Preliminary evaluation of monolithic column high-performance liquid chromatography with tris(2,2’-bipyridyl)ruthenium(II) chemiluminescence detection for the determination of quetiapine in human body fluids

High-performance liquid chromatography (HPLC) with tris(2,2-bipyridyl)ruthenium(II) chemiluminescence detection methodology is reported for the determination of the atypical antipsychotic drug quetiapine and the observation of its major active and inactive metabolites in human urine and serum. The method uses a monolithic chromatographic column allowing high flow rates of 3mL min−1 enabling rapid quantification. Flow injection analysis (FIA) with tris(2,2-bipyridyl)ruthenium(II) chemiluminescence detection and HPLC time of flight mass spectrometry (TOF-MS) were used for the determination of quetiapine in a pharmaceutical preparation to establish its suitability as a calibration standard. The limit of detection achieved with FIA was 2×10−11 mol L−1 in simple aqueous solution. The limits of detection achieved with HPLC were 7×10−8 and 2×10−10 mol L−1 in urine and serum, respectively. The calibration range for FIA was between 5×10−9 and 1×10−6 mol L−1. The calibration ranges for HPLC were between 1×10−7–1×10−4 and 1×10−8–1×10−4 mol L−1 in urine and serum, respectively. The quetiapine concentrations in patient samples were found to be 3×10−6 mol L−1 in urine and 7×10−7 mol L−1 in serum. Without the need for preconcentration, the HPLC detection limits compared favourably with those in previously published methodologies. The metabolites were identified using HPLC-TOF-MS.

Chemiluminescence detection of piperazine designer drugs and related compounds using tris(2,2?-bipyridine)ruthenium(III)

 An exploration of the chemiluminescence from reactions of a large number of benzyl and phenylpiperazine analytes with tris(2,2’-bipyridyl)ruthenium(III) was carried out providing information towards the emission intensity of this chemiluminescent reagent and the structure of analytes it interacts with.

A review of recent advances in chemiluminescence detection using nano-colloidal manganese(IV)

The application of 'soluble' (colloidal) manganese(IV) for chemiluminescence detection is reviewed, focussing on papers published since the last comprehensive review of the subject in 2008. Advances in this reagent system include: the on-line formation of manganese(IV); new insight into the light-producing pathway and selectivity of the reagent; its application to assess total antioxidants in plant derived samples and oxidative stress in biological fluids and tissues; and the replacement of the formaldehyde enhancer with ethanol.

Ethanol as an alternative to formaldehyde for the enhancement of manganese(IV) chemiluminescence detection.

Previous applications of manganese(IV) as a chemiluminescence reagent have required the use of formaldehyde to enhance the emission intensity to analytically useful levels. However, this known human carcinogen (by inhalation) is not ideal for routine application. A wide range of alternative enhancers have been examined but to date none have been found to provide the dramatic increase in chemiluminescence intensities obtained using formaldehyde. Herein, we demonstrate that ethanol offers a simple, safe and inexpensive alternative to the use of formaldehyde for manganese(IV) chemiluminescence detection, without compromising signal intensity or sensitivity. For example, chemiluminescence signals for opiate alkaloids using 50-100% ethanol were 0.8-1.6-fold those using 2M formaldehyde. This innocuous alternative enhancer is shown to be a particularly effective for the direct detection of thiols and disulfides by manganese(IV) chemiluminescence, which we have applied to a simple HPLC procedure to determine a series of biomarkers of oxidative stress.

Chemiluminescence detection of MDMA in street drug samples using tris (2,2'-bipyridine) ruthenium (III)

Tris(2,2'-bipyridine)ruthenium(II) chemiluminescence was investigated for the detection of 3,4-methylenedioxymethamphetamine (MDMA) and several related compounds in street drug samples. Optimization using flow injection analysis showed that the selectivity of the reagent can be targeted towards the detection of secondary amines by altering the pH of the reaction environment. The greater selectivity of this mode of detection, compared to UV-absorbance, reduces the probability of false positive results from interfering compounds. The detection limit for MDMA under these conditions was 0.48 μM. A HPLC method incorporating post-column tris(2,2'-bipyridine)ruthenium(II) chemiluminescence detection was applied to the determination of MDMA in five street drug samples. The results obtained were in good agreement with quantification performed using traditional UV-absorbance detection, which demonstrates the viability of this method for confirmatory analysis of drug samples. This is the first report of tris(2,2'-bipyridine)ruthenium(II) chemiluminescence for the detection of MDMA and related amphetamine derivatives.

Flow analysis based on a pulsed flow of solution: theory, instrumentation and applications

The increased demands placed on solution propulsion by programmed flow systems, such as sequential injection analysis, lab-on-value technology, bead injection and multi-commutation, has highlighted the inability of many conventional pumps to generate a smooth, consistent flow. A number of researchers have examined ways to overcome the inadvertent, uncontrolled pulsation caused by the mechanical action of peristaltic pumps. In contrast, we have developed instruments that exploit the characteristics of a reproducible pulsed flow of solution. In this paper, we discuss our instrumental approaches and some applications that have benefited from the use of a reproducible pulsed flow rather than the traditional linear flow approach. To place our approach in the context of the continuously developing field of flow analysis, an overview of other programmed flow systems is also presented.

Determination of sodium oxalate in Bayer liquor using flow-analysis incorporating an anion exchange column and tris(2,2`-bipyridyl)ruthenium(II) chemiluminescence detection

Semi-automated flow injection instrumentation, incorporating a small anion exchange column coupled with tris(2,2′-bipyridyl)ruthenium(II) (Ru(bipy)₃²⁺) chemiluminescence detection, was configured and utilised to develop rapid methodology for the determination of sodium oxalate in Bayer liquors. The elimination of both negative and positive interferences from aluminium(III) and, as yet, unknown concomitant organic species, respectively are discussed. The robustness of the methodology was considerably enhanced by using the temporally stable form of the chemiluminescence reagent, tris(2,2′-bipyridyl)ruthenium(III) perchlorate in dry acetonitrile. Real Bayer process samples were analysed and the results obtained compared well with those performed using standard methods within industrial laboratories.

Development of an automated DNA purification module using a micro-fabricated pillar chip

We present a fully automated DNA purification module comprised of a micro-fabricated chip and sequential injection analysis system that is designed for use within autonomous instruments that continuously monitor the environment for the presence of biological threat agents. The chip has an elliptical flow channel containing a bed (3.5 × 3.5 mm) of silica-coated pillars with height, width and center-to-center spacing of 200, 15, and 30 µm, respectively, which provides a relatively large surface area (ca. 3 cm²) for DNA capture in the presence of chaotropic agents. We have characterized the effect of various fluidic parameters on extraction performance, including sample input volume, capture flow rate, and elution volume. The flow-through design made the pillar chip completely reusable; carryover was eliminated by flushing lines with sodium hypochlorite and deionized water between assays. A mass balance was conducted to determine the fate of input DNA not recovered in the eluent. The device was capable of purifying and recovering Bacillus anthracis genomic DNA (input masses from 0.32 to 320 pg) from spiked environmental aerosol samples, for subsequent analysis using polymerase chain reaction-based assays.

Aqueous/micellar peroxyoxalate chemiluminescent detection for ion chromatography

This thesis covers the development of the traditionally fluorescent bis(8-quinolinol-5-sulfonic acid) magnesium (II) fluorophore as a chemiluminescent emitter. A brief description of luminescence spectroscopy and its application to analytical chemistry lays the foundation to the discussion of the results obtained herein. This includes the synthesis and identification of two so called ‘water soluble’ aryl oxamides 2,2’-oxalyl-bis(trifluoromethanesulfonyl) imino] ethylene-bis(N- methylpyridinium) trifluoromethane sulfonate (PETQ) and 2,2’-oxalyl-bis(trifluoromethanesulfonyl) imino]ethylene-bis(N-pyridinium) chloride (PETH), previously developed for the US navy as a possible emergency light source, yet the synthetic methodology were incomplete. The inconsistencies of the synthetic methods for PETQ and PETH were overcome with yields satisfactory for their preliminary analytical evaluation. The evaluation of these aryl oxamides, including 4,4’-oxalyI- bis[(trifluoromethanesulfonyl) imino]ethylene-bis(l-methyM-benzylpiperidinium) trifluoromethanesulfonate (BPTQ), 4,4’-oxalyl-bis [(trifluoromethylsulfonyl)imino] ethylene-bis(N-methylmorpholinium)trifluoromethanesulfonate (METQ) and the oxalate bis(2,4,6-trichlorophenyl) oxalate (TCPO) were performed with the peroxyoxalate chemiluminescent reaction using bis(8-quinolinol-5-sulfonic acid) magnesium (II) as the fluorophore. A univariate optimisation of this system resulted in 0,0082 mol 1-1 the detection limit of magnesium in the absence of cationic surfactants and 0.0041 mol 1-1 in their presence for the majority of these compounds. The oxamides were found to be insoluble in water with long ulrasonication periods required to dissolve the compound, with solvents such as acetonitrile preferred. The determination of other chemiluminescent metal-8HQS chelates to replace magnesium -8HQS in the peroxyoxalate were limited to Al (III), Cd (II), Ca (II), In (II) and Zn (II), unfortunately these metals all possessed poorer detection limits than those obtained using magnesium The base reaction conditions used for the flow injection system with chemiluminescent detection were transferred to an ion chromatographic configuration for the separation of magnesium from other cations on an exchange column. After a univariate and simplex optimisation of these conditions, the detection limit of magnesium was found to be 0.0411 mol 1-1 which was less than the limits that could be achieved with fluorescent detection, The further development of this reaction to incorporate the displacement of magnesium from Mg-EDTA by other metals that possessed a higher conditional stability constant than magnesium also proved to be problematic with interferences from not only EDTA but from the eluant (lactic acid) from the cation column. Using this system the detection limits of the displacing metals were found to be in the order of 10 mg 1-1 which was substantially less that what was observed when exactly the same configuration was used with fluorescent detection. The final component of the thesis entails the discussion of the background emission that results from the reaction of oxamides/oxalates with hydrogen peroxide. A detailed investigation into the reaction of TCPO and hydrogen peroxide in the presence of various additives, such as imidazole , heavy atoms and triethylamine illustrated the existence of a further intermediate in fee mechanism for this reaction. The species responsible for this emission was attributed to the degradation product 2,4,6-trichlorophenyi of TCPO, which was supported by the non-existent background present with the oxamides that do not contain this degradation product.

α2β1 integrin affects metastatic potential of ovarian carcinoma spheroids by supporting disaggregation and proteolysis

Background: Ovarian cancer is characterized by a wide-spread intra-abdominal metastases which represents a major clinical hurdle in the prognosis and management of the disease. A significant proportion of ovarian cancer cells in peritoneal ascites exist as multicellular aggregates or spheroids. We hypothesize that these cellular aggregates or spheroids are invasive with the capacity to survive and implant on the peritoneal surface. This study was designed to elucidate early inherent mechanism(s) of spheroid survival, growth and disaggregation required for peritoneal metastases.

Methods: In this study, we determined the growth pattern and adhesive capacity of ovarian cancer cell lines (HEY and OVHS1) grown as spheroids, using the well established liquid overlay technique, and compared them to a normal ovarian cell line (IOSE29) and cancer cells grown as a monolayer. The proteolytic capacity of these spheroids was compared with cells grown as a monolayer using a gelatin zymography assay to analyze secreted MMP-2/9 in conditioned serum-free medium. The disaggregation of cancer cell line spheroids was determined on extracellular matrices (ECM) such as laminin (LM), fibronectin (FN) and collagen (CI) and the expression of α2, α3, αv, α6 and β1 interin was determined by flow cytometric analysis. Neutralizing antibodies against α2, β1 subunits and α2β1 integrin was used to inhibit disaggregation as well as activation of MMPs in spheroids.

Results: We demonstrate that ovarian cancer cell lines grown as spheroids can sustain growth for 10 days while the normal ovarian cell line failed to grow beyond 2 days. Compared to cells grown as a monolayer, cancer cells grown as spheroids demonstrated no change in adhesion for up to 4 days, while IOSE29 cells had a 2–4-fold loss of adhesion within 2 days. Cancer cell spheroids disaggregated on extracellular matrices (ECM) and demonstrated enhanced expression of secreted pro-MMP2 as well as activated MMP2/MMP9 with no such activation of MMP's observed in monolayer cells. Flow cytometric analysis demonstrated enhanced expression of α2 and diminution of α6 integrin subunits in spheroids
versus monolayer cells. No change in the expression of α3, αv and β1 subunits was evident. Conversely, except for αv integrin, a 1.5–7.5-fold decrease in α2, α3, α6 and β1 integrin subunit expression was observed in IOSE29 cells within 2 days. Neutralizing antibodies against α2, β1 subunits and α2β1 integrin inhibited disaggregation as well as activation of
MMPs in spheroids.

Conclusion: Our results suggest that enhanced expression of α2β1 integrin may influence spheroid disaggregation and
proteolysis responsible for the peritoneal dissemination of ovarian carcinoma. This may indicate a new therapeutic target
for the suppression of the peritoneal metastasis associated with advanced ovarian carcinomas.