Liquid-phase microextraction for simultaneous chromatographic analysis of three antidepressant drugs in plasma

A method using Liquid Phase Microextraction for simultaneous detection of citalopram (CIT), paroxetine (PAR) and fluoxetine (FLU), using venlafaxine as internal standard, in plasma by high performance liquid chromatography with fluorescence detection was developed. The linearity was evaluated between 5.0 and 500 ng mL-1 (r > 0.99) and the limit of quantification was 2.0, 3.0 and 5.0 ng mL-1 for CIT, PAR and FLU, respectively. Therefore, it can be applied to therapeutic drug monitoring, pharmacokinetics or bioavailability studies and its advantages are that it necessary relatively inexpensive equipment and sample preparation techniques.

A new sensitive method for the quantification of glyoxal and methylglyoxal in snow and ice by stir bar sorptive extraction and liquid desorption-HPLC-ESI-MS

In this study, the development of a new sensitive method for the analysis of alpha-dicarbonyls glyoxal (G) and methylglyoxal (MG) in environmental ice and snow is presented. Stir bar sorptive extraction with in situ derivatization and liquid desorption (SBSE-LD) was used for sample extraction, enrichment, and derivatization. Measurements were carried out using high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). As part of the method development, SBSE-LD parameters such as extraction time, derivatization reagent, desorption time and solvent, and the effect of NaCl addition on the SBSE efficiency as well as measurement parameters of HPLC-ESI-MS/MS were evaluated. Calibration was performed in the range of 1–60 ng/mL using spiked ultrapure water samples, thus incorporating the complete SBSE and derivatization process. 4-Fluorobenzaldehyde was applied as internal standard. Inter-batch precision was <12 % RSD. Recoveries were determined by means of spiked snow samples and were 78.9 ± 5.6 % for G and 82.7 ± 7.5 % for MG, respectively. Instrumental detection limits of 0.242 and 0.213 ng/mL for G and MG were achieved using the multiple reaction monitoring mode. Relative detection limits referred to a sample volume of 15 mL were 0.016 ng/mL for G and 0.014 ng/mL for MG. The optimized method was applied for the analysis of snow samples from Mount Hohenpeissenberg (close to the Meteorological Observatory Hohenpeissenberg, Germany) and samples from an ice core from Upper Grenzgletscher (Monte Rosa massif, Switzerland). Resulting concentrations were 0.085–16.3 ng/mL for G and 0.126–3.6 ng/mL for MG. Concentrations of G and MG in snow were 1–2 orders of magnitude higher than in ice core samples. The described method represents a simple, green, and sensitive analytical approach to measure G and MG in aqueous environmental samples.

Simultaneous liquid chromatographic determination of doxorubicin and its major metabolite doxorubicinol in parrot plasma

A new microscale method is reported for the determination of doxorubicin and its active metabolite, doxorubicinol, in parrot plasma. Sample workup involved acetonitrile protein precipitation, ethyl acetate extraction, followed by back extraction into HCl. Separations were achieved on a phenyl-hexyl column at 30 degrees C using acetonitrile (17%, v/v) in 0.01 M orthophosphoric acid (83%, v/v) delivered via a linear flow program. Fluorometric detection wavelengths were 235 nm (excitation) and 550 nm (emission). Calibration plots were linear (1 2 > 0.999), and recoveries were 71-87% from 20 to 400 ng/mL. Assay imprecision was

Polymer fractionation by continuous chromatography

A review is given of general chromatographic theory, the factors affecting the performance of chromatographi c columns, and aspects of scale-up of the chromatographic process. The theory of gel permeation chromatography (g. p. c.) is received, and the results of an experimental study to optimize the performance of an analytical g.p.c. system are reported. The design and construction of a novel sequential continuous chromatographic refining unit (SCCR3), for continuous liquid-liquid chromatography applications, is described. Counter-current operation is simulated by sequencing a system of inlet and outlet port functions around a connected series of fixed, 5.1 cm internal diameter x 70 cm long, glass columns. The number of columns may be varied, and, during this research, a series of either twenty or ten columns was used. Operation of the unit for continuous fractionation of a dextran polymer (M. W. - 30,000) by g.p.c. is reported using 200-400 µm diameter porous silica beads (Spherosil XOB07S) as packing, and distilled water for the mobile phase. The effects of feed concentration, feed flow rate, and mobile and stationary phase flow rates have been investigated, by means of both product, and on-column, concentrations and molecular weight distributions. The ability to operate the unit successfully at on-column concentrations as high as 20% w/v dextran has been demonstrated, and removal of both high and low molecular weight ends of a polymer feed distribution, to produce products meeting commercial specifications, has been achieved. Equivalent throughputs have been as high as 2.8 tonnes per annum for ten columns, based on continuous operation for 8000 hours per annum. A concentration dependence of the equilibrium distribution coefficient, KD observed during continuous fractionation studies, is related to evidence in the literature and experimental results obtained on a small-scale batch column. Theoretical treatments of the counter-current chromatographic process are outlined, and a preliminary computer simulation of the SCCR3 unit t is presented.

Lipid-Based Liquid Crystals As Carriers for Antimicrobial Peptides: Phase Behavior and Antimicrobial Effect

International audience

Origin discrimination and quality evaluation of Gastrodiae rhizoma (Orchidaceae) by high-performance liquid chromatographic fingerprint

Purpose: To develop a high-performance liquid chromatography (HPLC) fingerprint method for the quality control and origin discrimination of Gastrodiae rhizoma . Methods: Twelve batches of G. rhizoma collected from Sichuan, Guizhou and Shanxi provinces in china were used to establish the fingerprint. The chromatographic peak (gastrodin) was taken as the reference peak, and all sample separation was performed on a Agilent C18 (250 mm×4.6 mmx5 μm) column with a column temperature of 25 °C. The mobile phase was acetonitrile/0.8 % phosphate water solution (in a gradient elution mode) and the flow rate of 1 mL/min. The detection wavelength was 270 nm. The method was validated as per the guidelines of Chinese Pharmacopoeia. Results: The chromatograms of the samples showed 11 common peaks, of which no. 4 was identified as that of Gastrodin. Data for the samples were analyzed statistically using similarity analysis and hierarchical cluster analysis (HCA). The similarity index between reference chromatogram and samples’ chromatograms were all > 0.80. The similarity index of G. rhizoma from Guizhou, Shanxi and Sichuan is evident as follows: 0.854 - 0.885, 0.915 - 0.930 and 0.820 - 0.848, respectively. The samples could be divided into three clusters at a rescaled distance of 7.5: S1 - S4 as cluster 1; S5 - S8 cluster 2, and others grouped into cluster 3. Conclusion: The findings indicate that HPLC fingerprinting technology is appropriate for quality control and origin discrimination of G. rhizoma.

Membrane associated transporter protein gene (SLC45A2) and the genetic basis of normal human pigmentation variation

This work is concerned with the genetic basis of normal human pigmentation variation. Specifically, the role of polymorphisms within the solute carrier family 45 member 2 (SLC45A2 or membrane associated transporter protein; MATP) gene were investigated with respect to variation in hair, skin and eye colour ― both between and within populations. SLC45A2 is an important regulator of melanin production and mutations in the gene underly the most recently identified form of oculocutaneous albinism. There is evidence to suggest that non-synonymous polymorphisms in SLC45A2 are associated with normal pigmentation variation between populations. Therefore, the underlying hypothesis of this thesis is that polymorphisms in SLC45A2 will alter the function or regulation of the protein, thereby altering the important role it plays in melanogenesis and providing a mechanism for normal pigmentation variation. In order to investigate the role that SLC45A2 polymorphisms play in human pigmentation variation, a DNA database was established which collected pigmentation phenotypic information and blood samples of more than 700 individuals. This database was used as the foundation for two association studies outlined in this thesis, the first of which involved genotyping two previously-described non-synonymous polymorphisms, p.Glu272Lys and p.Phe374Leu, in four different population groups. For both polymorphisms, allele frequencies were significantly different between population groups and the 272Lys and 374Leu alleles were strongly associated with black hair, brown eyes and olive skin colour in Caucasians. This was the first report to show that SLC45A2 polymorphisms were associated with normal human intra-population pigmentation variation. The second association study involved genotyping several SLC45A2 promoter polymorphisms to determine if they also played a role in pigmentation variation. Firstly, the transcription start site (TSS), and hence putative proximal promoter region, was identified using 5' RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE). Two alternate TSSs were identified and the putative promoter region was screened for novel polymorphisms using denaturing high performance liquid chromatography (dHPLC). A novel duplication (c.–1176_–1174dupAAT) was identified along with other previously described single nucleotide polymorphisms (c.–1721C>G and c.–1169G>A). Strong linkage disequilibrium ensured that all three polymorphisms were associated with skin colour such that the –1721G, +dup and –1169A alleles were associated with olive skin in Caucasians. No linkage disequilibrium was observed between the promoter and coding region polymorphisms, suggesting independent effects. The association analyses were complemented with functional data, showing that the –1721G, +dup and –1169A alleles significantly decreased SLC45A2 transcriptional activity. Based on in silico bioinformatic analysis that showed these alleles remove a microphthalmia-associated transcription factor (MITF) binding site, and that MITF is a known regulator of SLC45A2 (Baxter and Pavan, 2002; Du and Fisher, 2002), it was postulated that SLC45A2 promoter polymorphisms could contribute to the regulation of pigmentation by altering MITF binding affinity. Further characterisation of the SLC45A2 promoter was carried out using luciferase reporter assays to determine the transcriptional activity of different regions of the promoter. Five constructs were designed of increasing length and their promoter activity evaluated. Constitutive promoter activity was observed within the first ~200 bp and promoter activity increased as the construct size increased. The functional impact of the –1721G, +dup and –1169A alleles, which removed a MITF consensus binding site, were assessed using electrophoretic mobility shift assays (EMSA) and expression analysis of genotyped melanoblast and melanocyte cell lines. EMSA results confirmed that the promoter polymorphisms affected DNA-protein binding. Interestingly, however, the protein/s involved were not MITF, or at least MITF was not the protein directly binding to the DNA. In an effort to more thoroughly characterise the functional consequences of SLC45A2 promoter polymorphisms, the mRNA expression levels of SLC45A2 and MITF were determined in melanocyte/melanoblast cell lines. Based on SLC45A2’s role in processing and trafficking TYRP1 from the trans-Golgi network to stage 2 melanosmes, the mRNA expression of TYRP1 was also investigated. Expression results suggested a coordinated expression of pigmentation genes. This thesis has substantially contributed to the field of pigmentation by showing that SLC45A2 polymorphisms not only show allele frequency differences between population groups, but also contribute to normal pigmentation variation within a Caucasian population. In addition, promoter polymorphisms have been shown to have functional consequences for SLC45A2 transcription and the expression of other pigmentation genes. Combined, the data presented in this work supports the notion that SLC45A2 is an important contributor to normal pigmentation variation and should be the target of further research to elucidate its role in determining pigmentation phenotypes. Understanding SLC45A2’s function may lead to the development of therapeutic interventions for oculocutaneous albinism and other disorders of pigmentation. It may also help in our understanding of skin cancer susceptibility and evolutionary adaptation to different UV environments, and contribute to the forensic application of pigmentation phenotype prediction.

Dopamine and visually regulated eye growth in chick

Retinal image properties such as contrast and spatial frequency play important roles in the development of normal vision. For example, visual environments comprised solely of low contrast and/or low spatial frequencies induce myopia. The visual image is processed by the retina and it then locally controls eye growth. In terms of the retinal neurotransmitters that link visual stimuli to eye growth, there is strong evidence to suggest involvement of the retinal dopamine (DA) system. For example, effectively increasing retinal DA levels by using DA agonists can suppress the development of form-deprivation myopia (FDM). However, whether visual feedback controls eye growth by modulating retinal DA release, and/or some other factors, is still being elucidated. This thesis is chiefly concerned with the relationship between the dopaminergic system and retinal image properties in eye growth control. More specifically, whether the amount of retinal DA release reduces as the complexity of the image degrades was determined. For example, we investigated whether the level of retinal DA release decreased as image contrast decreased. In addition, the effects of spatial frequency, spatial energy distribution slope, and spatial phase on retinal DA release and eye growth were examined. When chicks were 8-days-old, a cone-lens imaging system was applied monocularly (+30 D, 3.3 cm cone). A short-term treatment period (6 hr) and a longer-term treatment period (4.5 days) were used. The short-term treatment tests for the acute reduction in DA release by the visual stimulus, as is seen with diffusers and lenses, whereas the 4.5 day point tests for reduction in DA release after more prolonged exposure to the visual stimulus. In the contrast study, 1.35 cyc/deg square wave grating targets of 95%, 67%, 45%, 12% or 4.2% contrast were used. Blank (0% contrast) targets were included for comparison. In the spatial frequency study, both sine and square wave grating targets with either 0.017 cyc/deg and 0.13 cyc/deg fundamental spatial frequencies and 95% contrast were used. In the spectral slope study, 30% root-mean-squared (RMS) contrast fractal noise targets with spectral fall-off of 1/f0.5, 1/f and 1/f2 were used. In the spatial alignment study, a structured Maltese cross (MX) target, a structured circular patterned (C) target and the scrambled versions of these two targets (SMX and SC) were used. Each treatment group comprised 6 chicks for ocular biometry (refraction and ocular dimension measurement) and 4 for analysis of retinal DA release. Vitreal dihydroxyphenylacetic acid (DOPAC) was analysed through ion-paired reversed phase high performance liquid chromatography with electrochemical detection (HPLC-ED), as a measure of retinal DA release. For the comparison between retinal DA release and eye growth, large reductions in retinal DA release possibly due to the decreased light level inside the cone-lens imaging system were observed across all treated eyes while only those exposed to low contrast, low spatial frequency sine wave grating, 1/f2, C and SC targets had myopic shifts in refraction. Amongst these treatment groups, no acute effect was observed and longer-term effects were only found in the low contrast and 1/f2 groups. These findings suggest that retinal DA release does not causally link visual stimuli properties to eye growth, and these target induced changes in refractive development are not dependent on the level of retinal DA release. Retinal dopaminergic cells might be affected indirectly via other retinal cells that immediately respond to changes in the image contrast of the retinal image.

Fingerprint analysis of eucommia bark by LC-DAD and LC-MS with the aid of chemometrics

Chromatographic fingerprints of 46 Eucommia Bark samples were obtained by liquid chromatography-diode array detector (LC-DAD). These samples were collected from eight provinces in China, with different geographical locations, and climates. Seven common LC peaks that could be used for fingerprinting this common popular traditional Chinese medicine were found, and six were identified as substituted resinols (4 compounds), geniposidic acid and chlorogenic acid by LC-MS. Principal components analysis (PCA) indicated that samples from the Sichuan, Hubei, Shanxi and Anhui—the SHSA provinces, clustered together. The other objects from the four provinces, Guizhou, Jiangxi, Gansu and Henan, were discriminated and widely scattered on the biplot in four province clusters. The SHSA provinces are geographically close together while the others are spread out. Thus, such results suggested that the composition of the Eucommia Bark samples was dependent on their geographic location and environment. In general, the basis for discrimination on the PCA biplot from the original 46 objects× 7 variables data matrix was the same as that for the SHSA subset (36 × 7 matrix). The seven marker compound loading vectors grouped into three sets: (1) three closely correlating substituted resinol compounds and chlorogenic acid; (2) the fourth resinol compound identified by the OCH3 substituent in the R4 position, and an unknown compound; and (3) the geniposidic acid, which was independent of the set 1 variables, and which negatively correlated with the set 2 ones above. These observations from the PCA biplot were supported by hierarchical cluster analysis, and indicated that Eucommia Bark preparations may be successfully compared with the use of the HPLC responses from the seven marker compounds and chemometric methods such as PCA and the complementary hierarchical cluster analysis (HCA).

Simultaneous kinetic-spectrophotometric determination of maltol and ethyl maltol in food samples by using chemometrics

A fast and accurate procedure has been researched and developed for the simultaneous determination of maltol and ethyl maltol, based on their reaction with iron(III) in the presence of o-phenanthroline in sulfuric acid medium. This reaction was the basis for an indirect kinetic spectrophotometric method, which followed the development of the pink ferroin product (λmax = 524 nm). The kinetic data were collected in the 370–900 nm range over 0–30 s. The optimized method indicates that individual analytes followed Beer’s law in the concentration range of 4.0–76.0 mg L−1 for both maltol and ethyl maltol. The LOD values of 1.6 mg L−1 for maltol and 1.4 mg L−1 for ethyl maltol agree well with those obtained by the alternative high performance liquid chromatography with ultraviolet detection (HPLC-UV). Three chemometrics methods, principal component regression (PCR), partial least squares (PLS) and principal component analysis–radial basis function–artificial neural networks (PC–RBF–ANN), were used to resolve the measured data with small kinetic differences between the two analytes as reflected by the development of the pink ferroin product. All three performed satisfactorily in the case of the synthetic verification samples, and in their application for the prediction of the analytes in several food products. The figures of merit for the analytes based on the multivariate models agreed well with those from the alternative HPLC-UV method involving the same samples.

Simultaneous kinetic spectrophotometric determination of norfloxacin and rifampicin in pharmaceutical formulation and human urine samples by use of chemometrics approaches

A kinetic spectrophotometric method with aid of chemometrics is proposed for the simultaneous determination of norfloxacin and rifampicin in mixtures. The proposed method was applied for the simultaneous determination of these two compounds in pharmaceutical formulation and human urine samples, and the results obtained are similar to those obtained by high performance liquid chromatography.

Biotribological assessment for artificial synovial joints : the role of boundary lubrication

Biotribology, the study of lubrication, wear and friction within the body, has become a topic of high importance in recent times as we continue to encounter debilitating diseases and trauma that destroy function of the joints. A highly successful surgical procedure to replace the joint with an artificial equivalent alleviates dysfunction and pain. However, the wear of the bearing surfaces in prosthetic joints is a significant clinical problem and more patients are surviving longer than the life expectancy of the joint replacement. Revision surgery is associated with increased morbidity and mortality and has a far less successful outcome than primary joint replacement. As such, it is essential to ensure that everything possible is done to limit the rate of revision surgery. Past experience indicates that the survival rate of the implant will be influenced by many parameters, of primary importance, the material properties of the implant, the composition of the synovial fluid and the method of lubrication. In prosthetic joints, effective boundary lubrication is known to take place. The interaction of the boundary lubricant and the bearing material is of utmost importance. The identity of the vital active ingredient within synovial fluid (SF) to which we owe the near frictionless performance of our articulating joints has been the quest of researchers for many years. Once identified, tribo tests can determine what materials and more importantly what surfaces this fraction of SF can function most optimally with. Surface-Active Phospholipids (SAPL) have been implicated as the body’s natural load bearing lubricant. Studies in this thesis are the first to fully characterise the adsorbed SAPL detected on the surface of retrieved prostheses and the first to verify the presence of SAPL on knee prostheses. Rinsings from the bearing surfaces of both hip and knee prostheses removed from revision operations were analysed using High Performance Liquid Chromatography (HPLC) to determine the presence and profile of SAPL. Several common prosthetic materials along with a novel biomaterial were investigated to determine their tribological interaction with various SAPLs. A pin-on-flat tribometer was used to make comparative friction measurements between the various tribo-pairs. A novel material, Pyrolytic Carbon (PyC) was screened as a potential candidate as a load bearing prosthetic material. Friction measurements were also performed on explanted prostheses. SAPL was detected on all retrieved implant bearing surfaces. As a result of the study eight different species of phosphatidylcholines were identified. The relative concentrations of each species were also determined indicating that the unsaturated species are dominant. Initial tribo tests employed a saturated phosphatidylcholine (SPC) and the subsequent tests adopted the addition of the newly identified major constituents of SAPL, unsaturated phosphatidylcholine (USPC), as the test lubricant. All tribo tests showed a dramatic reduction in friction when synthetic SAPL was used as the lubricant under boundary lubrication conditions. Some tribopairs showed more of an affinity to SAPL than others. PyC performed superior to the other prosthetic materials. Friction measurements with explanted prostheses verified the presence and performance of SAPL. SAPL, in particular phosphatidylcholine, plays an essential role in the lubrication of prosthetic joints. Of particular interest was the ability of SAPLs to reduce friction and ultimately wear of the bearing materials. The identification and knowledge of the lubricating constituents of SF is invaluable for not only the future development of artificial joints but also in developing effective cures for several disease processes where lubrication may play a role. The tribological interaction of the various tribo-pairs and SAPL is extremely favourable in the context of reducing friction at the bearing interface. PyC is highly recommended as a future candidate material for use in load bearing prosthetic joints considering its impressive tribological performance.

Androgen levels increase by intratumoral de novo steroidogenesis during progression of castration-resistant prostate cancer

Although systemic androgen deprivation prolongs life in advanced prostate cancer, remissions are temporary because patients almost uniformly progress to a state of a castration-resistant prostate cancer (CRPC) as indicated by recurring PSA. This complex process of progression does not seem to be stochastic as the timing and phenotype are highly predictable, including the observation that most androgen-regulated genes are reactivated despite castrate levels of serum androgens. Recent evidence indicates that intraprostatic levels of androgens remain moderately high following systemic androgen deprivation therapy, whereas the androgen receptor (AR) remains functional, and silencing the AR expression following castration suppresses tumor growth and blocks the expression of genes known to be regulated by androgens. From these observations, we hypothesized that CRPC progression is not independent of androgen-driven activity and that androgens may be synthesized de novo in CRPC tumors leading to AR activation. Using the LNCaP xenograft model, we showed that tumor androgens increase during CRPC progression in correlation to PSA up-regulation. We show here that all enzymes necessary for androgen synthesis are expressed in prostate cancer tumors and some seem to be up-regulated during CRPC progression. Using an ex vivo radiotracing assays coupled to high-performance liquid chromatography-radiometric/mass spectrometry detection, we show that tumor explants isolated from CRPC progression are capable of de novo conversion of [(14)C]acetic acid to dihydrotestosterone and uptake of [(3)H]progesterone allows detection of the production of six other steroids upstream of dihydrotestosterone. This evidence suggests that de novo androgen synthesis may be a driving mechanism leading to CRPC progression following castration.

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.