GC/MS Composition and Resistance Modulatory Inhibitory Activities of Three Extracts of Lemongrass: Citral Modulates the Activities of Five Antibiotics at Sub-Inhibitory Concentrations on Methicillin-Resistant Staphylococcus aureus.

We investigated whether three extractable fractions of lemongrass (Cymbopogon citratus): aqueous and ethanol extracts and lemongrass essential oil exhibited any antimicrobial resistance modulatory effects if used in combination with selected antibiotics ampicillin, tetracycline, streptomycin, cefloxacin and amoxicillin on methicillin-resistant Staphylococcus aureus (MRSA). MRSA growth inhibition (zones of inhibition) was greatest for the lemongrass oil at concentrations of 1, 2, 5, 10 and 20 % (wt/vol). The MIC for lemongrass oil was 0.5 mg/mL, while it was 4 mg/mL for both the aqueous and ethanol extracts. Evaluation of extracts for antibacterial resistance modifying activities when used in combination with either of the five antibiotics at sub-inhibitory concentrations, showed that lemongrass oil highly potentiated the activities of three antibiotics; amoxicillin, streptomycin and tetracycline. The ethanol extract enhanced the activity of tetracycline and ampicillin, while the aqueous extract only increased the activity of tetracycline against MRSA. The activity of cefloxacin with the extracts was either indifferent. Analysis of the lemongrass oil by GC/MS showed the prominence of three compounds: the two isomers neral and geranial of citral and, the acetate geranyl acetate, which together made up 94 % of the composition. The compounds were also observed in the ethanol and water extracts but to a lesser extent when analyzed by HPLC-UV (λ 233 nm). Our study confirms the antibacterial properties of the extracts especially, lemongrass oil. It also demonstrates that lemongrass oil potentiates the activities of three antibiotics against the biofilm-forming MRSA. This biocidal, anti-biofilm disruption and antibiotic potentiating abilities are mainly attributable to citral and geranyl acetate, further evidence of lemongrass oil as a very useful source of phytochemicals, especially citral for the fight against antibiotic resistance.

Evaluation of the Modulatory Effect of Annona muricata Extracts on the Activity of Some Selected Antibiotics against Biofilm-Forming MRSA.

The study investigated the influence of Annona muricata extracts on the action of selected antibiotics against biofilm-forming MRSA. The various parts of the plant were processed into powder and extracted with ethanol or hot water and then screened for the presence of phytochemicals. The modulatory effect of the Annona muricata extract was also tested on some antibiotics against Methicillin-resistant Staphylococcus aureus (MRSA). The findings from this study revealed that the various parts of the Annona muricata extract (ethanolic and aqueous) contained different proportions of secondary metabolites. Varied antimicrobial activities were observed when the extract of the A. muricata was exposed to MRSA strain at a concentration of 100 mg/mL. The stem recorded the highest (17.00 and 18.00 mm) inhibitory activity against MRSA for both the aqueous and the ethanolic extract, respectively, and this was not different from the control, tetracycline. Again, the results on the modulatory action indicated that out of the 10 extracts of A. muricata, 4 of them antagonized the activity of ampicillin against the tested MRSA by a factor of 0.5 folds and the rest potentiated the drug within 1-4 folds, respectively. On the other hand, the various test extracts significantly potentiated the efficacy of streptomycin and tetracycline against the MRSA by a range of 1-32 folds with the aqueous root extract recording the highest synergistic effect and ethanol seed extract with the least effect. The findings of this study support the antibacterial activities of the A. muricata plant parts.

Current perspectives on supercharging reagents in electrospray ionization mass spectrometry.

In electrospray ionization mass spectrometry (ESI-MS), analytes are introduced into the mass spectrometer in typically aqueous-organic solvent mixtures, including pH modifiers. One mechanism for improving the signal intensity and simultaneously increasing the generation of higher charge-state ions is the inclusion of small amounts (approx. <0.5% v/v mobile phase solution) of charge-inducing or supercharging reagents, such as m-nitrobenzyl alcohol, o-nitrobenzyl alcohol, m-nitrobenzonitrile, m-(trifluoromethyl)-benzyl alcohol and sulfolane. We explore the direct and indirect (colligative properties) that have been proposed as responsible for their modes of action during ESI. Of the many theorized mechanisms of ESI, we re-visit the three most popular and highlight how they are impacted by supercharging observations on small ions to large molecules including proteins. We then provide a comprehensive list of 34 supercharging reagents that have been demonstrated in ESI experiments. We include an additional 19 potential candidate isomers as supercharging reagents and comment on their broad physico-chemical properties. It is becoming increasingly obvious that advances in technology and improved ion source design, analyzers e.g. the use of ion mobility, ion trap, circular dichroism (CD) spectroscopy, together with computer modeling are increasing the knowledge base and, together with the untested isomers and yet-to-be unearthed ones, offer opportunities for further research and application in other areas of polymer research.

Determination of homocysteine in human saliva by liquid chromatography and electrospray ionization quadrupole time-of-flight mass spectrometry: profiles in healthy adults.

Homocysteine (Hcys) is a non-essential amino acid associated with a range of diseased and abnormal metabolic conditions. Hcys concentration in saliva is routinely determined by enzyme assays, which are broadly specific, but can be expensive and suffer from cross-reactivity. Total Hcys (tHcys) concentrations in eight healthy adults were determined to establish the inter-day variation during resting, normal and intensive physical activity, using the more sensitive analytical techniques of liquid chromatography and tandem mass spectrometry without prior derivatization. Saliva (~ 1.5 mL) was collected over four days; early morning (EA), normal activity (NA) and during physical activity (PA). Samples were processed by disulphide reduction, acetonitrile precipitation and then centrifugation-filtration. Extracts were chromatographically resolved and analysed on a quadrupole time- of-flight (QToF) mass spectrometer. The protonated [M+H]+, m/z 136.101 and product ([M+H]+- HCOOH) m/z 90.103 ions were then monitored against an internal standard (13CHcys) and external set of calibration standards. Mean tHcys concentration for the whole group, including exercise was 6.6 ± 8.0 (range 0.2 - 29.6 nmol/mL). Overall, concentration of tHcys was greater in males than the females but not significantly (p > 0.05). The mean EA concentration was significantly (p < 0.05) greater than NA for both males (p = 0340) and females (p = 0.0045). There were large within-subject variations (coefficient of variation; CV%; 24% to 103%). The limits of detection (LOD) and quantification (LOQ) were 0.07 and 0.22 nmol/mL, respectively. The procedure potentially provides a convenient means of analyzing salivary Hcys as a diagnostic disease marker.

Influence of electrolytes and a supercharging reagent on charge state distribution and response of neuropeptide ions generated during positive electrospray ionisation mass spectrometry.

To aid in the detection of trace quantities of neuropeptides in a biological matrix (as saliva), the influence of different electrolytes and a supercharging reagent on the positive electrospray ionisation mass spectrometry (ESI-MS) response was investigated. Ammonium acetate, sodium chloride and sodium bicarbonate (10(-7) M to 10(-3) M) and the supercharging reagent m-nitrobenzyl alcohol (m-NBA) was added to the mobile phase and the effect on the ESI response and charge-states distribution (CSD) was studied in a group of peptides (molecular weight range 2.2kDa to 3.5kDa; CGRP, VIP, GLP1, CRF and PrRP). As expected, the result indicates that the ESI response is affected by the presence of additives: ammonium acetate shifted the observed charge states ratio whereas the addition of m-NBA resulted in the appearance of higher maximum charge state ions. This increase in higher charge state for all the peptide ions, [M + nH]n+ to [M + (n + 1)H](n+1), was atttributed to protonation of the C-terminal. However, when the composition of MeCN in a mobile phase containing m-NBA was increased, an enhancement of the total ion signal was observed for non-polar peptide samples. This is a very interesting observation as this is not observed in samples without m-NBA and could be a result of how these peptide ions are solubilised and positioned relative to the droplet surface/air interface.

Peptide polarity and the position of arginine as sources of selectivity during positive electrospray ionisation mass spectrometry.

Electrospray ionisation (ESI) is a selective process and, for similar sized analytes, the intrinsic properties of the molecules affect the ionisation process and their response. This research sets out to determine the effect of some of these properties in peptides: peptide polarity and the presence of arginine at positions 1 and 4 in the amino acid sequence on the ESI response. Six peptides; molecular mass ranges 1.3-1.6 kDa; substance P (SP) and glutamate fibrinopeptide (GFP) and 3.2-3.7 kDa; calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP), glucagon-like peptide 1 (GLP1) and defensin human neutropeptide 2 (DHNP2), were investigated. We have demonstrated that in positive ESI, for similar sized peptides and the same charge state, the responsiveness is in the order: Peptides with N or C terminal arginine > most non-polar peptides > least non-polar peptides. Therefore, arginine at the terminal position is a source of selectivity. Data from matrix-assisted laser desorption ionisation (MALDI) analysis supports that of the ESI experiments: Peptides with a terminal arginine residue generated higher signal intensities. Our observations extend our understanding of the ESI process and provide a rational approach to optimising sensitivity of electrospray conditions where a narrow mass range of peptides are poorly chromatographically resolved. This information will provide for a more effective method development process, especially during label-free quantitative determination of peptides extracted in solution.

Practical considerations in analysing neuropeptides, calcitonin gene-related peptide and vasoactive intestinal peptide, by nano-electrospray ionisation and quadrupole time-of-flight mass spectrometry: monitoring multiple protonations.

We investigated the pre-electrospray ionisation (pre-ESI) factors; analyte concentration (1-2500 ng/mL), concentration of formic acid (FA) in the mobile phase (0.01, 0.1 and 1%), concentration of the organic modifier (acetonitrile 50-90%) and flow rate (<10 µL/min) on the number of multiple protonations and ESI response for two neuropeptides (of ~3.3 kDa molecular mass); calcitonin gene-related peptide (CGRP) and vasoactive intestinal peptide (VIP). A pH of 3.23 (0.1% FA), nano-flow rate range of 350-750 nL/min and acetonitrile concentration of 50% were optimum for both neuropeptides where the highest intensities were observed. An inverse relationship between decreasing flow rate and ESI response for both peptides was also observed. The quadruply charged ([M+4H](4+)) ion was dominant for CGRP at all analyte concentrations, and also for VIP, but only at the higher analyte concentrations (250-2500 ng/mL); none of the [M+4H](4+), [M+5H](5+) or [M+6H](6+) ions were dominant at the lower concentrations. Linear correlations were obtained for the protonated states and ESI response at analyte concentrations (1-750 ng/mL). Acetonitrile concentration was critical; severe ion suppression was observed for VIP when the concentration of acetonitrile was ≥60%. Ion suppression was also observed for both peptides in an equimolar mixture, with the extent of ion suppression more severe for VIP. Our study concludes that it is important to monitor several protonated species when a single protonated state does not dominate, especially during label-free peptide quantitations.

Strong anion exchange liquid chromatographic separation of protein amino acids for natural 13C-abundance determination by isotope ratio mass spectrometry.

Amino acids are the building blocks of proteins and the analysis of their (13)C abundances is greatly simplified by the use of liquid chromatography (LC) systems coupled with isotope ratio mass spectrometry (IRMS) compared with gas chromatography (GC)-based methods. To date, various cation exchange chromatography columns have been employed for amino acid separation. Here, we report strong anion exchange chromatography (SAX) coupled to IRMS with a Liquiface interface for amino acid δ(13)C determination. Mixtures of underivatised amino acids (0.1-0.5 mM) and hydrolysates of representative proteins (prawns and bovine serum albumin) were resolved by LC/IRMS using a SAX column and inorganic eluents. Background inorganic carbon content was minimised through careful preparation of alkaline reagents and use of a pre-injector on-line carbonate removal device. SAX chromatography completely resolved 11 of the 16 expected protein amino acids following acid hydrolysis in underivatised form. Basic and neutral amino acids were resolved with 35 mM NaOH in isocratic mode. Elution of the aromatic and acidic amino acids required a higher hydroxide concentration (180 mM) and a counterion (NO 3-, 5-25 mM). The total run time was 70 min. The average δ(13)C precision of baseline-resolved peaks was 0.75‰ (range 0.04 to 1.06‰). SAX is a viable alternative to cation chromatography, especially where analysis of basic amino acids is important. The technology shows promise for (13)C amino acid analysis in ecology, archaeology, forensic science, nutrition and protein metabolism.

Blood volume and red cell mass in children with moderate and severe malaria measured by chromium-53 dilution and gas chromatography/mass spectrometric analysis.

Understanding blood volume changes in children with malaria is important for managing fluid status. Traditionally, blood/red cell volume measurements have used radioactive chromium isotopes. We applied an alternative approach, using non-radioactive chromium-53 labelling and mass spectrometry to investigate red cell volume (RCV) in Gabonese children with malaria. Nineteen children with malaria participated (10 severe, 9 moderately severe; ages 15 months to 7 years). Blood labelled with (53)Cr-chromate ex vivo was re-injected, then sampled 30 min later. Pre- and post-injection (53)Cr content were measured by gas chromatography/electron ionisation mass spectrometry of the chromium-trifluoroacetylacetone (TFA) chelate, calibrated against (50)Cr standards. Blood and red cell volumes were calculated from isotopic dilution in 15 of 19 children (in four, insufficient signal mitigated analysis). In this small pilot study, there were no significant differences between moderate and severe cases. Including all subjects, the mean RCV was reduced compared with predicted values (184 vs. 269 mL; p = 0.016) but blood volume, 71 +/- 33 mL/kg (normalised for weight), was close to predicted, approximately 77 mL/kg, commensurate with reduced haematocrit. Blood lactate concentration correlated negatively with RCV/weight (r = -0.56, p = 0.028), consistent with anaemia. In one case, sequential samples over 42 days gave an estimated rate of (53)Cr disappearance of 1.4%/day (equivalent half-life: 70 days). (53)Cr-labelling of red cells may be used to estimate blood and red cell volumes and can be used as an investigative tool in situations such as childhood diseases and resource-constrained settings. Although the red cell mass is depleted in malaria, the blood volume appears relatively well preserved.