Amitha Hewavitharana | The University of Queensland, Australia (original) (raw)

Papers by Amitha Hewavitharana

Journal of Bioanalysis & Biomedicine, 2009

Journal of Chromatography A, 2004

A simple and reliable method for the simultaneous determination of all eight homologs of Vitamin ... more 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 (␣-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 ␣-tocopherol, ␥-tocopherol, ␣-tocotrienol, ␤-tocotrienol, ␥-tocotrienol and ␦-tocotrienol were 0.73, 0.86, 1.0, 1.2, 1.7 and 1.3 ng, respectively.

Canadian Journal of Chemistry-revue Canadienne De Chimie, 1993

ABSTRACT A quantitative ion-exchange/atomic absorption method is described for measuring the conc... more ABSTRACT A quantitative ion-exchange/atomic absorption method is described for measuring the concentration of free (hydrated) calcium and magnesium in solution at micromolar levels. Sample solutions are pumped through a micro-column of strong acid-type cation-exchange resin until equilibrium has been achieved between resin and solution. After removal of interstitial solution by first air, then water, the sorbed metal ion is eluted from the resin with nitric acid directly into an atomic absorption spectrophotometer. In a 0.1 M 1:1 electrolyte, here KNO3, the amount of metal ion sorbed on the resin is directly proportional to the free metal ion concentration in solution over a concentration range of 1.25 to 5 × 10−5 mol/L (12–50 μmol/L). Selectivities for free calcium and magnesium in the presence of complexing ligands such as citrate and phosphate compare well with calculated values.

Analytical Biochemistry, 2008

An HPLC-MS/MS method has been developed for the selective quantitative analysis of paracetamol an... more 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.

International Dairy Journal, 1996

ABSTRACT

Journal of Chromatography B-analytical Technologies in The Biomedical and Life Sciences, 2006

During the analytical method development for BAY 11-7082 ((E)-3-[4-methylphenylsulfonyl]-2-propen... more 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.

Journal of Chromatography B-analytical Technologies in The Biomedical and Life Sciences, 2003

A simple liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and vali... more A simple liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated to simultaneously determine creatinine (Cr) and uric acid (UA) levels as a confirmatory method for adulteration or dilution of urine. Centrifuged urine samples (10 mL) were diluted with 390 mL of distilled water. 30 mL of internal standard solution (Cr-d 3 , 5 mg/mL) and 10 mL of acetonitrile were added to 20 mL aliquots of diluted urine samples and filtered. The samples (1 mL) were introduced into LC-MS/ MS with no further pretreatment. Cr and UA were separated on a multi-mode ODS column (Scherzo SM-C18, 75 mm Â 2.0 mm I.D., 3 mm) and quantified by LC-MS/MS with polarity-switching electrospray ionization. Cr requires the positive-ion mode, whereas the negative-ion mode is required for the analysis of UA. The linear ranges were 1.0-300 mg/dL for Cr and 0.5-300 mg/dL for UA, with good determination coefficients (R 2 ! 0.9988). The intra-day and inter-day precision of the analytes was within 13.0% and 14.4%, respectively. The intra-day and inter-day accuracy was À8.8 to 3.7% and À0.3 to 6.6%, respectively. The lower limits of detection (LLODs) were 0.3 mg/dL for Cr and 0.07 mg/dL for UA. The applicability of the developed method was examined by analyzing urine samples from suspected drug abusers (n = 46). ß

Current Drug Delivery, 2006

ISCOMs have received much attention as vaccine adjuvants due to their immunostimulatory effects. ... more ISCOMs have received much attention as vaccine adjuvants due to their immunostimulatory effects. They are colloidal particles typically comprised of phospholipids, cholesterol and Quil A, a crude mixture of saponins extracted from the bark of Quillaja saponaria Molina. We have previously shown that ISCOMs can be prepared by ether injection wherein an ether solution of phospholipids and cholesterol in a mass ratio of 5:2 is injected into a solution of Quil A at a mass ratio of 7 lipids: 3 Quil A. The aim of this study was firstly to isolate and characterise discrete fractions of Quil A and secondly to investigate which of these fractions were able to form ISCOMs by the method of ether injection. Six fractions of Quil A were isolated by semi-preparative reverse phase high performance liquid chromatography (RP-HPLC) and characterised by analytical HPLC, liquid chromatography tandem mass spectrometry (LC-MS) and the qualitative Liebermann-Burchard and Molisch tests for triterpenoids and carbohydrates respectively. ISCOMs were subsequently prepared from the isolated fractions by the method of ether injection and the resulting preparations characterized by photon correlation spectroscopy (PCS) and negative stain transmission electron microscopy (TEM). The molecular weights of the major compounds in the fractions ranged from approximately 1200 to approximately 2300 Da; all fractions tested positive for triterpenoids and saccharides and four of the fractions were identified as QS-7, QS-17, QS-18 and QS-21 by analysis (LC-MS and analytical HPLC). Injection of ether solutions of lipids into aqueous solutions of QS-17, QS-18 or QS-21 all resulted in homogeneous ISCOM dispersions. The combination of lipids and QS-7 by ether injection produced lamellae and liposomes as the prominent structures and a minor amount of ISCOMs. The remaining two hydrophilic, low molecular weight fractions of Quil A did not produce ISCOMs, instead liposomes and helical structures predominated in the samples.

Analytical Biochemistry, 2003

Natural vitamin E is an important antioxidant in foods and is composed of eight chemical compound... more Natural vitamin E is an important antioxidant in foods and is composed of eight chemical compounds; four isomeric forms of tocopherol and four isomeric forms of tocotrienol. As different forms of tocopherols and tocotrienols have different antioxidant activities, separation and quantification of individual isomeric forms of tocopherols and tocotrienols in natural sources are important and many chromatographic methods have been developed for this purpose [1]. Silica is a commonly used stationary phase in these methods as reversed-phase columns are not capable of separating b and c isomers of tocopherols and tocotrienols .

Meat Science, 2004

We determined the effect of dietary tocopherols and tocotrienols on the lipid stability of pre-co... more 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 a-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 a-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 a-and c-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.

Journal of Agricultural and Food Chemistry, 2010

This study tested the hypothesis that mango extracts contain bioactive molecules capable of modul... more This study tested the hypothesis that mango extracts contain bioactive molecules capable of modulating endothelial cell migration, an essential step in the formation of new blood vessels or angiogenesis. The formation of new blood vessels is an important therapeutic target for diseases such as limb ischemia, coronary infarction or stroke. We examined the effect of mango peel and flesh extracts as well as the individual polyphenolic molecules, mangiferin and quercetin, on bovine aortic cell migration using a modified Boyden chamber assay. Our results show that mangiferin, and extracts rich in mangiferin, increase endothelial cell migration. The dose-effect relationship for various extracts further suggests that this action of mangiferin is modulated by other components present in the extracts. The promigratory effect of mango extracts or mangiferin was unrelated to an effect on cell proliferation, and did not involve a change in the production of matrix metalloprotease-2 or -9 by the endothelial cells. Taken together, these results suggest that mangiferin present in mango extracts may have health promoting effects in diseases related to the impaired formation of new blood vessels.

Analytical and Bioanalytical Chemistry, 2005

A common problem encountered during the development of MS methods for the quantitation of small o... more 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+, MNH 4+, 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.

Journal of Agricultural and Food Chemistry, 2003

Journal of Chromatography B-analytical Technologies in The Biomedical and Life Sciences, 2009

Aim: A simple, accurate, precise, and reproducible reversed-phase high-performance liquid chromat... more Aim: A simple, accurate, precise, and reproducible reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the determination of rizatriptan benzoate in oral strip formulations. Methodology: Separation was achieved under optimized chromatographic condition on a Hiper C 18 column (250 mm × 4.6 mm, 5 m) using Shimadzu HPLC. The mobile phase consisted of phosphate buffer (20 mM pH adjusted to 3.2 ± 0.005 with ortho phosphoric acid): Methanol in the ratio of 70:30 v/v with isocratic elution at a flow rate of 1 ml/min at ambient temperature was performed. The detection was carried out at 225 nm using photodiode array detector. The method was validated as per Q1A (R2) guidelines and suitability of developed method was ascertained by using optimized oral strip formulation. Results: The retention time of rizatriptan benzoate was found to be 5.17 min, and the calibration curve was linear in the concentration range of 0.20-20 mg/mL (r 2 = 0.9998). The limit of detection and the limit of quantitation were found to be 0.016 mg/mL and 0.0528 mg/mL, respectively. Method validation parameters were found to be within the specified limits. The percentage drug content of oral strips formulation was found to be 98.96 ± 1.37. Conclusion: The proposed HPLC method may be used efficiently for routine and quality control analysis of rizatriptan benzoate in pharmaceutical formulations.

Livestock Science, 2008

Metabolic state as influenced by growth rate may influence meat toughness and can be estimated fr... more Metabolic state as influenced by growth rate may influence meat toughness and can be estimated from metabolites excreted in urine. Urine collection over 24 h requires animals to be constrained in metabolism crates for many days. Single blood sampling to estimate metabolites in plasma would be less stressful on animals than collecting 24 h urine excretion. This study investigated the hypothesis that the plasma concentrations of pseudouridine and creatinine were representative of those found in 24 h urine excretions in steers fed different quality diets. Eleven Brahman-cross steers were fed a high (n = 3), medium (n = 4) or low (n = 4) quality hay diet for three weeks. Steers were catheterized and housed in metabolism crates for 6 days. Urine was collected every 24 h and total volume sub-sampled for analyses of creatinine and pseudouridine. Jugular blood was collected from each steer every 3 h from 07:30 to 16:30 h. Plasma was separated from red blood cells by centrifugation and frozen for creatinine and pseudouridine analyses. No time of day effect was apparent for pseudouridine or creatinine so daily means were used to test for effect of diet and to relate to urinary concentrations. Nutritional restriction halted live weight gain but had no effect on urinary or plasma pseudouridine, suggesting that diet did not affect tRNA turnover. Increased plasma creatinine concentrations and reduced urinary creatinine concentration in steers experiencing nutritional restriction indicated that their renal clearance rate decreased. As a result, the ratio of plasma pseudouridine to creatinine concentration was not directly proportional to that of 24 h urinary excretion.

Hepatology, 2006

Sulfate is required for detoxification of xenobiotics such as acetaminophen (APAP), a leading cau... more Sulfate is required for detoxification of xenobiotics such as acetaminophen (APAP), a leading cause of liver failure in humans. The NaS1 sulfate transporter maintains blood sulfate levels sufficiently high for sulfonation reactions to work effectively for drug detoxification. In the present study, we identified two loss-of-function polymorphisms in the human NaS1 gene and showed the Nas1-null mouse to be hypersensitive to APAP hepatotoxicity. APAP treatment led to increased liver damage and decreased hepatic glutathione levels in the hyposulfatemic Nas1-null mice compared with that in normosulfatemic wild-type mice. Analysis of urinary APAP metabolites revealed a significantly lower ratio of APAP-sulfate to APAP-glucuronide in the Nas1-null mice. These results suggest hyposulfatemia increases sensitivity to APAP-induced hepatotoxicity by decreasing the sulfonation capacity to metabolize APAP. In conclusion, the results of this study highlight the importance of plasma sulfate level as a key modulator of acetaminophen metabolism and suggest that individuals with reduced NaS1 sulfate transporter function would be more sensitive to hepatotoxic agents. (HEPATOLOGY 2006;43: 1241–1247.)

Canadian Journal of Chemistry-revue Canadienne De Chimie, 1991

Journal of Chromatography A, 2011

Matrix matching is used in analysis to compensate for matrix effects that influence analytical re... more Matrix matching is used in analysis to compensate for matrix effects that influence analytical response. It has been a widely discussed topic in electro-spray mass spectrometry where the ionization suppression is a major problem in accurate quantitative analysis. However, the unique strength of mass spectrometry to detect and quantify accurately a co-eluting stable isotope labelled internal standard offers an easy solution to the ionization suppression problem. Given the fact that it is impossible to match the matrix of the calibration standards with all samples, mass spectrometry allows accurate quantitation without the need for matrix matching, as long as the internal standard co-elutes with the analyte of interest. If the analyte and internal standard co-elute, the slope of the calibration curve analyte response/internal standard vs. analyte concentration is independent of the matrix composition, eliminating the need for matrix matching.

European Urology, 2010

We would like to thank Dr. Hewavitharana [1] for his pensive comments regarding the potential val... more We would like to thank Dr. Hewavitharana [1] for his pensive comments regarding the potential validity of urinary sarcosine as marker in prostate cancer (PCa) diagnostics as reported in our article [2] and the original paper of Sreekumar et al. . We discussed the limitations of our study and the differences between our study and the study of Sreekumar et al in replies to the editorial to our article [6] and to a letter to the editor [7]. These differences concerning the measuring method, test samples, and study cohorts might explain some discrepant results [4-7] but should not be discussed again here. However, more importantly, Dr Hewavitharana raised fundamental objections regarding the analytic performance of the measurement of sarcosine in urine in both studies. He assumed that analytic issues significantly contributed to the observed different results and that the methods in both studies are unvalidated.