A novel, quorum sensor-infused liposomal drug delivery system suppresses Candida albicans biofilms.

In contrast to the plethora of antibacterial agents, only a handful of antifungals are currently available to treat Candida albicans biofilm-associated infections. Additional novel antibiofilm strategies to eliminate C. albicans biofilm infections are needed. This study aims to improve the efficacy of a widely used azole, fluconazole by co-delivering it with a Pseudomonas aeruginosa quorum sensing molecule (QSM), N-3-oxo-dodecanoyl-L-homoserine lactone (C12AHL) in a liposomal formulation. C12AHL is known to inhibit C. albicans' morphological transition and biofilm formation. Four different formulations of liposomes with fluconazole (L-F), with C12AHL (L-H), with fluconazole and C12AHL (L-HF), and a drug-free control (L-C) were prepared using a thin-film hydration followed by extrusion method, and characterised. The effect of liposomes on colonising (90 min-24 h) and preformed (24 h) C. albicans biofilms were assessed using a standard biofilm assay. Biofilm viability (XTT reduction assay), biomass (Safranin-O staining) and architecture (confocal laser scanning microscopy, CLSM) were determined. Similar efficiencies of fluconazole entrapment were noticed in L-HF and L-F (11.74% vs 10.2%), however, L-HF released greater quantities of fluconazole compared to L-F during 24 h (4.27% vs 0.97%, P < 0.05). The entrapment and release of C12AHL was similar for L-H and L-HF liposomes (33.3% vs 33% and 88.9% vs 92.3% respectively). L-HF treated colonising, and preformed biofilms exhibited >80%, and 60% reduction in their respective viabilities at a fluconazole concentration as low as 5.5 µg/mL compared to 12% and 36%, respective reductions observed in L-F treated biofilms (P < 0.05). CLSM confirmed biofilm disruption, lack of hyphae, and reduction in biomass when treated with L-HF compared to other liposomal preparations. Liposomal co-delivery of C12AHL and fluconazole appears to suppress C. albicans biofilms through efficacious disruption of the biofilm, killing of constituent yeasts, and diminishing their virulence at a significantly lower antifungal dose. Therefore, liposomal co-formulation of C12AHL and fluconazole appears to be a promising approach to improve the efficacy of this common triazole against biofilm-mediated candidal infections.

The efficacy of Dynorphin fragments at the κ, µ and δ opioid receptor in transfected HEK cells and in an animal model of unilateral peripheral inflammation.

BACKGROUND: Dynorphin 1-17 is an endogenous peptide that is released at sites of inflammation by leukocytes, binding preferentially to κ-opioid receptors (KOP) to mediate nociception. We have previously shown that dynorphin 1-17 is rapidly biotransformed to smaller peptide fragments in inflamed tissue homogenate. This study aimed to determine the efficacy and potency of selected dynorphin fragments produced in an inflamed environment at the KOP, µ and δ-opioid receptors (MOP and DOP respectively) and in a model of inflammatory pain. Functional activity of Dynorphin 1-17 and fragments (1-6, 1-7 and 1-9) were screened over a range of concentrations against forskolin stimulated human embryonic kidney 293 (HEK) cells stably transfected with one of KOP, MOP or DOP. The analgesic activity of dynorphin 1-7 in a unilateral model of inflammatory pain was subsequently tested. Rats received unilateral intraplantar injections of Freund's Complete Adjuvant to induce inflammation. After six days rats received either dynorphin 1-7, 1-17 or the selective KOP agonist U50488H and mechanical allodynia determined. Dynorphin 1-7 and 1-9 displayed the greatest activity across all receptor subtypes, while dynorphin 1-7, 1-9 and 1-17 displaying a potent activation of both KOP and DOP evidenced by cAMP inihibition. Administration of dynorphin 1-7 and U50488H, but not dynorphin 1-17 resulted in a significant increase in paw pressure threshold at an equimolar dose suggesting the small peptide dynorphin 1-7 mediates analgesia. These results show that dynorphin fragments produced in an inflamed tissue homogenate have changed activity at the opioid receptors and that dynorphin 1-7 mediates analgesia.

The effect of pasteurization on trace elements in donor breast milk.

OBJECTIVE: Premature infants often receive pasteurized donor human milk when mothers are unable to provide their own milk. This study aims to establish the effect of the pasteurization process on a range of trace elements in donor milk. STUDY DESIGN: Breast milk was collected from 16 mothers donating to the milk bank at the Royal Brisbane and Women's Hospital. Samples were divided into pre- and post-pasteurization aliquots and were Holder pasteurized. Inductively coupled plasma mass spectrometry was used to analyze the trace elements zinc (Zn), copper (Cu), selenium (Se), manganese (Mn), iodine (I), iron (Fe), molybdenum (Mo) and bromine (Br). Differences in trace elements pre- and post-pasteurization were analyzed. RESULTS: No significant differences were found between the trace elements tested pre- and post-pasteurization, except for Fe (P<0.05). The median (interquartile range, 25 to 75%; µg l(-1)) of trace elements for pre- and post- pasteurization aliquots were-Zn: 1639 (888-4508), 1743 (878-4143), Cu: 360 (258-571), 367 (253-531), Se: 12.34 (11.73-17.60), 12.62 (11.94-16.64), Mn: (1.48 (1.01-1.75), 1.49 (1.11-1.75), I (153 (94-189), 158 (93-183), Fe (211 (171-277), 194 (153-253), Mo (1.46 (0.37-2.99), 1.42 (0.29-3.73) and Br (1066 (834-1443), 989 (902-1396). CONCLUSIONS: Pasteurization had minimal effect on several trace elements in donor breast milk but high levels of inter-donor variability of trace elements were observed. The observed decrease in the iron content of pasteurized donor milk is, however, unlikely to be clinically relevant.

Effects of salinity on antibiotic production in sponge-derived Salinispora actinobacteria.

AIMS: To investigate the effects of growth conditions related to marine habitat on antibiotic production in sponge-derived Salinispora actinobacteria. METHODS AND RESULTS: Media with varying salt concentration were used to investigate the effects of salinity in relation to Salinispora growth and rifamycin production. The chemotypic profiles of the model strain Salinispora arenicola M413 was then assessed using metabolomic fingerprints from high-pressure liquid chromatography with diode array detection (HPLC-DAD) and multivariate data analysis, before extending this approach to two other strains of S. arenicola. Fingerprint data were generated from extracts of S. arenicola broth cultures grown in media of varying salt (NaCl) concentrations. These fingerprints were then compared using multivariate analysis methods such as principal components analysis (PCA) and orthogonal projection to latent structures discriminant analysis (OPLS-DA). From the analysis, a low-sodium growth condition (1% NaCl) was found to delay the onset of growth of the model S. arenicola M413 strain when compared to growth in media with either 3% artificial sea salt or 3% NaCl. However, low-sodium growth conditions also increased cell mass yield and contributed to at least a significant twofold increase in rifamycin yield when compared to growth in 3% artificial sea salt and 3% NaCl. CONCLUSIONS: The integration of HPLC-DAD and multivariate analysis proved to be an effective method of assessing chemotypic variations in Salinispora grown in different salt conditions, with clear differences between strain-related chemotypes apparent due to varying salt concentrations. SIGNIFICANCE AND IMPACT OF THE STUDY: The observed variation in S. arenicola chemotypic profiles further suggests diversity in secondary metabolites in this actinomycete in response to changes in the salinity of its environment.

Dynorphin A 1-17 biotransformation in inflamed tissue, serum and trypsin solution analysed by liquid chromatography-tandem mass spectrometry.

Dynorphin A 1-17 (DYN A) is an endogenous neuropeptide that is of interest due to its diverse roles in analgesia, inflammation and addiction. Upon release, DYN A is subject to metabolism by a range of enzymes and its biotransformation is dependent on the site and environment into which it is released. In this study, we investigated the biotransformation of DYN A in rat inflamed tissue at pH 7.4 and 5.5, in rat serum and in trypsin solution. DYN A-porcine was incubated at 37 °C in each matrix over a range of incubation periods. The resultant fragments were separated using a C4 column and detected by mass spectrometry using total ion current mode. Incubation of DYN A in trypsin solution and in rat serum resulted in 6 and 14 fragments, respectively. Incubation in inflamed rat paw tissue occasioned 21 fragments at pH 7.4 and 31 fragments at pH 5.5. Secondary breakdown of some larger primary fragments was also observed in this study.

Study of beta endorphin metabolism in inflamed tissue, serum and trypsin solution by liquid chromatography-tandem mass spectrometric analysis.

Beta endorphin (ß-END) is recognised as one of the most significant endogenous neuropeptides, responsible for a wide range of biological activities in the body. However, within the body ß-END is exposed to hydrolysis by a variety of enzymes. In this study, we investigated the metabolism and fragmentation pattern of ß-END in rat inflamed tissue, in rat serum and in trypsin solution. ß-END (1-31)-rat was incubated at 37 °C in each matrix for different incubation times. The resultant fragments were separated using a C4 column and detected by mass spectrometry using total ion current mode. Structural information for the fragments was elucidated using tandem mass spectrometry. Incubation of ß-END (1-31)-rat in trypsin solution and in rat serum resulted in 8 and 13 fragments, respectively. Incubation in inflamed rat paw tissue resulted in 22 fragments at pH 7.4 and 26 fragments at pH 5.5. Some of these fragments were common to both pH values. The degradation of ß-END (1-31)-rat in inflamed tissue at pH 5.5 was faster than that at pH 7.4. Secondary fragmentation of some larger primary fragments was also observed in this study.

Effect of solvent and electrospray mass spectrometer parameters on the charge state distribution of peptides--a case study using liquid chromatography/mass spectrometry method development for beta-endorphin assay.

Beta-endorphin was used as a model peptide to study the effect of solvent and electrospray mass spectrometer parameters in the optimisation of an assay method for multiply charged compounds using liquid chromatography/mass spectrometry (LC/MS). Unlike with singly charged compounds, the charge state distribution has a significant impact in the method development of multiply charged compounds such as peptides. Using a 50% acetonitrile/water solvent mixture, we found that the ion spray voltage had no influence on the charge state distribution. However, increasing declustering potential led to deprotonation of the higher charge states of the peptide thus causing a shift to lower charge states. The mechanism leading to the deprotonation was examined. It was concluded that the deprotonation is due to endoergic proton transfer from the peptide to solvent molecules clustered to the peptide that occurs in the declustering region. The extent of deprotonation increases with increasing proton affinity of the molecules of the non-aqueous solvent component used. Thus, if desired, deprotonation can be avoided by selecting a low proton affinity solvent such as methanol. The focusing potential was also found to have a great influence on the charge state distribution observed. The results of this study enabled us to select the optimum ion to be used in single ion/reaction monitoring mode. They also provided the most favourable parameter values to be used in the method to obtain the best sensitivity for the ion of choice. The results demonstrate the importance of considering the charge state distribution in the optimisation of electrospray LC/MS methods for multiply charged compounds.

Effect of ionization suppression by trace impurities in mobile phase water on the accuracy of quantification by high-performance liquid chromatography/mass spectrometry.

The high-performance liquid chromatography (HPLC) column is capable of enrichment/pre-concentration of trace impurities in the mobile phase during the column equilibration, prior to sample injection and elution. These impurities elute during gradient elution and result in significant chromatographic peaks. Three types of purified water were tested for their impurity levels, and hence their performances as mobile phase, in HPLC followed by total ion current (TIC) mode of MS. Two types of HPLC-grade water produced 3-4 significant peaks in solvent blanks while LC/MS-grade water produced no peaks (although peaks were produced by LC/MS-grade water also after a few days of standing). None of the three waters produced peaks in HPLC followed by UV-Vis detection. These peaks, if co-eluted with analyte, are capable of suppressing or enhancing the analyte signal in a MS detector. As it is not common practice to run solvent blanks in TIC mode, when quantification is commonly carried out using single ion monitoring (SIM) or single or multiple reaction monitoring (SRM or MRM), the effect of co-eluting impurities on the analyte signal and hence on the accuracy of the results is often unknown to the analyst. Running solvent blanks in TIC mode, regardless of the MS mode used for quantification, is essential in order to detect this problem and to take subsequent precautions.

Development and validation of a reversed-phase high-performance liquid chromatographic method for quantification of peptide dendrimers in human skin permeation experiments.

The aim of the present work was to develop and validate a simple RP-HPLC method with UV detection to quantify peptide dendrimers in skin permeation experiments. Six dendrimers of varying positive charges (4(+), 8(+) and 16(+)) containing either histidine or arginine as terminal aminoacids were prepared by solid phase peptide synthesis. Mobile phase containing 0.02% (v/v) heptafluorobutyric acid in 90% acetonitrile-water was capable of separating all dendrimers from interfering peaks of receptor fluid. For the calibration of each dendrimer, a different dendrimer from the same class was selected as the internal standard. The results of preliminary human skin permeation studies showed that the developed analytical method can be successfully used for the quantification of cationic poly(aminoacid)-based dendrimers in skin permeation experiments.

Development of an HPLC-MS/MS method for the selective determination of paracetamol metabolites in mouse urine.

An HPLC-MS/MS method has been developed for the selective quantitative analysis of paracetamol and its two major metabolites. The use of tandem MS enabled the detection and quantitation of metabolites in small sample sizes with high sensitivity and selectivity. Isocratic elution using acetonitrile and water containing formic acid combined with electrospray-tandem MS enabled the separation and accurate quantitation of each analyte and the internal standard 3-acetamidophenol. The on-column limits of detection for paracetamol, paracetamol sulfate, and paracetamol glucuronide were 2.4, 1.2, and 1.2 pmol, respectively. The method was applied to quantitate paracetamol and its metabolites in mouse urine. It is highly specific, sensitive, and easily adaptable to measure these analytes in biological fluids of other animals.

Shortcomings of protein removal prior to high performance liquid chromatographic analysis-A case study using method development for BAY 11-7082.

During the analytical method development for BAY 11-7082 ((E)-3-[4-methylphenylsulfonyl]-2-propenenitrile), using HPLC-MS-MS and HPLC-UV, we observed that the protein removal process (both ultrafiltration and precipitation method using organic solvents) prior to HPLC brought about a significant reduction in the concentration of this compound. The use of a structurally similar internal standard, BAY 11-7085 ((E)-3-[4-t-butylphenylsulfonyl]-2-propenenitrile), was not effective in compensating for the loss of analyte as the extent of reduction was different to that of the analyte. We present here a systematic investigation of this problem and a new validated method for the determination of BAY 11-7082.

Quantitative GC-MS analysis of D(9)-tetrahydrocannabinol in fiber hemp varieties.

A sensitive and specific method for the determination of total available D(9)-tetrahydrocannabinol in fiber hemp varieties is described. The method was used for the regulatory purposes in which the detection of higher than the maximum allowed concentration of the psychoactive cannabinoid, D(9)-tetrahydrocannabinol, in industrial fiber hemp would result in cancellation of the grower's license. Cannabinoids were extracted from dry leaf powder into hexane containing internal standard chrysene-d(12) using sonication. D(9)-Tetrahydrocannabinol in the extract was separated by gas chromatography and quantitated by mass spectroscopy. A linear calibration range extending to 40 ppm and a limit of detection of 0.2 ng were obtained by using the total ion current mode of detection.

Enhancing the ratio of molecular ions to non-covalent compounds in the electrospray interface of LC-MS in quantitative analysis.

A common problem encountered during the development of MS methods for the quantitation of small organic molecules by LC-MS is the formation of non-covalently bound species or adducts in the electrospray interface. Often the population of the molecular ion is insignificant compared to those of all other forms of the analyte produced in the electrospray, making it difficult to obtain the sensitivity required for accurate quantitation. We have investigated the effects of the following variables: orifice potential, nebulizer gas flow, temperature, solvent composition and the sample pH on the relative distributions of ions of the types MH+, MNa+, MNH4+, and 2MNa+, where M represents a small organic molecule: BAY 11-7082 ((E)-3-[4-methylphenylsulfonyl]-2-propenenitrile). Orifice potential, solvent composition and the sample pH had the greatest influence on the relative distributions of these ions, making these parameters the most useful for optimizing methods for the quantitation of small molecules.

Simultaneous determination of vitamin E homologs in chicken meat by liquid chromatography with fluorescence detection.

A simple and reliable method for the simultaneous determination of all eight homologs of Vitamin E in chicken meat is described. All analytes, including the internal standard (alpha-tocopherol acetate), were eluted within 35 min and detected using their native fluorescence (295 nm excitation and 330 nm emission). Chromatography using hexane based eluent on a normal phase silica column included an initial column conditioning step to prevent irreversible adsorption of tocopherols and tocotrienols on silica. Lowest detectable levels of alpha-tocopherol, gamma-tocopherol, alpha-tocotrienol, beta-tocotrienol, gamma-tocotrienol and delta-tocotrienol were 0.73, 0.86, 1.0, 1.2, 1.7 and 1.3 ng, respectively.

Effect of dietary tocopherols and tocotrienols on the antioxidant status and lipid stability of chicken.

We determined the effect of dietary tocopherols and tocotrienols on the lipid stability of pre-cooked chicken breast and thigh. The birds were supplemented with one of two doses of a commercial mixture of tocopherols and tocotrienols (Oryza1, Oryza2) or one of two doses of all-rac α-tocopherol acetate (Toc1, Toc2). Diets were formulated so that Oryza1 and Toc1 and Oryza2 and Toc2 contained similar tocopherol concentrations. No quantifiable amounts of tocotrienols were found in either breast or thigh muscles. Tocotrienols present in the diet reduced muscle α-tocopherol concentration. The effect of Oryza1 on the tocopherol content in muscle and on its lipid stability was not significant. The Oryza2, Toc1 and Toc2 diets increased the α- and γ-tocopherol in breast and thigh muscles and enhanced their lipid stability. This improvement was only due to the antioxidant action of the tocopherols. Lipid stability of pre-cooked chicken was not enhanced by adding tocotrienols to a tocopherol supplement.

Simultaneous determination of creatinine and pseudouridine concentrations in bovine plasma by reversed-phase liquid chromatography with photodiode array detection.

A simple, rapid method for the simultaneous determination of creatinine and pseudouridine in bovine plasma is described. Plasma was de-proteinised, concentrated, and chromatographed for 15 min on a C(18) column. Analytes were detected at an optimum wavelength (262 nm) and the internal standard (cimetidine) was detected at 220 nm. The pH of analysis was between 6.5 and 7 where both analytes exist in single chemical forms giving maximum accuracy. Recoveries of both analytes were above 96%. Lowest detectable amounts of creatinine and pseudouridine were 0.28 nmol and 9.0 pmol, and the typical levels detected (+/-SD) were 60 (+/-2.8) and 2.3 (+/-0.10) micromol/L, respectively.

Method for the extraction of riboflavin for high-performance liquid chromatography and application to casein.

Three different approaches, including the commonly used acid digestion, were compared for the efficient extraction of very low concentrations of riboflavin (vitamin B2) from casein. An extraction method that used pepsin to release riboflavin was the most efficient. The pepsin extraction method was then further optimized using a factorial design to yield the maximum amount of riboflavin. The enzyme takadiastase was used for the conversion of all forms of riboflavin to the free form, and this conversion was found to be time dependent. A reversed-phase HPLC system with fluorescence detection and a methanol-water mobile phase containing acetate buffer were used for the separation and quantification of riboflavin in the extracts. A limit of detection of less than 0.1 mg kg-1 casein and a repeatability RSD of 3% were achieved. The recovery of the method was satisfactory.