Development of descriptor tools for the characterization of Australian sugar mill evaporator scale

Cleaning of sugar mill evaporators is an expensive exercise. Identifying the scale components assists in determining which chemical cleaning agents would result in effective evaporator cleaning. The current methods (based on x-ray diffraction techniques, ion exchange/high performance liquid chromatography and thermogravimetry/differential thermal analysis) used for scale characterisation are difficult, time consuming and expensive, and cannot be performed in a conventional analytical laboratory or by mill staff. The present study has examined the use of simple descriptor tests for the characterisation of Australian sugar mill evaporator scales. Scale samples were obtained from seven Australian sugar mill evaporators by mechanical means. The appearance, texture and colour of the scale were noted before the samples were characterised using x-ray fluorescence and x-ray powder diffraction to determine the compounds present. A number of commercial analytical test kits were used to determine the phosphate and calcium contents of scale samples. Dissolution experiments were carried out on the scale samples with selected cleaning agents to provide relevant information about the effect the cleaning agents have on different evaporator scales. Results have shown that by simply identifying the colour and the appearance of the scale, the elemental composition and knowing from which effect the scale originates, a prediction of the scale composition can be made. These descriptors and dissolution experiments on scale samples can be used to provide factory staff with an on-site rapid process to predict the most effective chemicals for chemical cleaning of the evaporators.

A longitudinal assessment of chronic wound fluid to detect biochemical indicators of healing

Chronic venous leg ulcers are a detrimental health issue plaguing our society, resulting in long term pain, immobility and decreased quality of life for a large proportion of sufferers. The frequency of these chronic wounds has led current research to focus on the wound environment to provide important information regarding the prolonged, fluctuated or static healing patterns of these wounds. Disruption to the normal wound healing process results in release of multiple factors in the wound environment that could correlate to wound chronicity. These biochemical factors can often be detected through non-invasively sampling chronic wound fluid (CWF) from the site of injury. Of note, whilst there are numerous studies comparing acute and chronic wound fluids, there have not been any reports in the literature employing a longitudinal study in order to track biochemical changes in wound fluid as patients transition from a non-healing to healed state. Initially the objective of this study was to identify biochemical changes in CWF associated with wound healing using a proteomic approach. The proteomic approach incorporated a multi-dimensional liquid chromatography fractionation technique coupled with mass spectrometry (MS) to enable identification of proteins present in lower concentrations in CWF. Not surprisingly, many of the proteins identified in wound fluid were acute phase proteins normally expressed during the inflammatory phase of healing. However, the number of proteins positively identified by MS was quite low. This was attributed to the diverse range in concentration of protein species in CWF making it challenging to detect the diagnostically relevant low molecular weight proteins. In view of this, SELDI-TOF MS was also explored as a means to target low molecular weight proteins in sequential patient CWF samples during the course of healing. Unfortunately, the results generated did not yield any peaks of interest that were altered as wounds transitioned to a healed state. During the course of proteomic assessment of CWF, it became evident that a fraction of non-proteinaceous compounds strongly absorbed at 280 nm. Subsequent analyses confirmed that most of these compounds were in fact part of the purine catabolic pathway, possessing distinctive aromatic rings and which results in high absorbance at 254 nm. The accumulation of these purinogenic compounds in CWF suggests that the wound bed is poorly oxygenated resulting in a switch to anaerobic metabolism and consequently ATP breakdown. In addition, the presence of the terminal purine catabolite, uric acid (UA), indicates that the enzyme xanthine oxidoreductase (XOR) catalyses the reaction of hypoxanthine to xanthine and finally to UA. More importantly, the studies provide evidence for the first time of the exogenous presence of XOR in CWF. XOR is the only enzyme in humans capable of catalysing the production of UA in conjunction with a burst of the highly reactive superoxide radical and other oxidants like H2O2. Excessive release of these free radicals in the wound environment can cause cellular damage disrupting the normal wound healing process. In view of this, a sensitive and specific assay was established for monitoring low concentrations of these catabolites in CWF. This procedure involved combining high performance liquid chromatography (HPLC) with tandem mass spectrometry and multiple reaction monitoring (MRM). This application was selective, using specific MRM transitions and HPLC separations for each analyte, making it ideal for the detection and quantitation of purine catabolites in CWF. The results demonstrated that elevated levels of UA were detected in wound fluid obtained from patients with clinically worse ulcers. This suggests that XOR is active in the wound site generating significant amounts of reactive oxygen species (ROS). In addition, analysis of the amount of purine precursors in wound fluid revealed elevated levels of purine precursors in wound fluid from patients with less severe ulcers. Taken together, the results generated in this thesis suggest that monitoring changes of purine catabolites in CWF is likely to provide valuable information regarding the healing patterns of chronic venous leg ulcers. XOR catalysis of purine precursors not only provides a method for monitoring the onset, prognosis and progress of chronic venous leg ulcers, but also provides a potential therapeutic target by inhibiting XOR, thus blocking UA and ROS production. Targeting a combination of these purinogenic compounds and XOR could lead to the development of novel point of care diagnostic tests. Therefore, further investigation of these processes during wound healing will be worthwhile and may assist in elucidating the pathogenesis of this disease state, which in turn may lead to the development of new diagnostics and therapies that target these processes.

Challenges in mass spectrometry-based quantification of bioactive peptides: A case study exploring the neuropeptide Y family

The study of biologically active peptides is critical to the understanding of physiological pathways, especially those involved in the development of disease. Historically, the measurement of biologically active endogenous peptides has been undertaken by radioimmunoassay, a highly sensitive and robust technique that permits the detection of physiological concentrations in different biofluid and tissue extracts. Over recent years, a range of mass spectrometric approaches have been applied to peptide quantification with limited degrees of success. Neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP) belong to the NPY family exhibiting regulatory effects on appetite and feeding behavior. The physiological significance of these peptides depends on their molecular forms and in vivo concentrations systemically and at local sites within tissues. In this report, we describe an approach for quantification of individual peptides within mixtures using high-performance liquid chromatography electrospray ionization tandem mass spectrometry analysis of the NPY family peptides. Aspects of quantification including sample preparation, the use of matrix-matched calibration curves, and internal standards will be discussed. This method for the simultaneous determination of NPY, PYY, and PP was accurate and reproducible but lacks the sensitivity required for measurement of their endogenous concentration in plasma. The advantages of mass spectrometric quantification will be discussed alongside the current obstacles and challenges. © 2012 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 98: 357–366, 2012.

Studies on the surface properties of biodegradable polymer carriers in respiratory delivery of drug from Dry Powder Inhaler formulations

Dry Powder Inhaler (DPI) technology has a significant impact in the treatment of various respiratory disorders. DPI formulations consist of a micronized drug (<5ìm) blended with an inert coarse carrier, for which lactose is widely used to date. DPIs are one of the inhalation devices which are used to target the delivery of drugs to the lungs. Drug delivery via DPI formulations is influenced by the physico-chemical characteristics of lactose particles such as size, shape, surface roughness and adhesional forces. Commercially available DPI formulations, which utilise lactose as the carrier, are not efficient in delivering drug to the lungs. The reasons for this are the surface morphology, adhesional properties and surface roughness of lactose. Despite several attempts to modify lactose, the maximum efficient drug delivery to the lungs remains limited; hence, exploring suitable alternative carriers for DPIs is of paramount importance. Therefore, the objective of the project was to study the performance of spherical polymer microparticles as drug carriers and the factors controlling their performance. This study aimed to use biodegradable polymer microspheres as alternative carriers to lactose in DPIs for achieving efficient drug delivery into the lungs. This project focused on fabricating biodegradable polymer microparticles with reproducible surface morphology and particle shape. The surface characteristics of polymeric carriers and the adhesional forces between the drug and carrier particles were investigated in order to gain a better understanding of their influence on drug dispersion. For this purpose, two biodegradable polymers- polycaprolactone (PCL) and poly (DL-lactide-co-glycolide) (PLGA) were used as the carriers to deliver the anti-asthmatic drug - Salbutamol Sulphate (SS). The first study conducted for this dissertation was the aerosolization of SS from mixtures of SS and PCL or PLGA microparticles. The microparticles were fabricated using an emulsion technique and were characterized by laser diffraction for particle size analysis, Scanning Electron Microscopy (SEM) for surface morphology and X-ray Photoelectron Spectroscopy (XPS) to obtain surface elemental composition. The dispersion of the drug from the DPI formulations was determined by using a Twin Stage Impinger (TSI). The Fine particle Fraction (FPF) of SS from powder mixtures was analyzed by High Performance Liquid Chromatography (HPLC). It was found that the drug did not detach from the surface of PCL microspheres. To overcome this, the microspheres were coated with anti-adherent agents such as magnesium stearate and leucine to improve the dispersion of the drug from the carrier surfaces. It was found that coating the PCL microspheres helped in significantly improving the FPF of SS from the PCL surface. These results were in contrast to the PLGA microspheres which readily allowed detachment of the SS from their surface. However, coating PLGA microspheres with antiadherent agents did not further improve the detachment of the drug from the surface. Thus, the first part of the study demonstrated that the surface-coated PCL microspheres and PLGA microspheres can be potential alternatives to lactose as carriers in DPI formulations; however, there was no significant improvement in the FPF of the drug. The second part of the research studied the influence of the size of the microspheres on the FPF of the drug. For this purpose, four different sizes (25 ìm, 48 ìm, 100 ìm and 150 ìm) of the PCL and PLGA microspheres were fabricated and characterized. The dispersion of the drug from microspheres of different sizes was determined. It was found that as the size of the carrier increased there was a significant increase in the FPF of SS. This study suggested that the size of the carrier plays an important role in the dispersion of the drug from the carrier surface. Subsequent experiments in the third part of the dissertation studied the surface properties of the polymeric carrier. The adhesion forces existing between the drug particle and the polymer surfaces, and the surface roughness of the carriers were quantified using Atomic Force Microscopy (AFM). A direct correlation between adhesion forces and dispersion of the drug from the carrier surface was observed suggesting that adhesion forces play an important role in determining the detachment potential of the drug from the carrier surface. However, no direct relationship between the surface roughness of the PCL or PLGA carrier and the FPF of the drug was observed. In conclusion, the body of work presented in this dissertation demonstrated the potential of coated PCL microspheres and PLGA microspheres to be used in DPI formulations as an alternative carrier to sugar based carriers. The study also emphasized the role of the size of the carrier particles and the forces of interaction prevailing between the drug and the carrier particle surface on the aerosolization performances of the drug.

Pharmacokinetic studies of a Chinese triple herbal drug formula

Yin Chen Hao Tang preparation (YCHTP) is a classic traditional Chinese medicine formula, which is commonly used for clinical treatment of hepatological diseases. In this study, a rapid and validated high-performance liquid chromatography (HPLC) method was developed to simultaneously identify 6,7-dimethylesculetin and geniposide in rat plasma. This assay was performed on a Dikma Diamonsil RP(18) column (200 mmx4.6 mm, 5 mum) with acetonitrile-methanol-water (0.1% formic acid) as the mobile phase, showing acceptable linearity, intra- and inter-day precision and accuracy (R.S.D.=5%), and absolute recovery for two analytes (74%); the limits of quantitation were 0.4 and 1.12 mug/ml, and the limits of detection were 0.06 and 0.09 mug/ml for two analytes. The developed method was successfully applied to study the effect of formula compatibility on the pharmacokinetics of 6,7-dimethylesculetin and geniposide in YCHTP when orally administrating an effective human daily dose of YCHTP to rats. We surmise that formula compatibility can significantly influence the pharmacokinetics of YCHTP, and we have elucidated and validated the compatible administration of YCHTP.

Pharmacokinetics of isofraxidin in rat plasma after oral administration of the extract of acanthopanax senticosus using HPLC with solid phase extraction method

High-performance liquid chromatography coupled with solid phase extraction method was developed for determination of isofraxidin in rat plasma after oral administration of Acanthopanax senticosus extract (ASE), and pharmacokinetic parameters of isofraxidin either in ASE or pure compound were measured. The HPLC analysis was performed on a Dikma Diamonsil RP(18) column (4.6 mm x 150 mm, 5 microm) with the isocratic elution of solvent A (acetonitrile) and solvent B (0.1% aqueous phosphoric acid, v/v) (A : B = 22 : 78) and the detection wavelength was set at 343 nm. The calibration curve was linear over the range of 0.156-15.625 microg/ml. The limit of detection was 60 ng/ml. The intra-day precision was 5.8%, and the inter-day precision was 6.0%. The recovery was 87.30+/-1.73%. When the dosage of ASE is equal to pure compound caculated by the amount of isofraxidin, it has been found to have two maximum concentrations in plasma while the pure compound only showed one peak in the plasma concentration-time curve. The determined content of isofraxidin in plasma after oral administration of ASE is the total contents of free isofraxidin and its precursors in ASE in vitro. The pharmacokinetic characteristics of ASE showed the priority of the extract and the properities of traditional Chinese medicine.

Quality evaluation of Yin Chen Hao Tang extract based on fingerprint chromatogram and simultaneous determination of five bioactive constituents

A completely validated method based on HPLC coupled with photodiode array detector (HPLC-UV) was described for evaluating and controlling quality of Yin Chen Hao Tang extract (YCHTE). First, HPLC-UV fingerprint chromatogram of YCHTE was established for preliminarily elucidating amount and chromatographic trajectory of chemical constituents in YCHTE. Second, for the first time, five mainly bioactive constituents in YCHTE were simultaneously determined based on fingerprint chromatogram for furthermore controlling the quality of YCHTE quantitatively. The developed method was applied to analyze 12 batches of YCHTE samples which consisted of herbal drugs from different places of production, showed acceptable linearity, intraday (RSD <5%), interday precision (RSD <4.80%), and accuracy (RSD <2.80%). As a result, fingerprint chromatogram determined 15 representative general fingerprint peaks, and the fingerprint chromatogram resemblances are all better than 0.9996. The contents of five analytes in different batches of YCHTE samples do not indicate significant difference. So, it is concluded that the developed HPLC-UV method is a more fully validated and complete method for evaluating and controlling the quality of YCHTE.

Simultaneous determination of 6,7-dimethylesculetin and geniposide in rat plasma and its application to pharmacokinetic studies of Yin Chen Hao Tang preparation

A method for the rapid and simultaneous determination of 6,7-dimethylesculetin (CAS 120-08-1) and geniposide (CAS 24512-63-8) in rat plasma has been developed, using validated high performance liquid chromatography (HPLC) with solid phase extraction (SPE). The HPLC analysis was performed on a commercially available column (200 mm x 4.6 mm, 5 microm) with acetonitrile-methanol-0.1% aqueous formic acid as mobile phase and the UV detection at 343 nm and 238 nm for 6,7-dimethylesculetin and geniposide, respectively. The calibration curves for 6,7-dimethylesculetin and geniposide were linear over the range 0.4-25.6 microg/mL and 1.12-71.68 microg/mL, respectively. The lower limits of quantitation were 0.40 microg/ mL and 1.12 microg/mL, and the lower limits of detection were 0.06 microg/mL and 0.09 microg/ mL, respectively. The intra-day and inter-day precision for 6,7-dimethylesculetin and geniposide were < 5%, whereas the absolute recovery percentages were > 74%. A successful application of the developed HPLC analysis was demonstrated for the pharmacokinetic study of a Traditional Chinese Medicine formula of Yin Chen Hao Tang preparation.

Evaluation of brief dietary questions to estimate vegetable and fruit consumption – using serum carotenoids and red-cell folate

Objective: To evaluate responses to self-administered brief questions regarding consumption of vegetables and fruit by comparison with blood levels of serum carotenoids and red-cell folate. Design: A cross-sectional study in which participants reported their usual intake of fruit and vegetables in servings per day, and serum levels of five carotenoids (α-carotene, β-carotene, β-cryptoxanthin, lutein/zeaxanthin and lycopene) and red-cell folate were measured. Serum carotenoid levels were determined by high-performance liquid chromatography, and red-cell folate by an automated immunoassay system. Settings and subjects: Between October and December 2000, a sample of 1598 adults aged 25 years and over, from six randomly selected urban centres in Queensland, Australia, were examined as part of a national study conducted to determine the prevalence of diabetes and associated cardiovascular risk factors. Results: Statistically significant (P<0.01) associations with vegetable and fruit intake (categorised into groups: ≤1 serving, 2–3 servings and ≥4 servings per day) were observed for α-carotene, β-carotene, β-cryptoxanthin, lutein/zeaxanthin and red-cell folate. The mean level of these carotenoids and of red-cell folate increased with increasing frequency of reported servings of vegetables and fruit, both before and after adjusting for potential confounding factors. A significant association with lycopene was observed only for vegetable intake before adjusting for confounders. Conclusions: These data indicate that brief questions may be a simple and valuable tool for monitoring vegetable and fruit intake in this population.

Development of descriptor tools for the characterisation of Australian sugar mill evaporator scale

Cleaning of sugar mill evaporators is an expensive exercise. Identifying the scale components assists in determining which chemical cleaning agents would result in effective evaporator cleaning. The current methods (based on x-ray diffraction techniques, ion exchange/high performance liquid chromatography and thermogravimetry/differential thermal analysis) used for scale characterisation are difficult, time consuming and expensive, and cannot be performed in a conventional analytical laboratory or by mill staff. The present study has examined the use of simple descriptor tests for the characterisation of Australian sugar mill evaporator scales. Scale samples were obtained from seven Australian sugar mill evaporators by mechanical means. The appearance, texture and colour of the scale were noted before the samples were characterised using x-ray fluorescence and x-ray powder diffraction to determine the compounds present. A number of commercial analytical test kits were used to determine the phosphate and calcium contents of scale samples. Dissolution experiments were carried out on the scale samples with selected cleaning agents to provide relevant information about the effect the cleaning agents have on different evaporator scales. Results have shown that by simply identifying the colour and the appearance of the scale, the elemental composition and knowing from which effect the scale originates, a prediction of the scale composition can be made. These descriptors and dissolution experiments on scale samples can be used to provide factory staff with an on-site rapid process to predict the most effective chemicals for chemical cleaning of the evaporators.

Environmental regulation of leaf colour in red 35S : PAP1 Arabidopsis thaliana

High-temperature, low-light (HTLL) treatment of 35S:PAP1 Arabidopsis thaliana over-expressing the PAP1 (Production of Anthocyanin Pigment 1) gene results in reversible reduction of red colouration, suggesting the action of additional anthocyanin regulators. High-performance liquid chromatography (HPLC), liquid chromatography mass spectrometry (LCMS) and Affimetrix®-based microarrays were used to measure changes in anthocyanin, flavonoids, and gene expression in response to HTLL. HTLL treatment of control and 35S:PAP1 A. thaliana resulted in a reversible reduction in the concentrations of major anthocyanins despite ongoing over-expression of the PAP1 MYB transcription factor. Twenty-one anthocyanins including eight cis-coumaryl esters were identified by LCMS. The concentrations of nine anthocyanins were reduced and those of three were increased, consistent with a sequential process of anthocyanin degradation. Analysis of gene expression showed down-regulation of flavonol and anthocyanin biosynthesis and of transport-related genes within 24 h of HTLL treatment. No catabolic genes up-regulated by HTLL were found. Reductions in the concentrations of anthocyanins and down-regulation of the genes of anthocyanin biosynthesis were achieved by environmental manipulation, despite ongoing over-expression of PAP1. Quantitative PCR showed reduced expression of three genes (TT8, TTG1 and EGL3) of the PAP1 transcriptional complex, and increased expression of the potential transcriptional repressors AtMYB3, AtMYB6 and AtMYBL2 coincided with HTLL-induced down-regulation of anthocyanin biosynthesis. HTLL treatment offers a model system with which to explore anthocyanin catabolism and to discover novel genes involved in the environmental control of anthocyanins.

Determination of urinary thymidine glycol using affinity chromatography, HPLC and post-column reaction detection : a biomarker of oxidative DNA damage upon kidney transplantation

Reactive oxygen species are generated during ischaemia-reperfusion of tissue. Oxidation of thymidine by hydroxyl radicals (HO) leads to the formation of 5,6-dihydroxy-5,6-dihydrothymidine (thymidine glycol). Thymidine glycol is excreted in urine and can be used as biomarker of oxidative DNA damage. Time dependent changes in urinary excretion rates of thymidine glycol were determined in six patients after kidney transplantation and in six healthy controls. A new analytical method was developed involving affinity chromatography and subsequent reverse-phase high-performance liquid chromatography (RP-HPLC) with a post-column chemical reaction detector and endpoint fluorescence detection. The detection limit of this fluorimetric assay was 1.6 ng thymidine glycol per ml urine, which corresponds to about half of the physiological excretion level in healthy control persons. After kidney transplantation the urinary excretion rate of thymidine glycol increased gradually reaching a maximum around 48 h. The excretion rate remained elevated until the end of the observation period of 10 days. Severe proteinuria with an excretion rate of up to 7.2 g of total protein per mmol creatinine was also observed immediately after transplantation and declined within the first 24 h of allograft function (0.35 + 0.26 g/mmol creatinine). The protein excretion pattern, based on separation of urinary proteins on sodium dodecyl sulphate-polyacrylamide gel electrophorosis (SDS-PAGE), as well as excretion of individual biomarker proteins, indicated nonselective glomerular and tubular damage. The increased excretion of thymidine glycol after kidney transplantation may be explained by ischaemia-reperfusion induced oxidative DNA damage of the transplanted kidney.

Gold nanoparticles modified electrodes for biosensors

Biomolecules are chemical compounds found in living organisms which are the building blocks of life and perform important functions. Fluctuation from the normal concentration of these biomolecules in living system leads to several disorders. Thus the exact determination of them in human fluids is essential in the clinical point of view. High performance liquid chromatography, flow injection analysis, capillary electrophoresis, fluorimetry, spectrophotometry, electrochemical and chemiluminescence techniques were usually used for the determination of biologically important molecules. Among these techniques, electrochemical determination of biomolecules has several advantages over other methods viz., simplicity, selectivity and sensitivity. In the past two decades, electrodes modified with polymer films, self-assembled monolayers containing different functional groups and carbon paste have been used as electrochemical sensors. But in recent years, nanomaterials based electrochemical sensors play an important role in the improvement of public health because of its rapid detection, high sensitivity and specificity in clinical diagnostics. To date gold nanoparticles (AuNPs) have received arousing attention mainly due to their fascinating electronic and optical properties as a consequence of their reduced dimensions. These unique properties of AuNPs make them as an ideal candidate for the immobilization of enzymes for biosensing. Further, the electrochemical properties of AuNPs reveal that they exhibit interesting properties by enhancing the electrode conductivity, facilitating electron transfer and improving the detection limit of biomolecules. In this chapter, we summarized the different strategies used for the attachment of AuNPs on electrode surfaces and highlighted the electrochemical determination of glucose, ascorbic acid (AA), uric acid (UA) and dopamine derivatives using the AuNPs modified electrodes.