobile phase consisted of methanol:water (60:40, v/v) pumped at a flow rate of 0.3 ml/min. LC-APCI-MS detection was performed in the selected-ion monitoring (SIM) mode using target ions at m/z 341.1 and 303.3 for canrenone and internal standard respectively. The selectivity of the method was established by analysing DBS samples from 6 different sources (individuals). The calibration curve for canrenone was found to be linear over 25-1000 ng/ml (r >0.994). Accuracy (% RE) and precision (% CV) values for within and between day were

Freeze-dried, mucoadhesive system for vaginal delivery of the HIV microbicide, dapivirine: Optimisation by an artificial neural network

Dapivirine mucoadhesive gels and freeze-dried tablets were prepared using a 3 x 3 x 2 factorial design. An artificial neural network (ANN) with multi-layer perception was used to investigate the effect of hydroxypropyl-methylcellulose (HPMC): polyvinylpyrrolidone (PVP) ratio (XI), mucoadhesive concentration (X2) and delivery system (gel or freeze-dried mucoadhesive tablet, X3) on response variables; cumulative release of dapivirine at 24 h (Q(24)), mucoadhesive force (F-max) and zero-rate viscosity. Optimisation was performed by minimising the error between the experimental and predicted values of responses by ANN. The method was validated using check point analysis by preparing six formulations of gels and their corresponding freeze-dried tablets randomly selected from within the design space of contour plots. Experimental and predicted values of response variables were not significantly different (p > 0.05, two-sided paired t-test). For gels, Q(24) values were higher than their corresponding freeze-dried tablets. F-max values for freeze-dried tablets were significantly different (2-4 times greater, p > 0.05, two-sided paired t-test) compared to equivalent gets. Freeze-dried tablets having lower values for X1 and higher values for X2 components offered the best compromise between effective dapivirine release, mucoadhesion and viscosity such that increased vaginal residence time was likely to be achieved. (C) 2009 Elsevier B.V. All rights reserved.