Raymond G. Reid

Capillary electrochromatography of steroids: Increased sensitivity by on-line concentration and comparison with high-performance liquid chromatography

A reversed-phase HPLC method previously developed for the analysis of progesterone and its major metabolites has been transferred successfully to a capillary electrochromatography (CEC) system. Procedures for fabricating packed capillaries and the modifications made to the capillary electropherograph which allow operation in the CEC mode without pressurisation are described. The dependence of electroosmotic flow on electric field strength, pH and organic modifier content is discussed. Direct comparison with HPLC shows that CEC provides useful gains in efficiency and speed of analysis and requires vastly reduced amounts of both chromatographic phases and material for analysis. On-line concentration is described which allows the lower sensitivity of CEC to be offset by injecting analytes from a non-eluting solution. Examination of steroids in plasma demonstrates that the superior separation by CEC is maintained in a complex biological matrix.

Gc-ms Analysis Of The Essential Oil From The Oleoresin Of pistacia atlantica Var. mutica

The oleoresin of Pistacia atlantica var. mutica, growing in different regions of Iran, is a popular naturally occurring chewing gum and has been used traditionally in the treatment of peptic ulcer. The GC-MS analysis of the essential oil, obtained from steam distillation of the oleoresin of P. Atlantica var. mutica, has led to the identification and quantification of eleven terpenoids, α-pinene (70%), β-pinene (1.94%), 3-carene (0.2%), carveol (2.18%), epoxypinene (2.15%), limonene oxide (9%), myrtenol (5.31%), limonene (0.62%), citral (5.72%), α-phellandrene (0.2%), and β-myrcene (0.3%). The total amount of essential oil obtained was 22% v/w which is higher than any other species of the genus Pestacia.

Determination of opiates in urine by capillary electrophoresis.

A method for the separation of a mixture of opiates comprising pholcodine, 6-monoacetylmorphine, morphine, heroin, codeine and dihydrocodeine by capillary electrophoresis using a running buffer of 100 mM disodium hydrogenphosphate at pH 6 is described. The characteristics of an analytical method based on this separation for the determination of these drugs following extraction from urine and using levallorphan as internal standard are reported. Detection limits in the region of 10 ng cm-3 are achieved when using electrokinetic injection. A comparison is made of the sensitivity and reproducibility of electrokinetic and hydrodynamic injection for these drugs. Data are presented to show the results obtained when the proposed method is applied to urine spiked with all the above opiates and also to urine from a subject following consumption of dihydrocodeine and pholcodine. The concentrations found are compared with those obtained by LC.

A specific, accurate, and sensitive measure of total plasma malondialdehyde by HPLC

Malondialdehyde (MDA) is one of the most commonly reported biomarkers of lipid peroxidation in clinical studies. The reaction of thiobarbituric acid (TBA) with MDA to yield a pink chromogen attributable to an MDA-TBA2 adduct is a common assay approach with products being quantified by ultraviolet-Vis assay as nonspecific TBA-reactive substances (TBARS) or chromatographically as MDA. The specificity of the TBARS assay was compared with both chromatographic assays for total plasma MDA. The levels of total plasma MDA were significantly lower than the plasma TBARS in each of the samples examined, and interestingly, the interindividual variation apparent in the level of plasma MDA was not evident in the plasma TBARS assay. Each of the four online chromatographic detectors yielded a precise, sensitive, and accurate determination of total plasma MDA, and selected-ion monitoring was the most-accurate assay (101.3%, n = 4). The online diode array detectors provided good assay specificity (peak purity index of 999), sensitivity, precision, and accuracy. This research demonstrates the inaccuracy that is inherent in plasma TBARS assays, which claim to quantify MDA, and it is proposed that the TBARS approach may limit the likelihood of detecting true differences in the level of lipid peroxidation in clinical studies.

Biological activity of serotonin conjugates from the seeds of Centaurea nigra

Two serotonin conjugates, N-(trans-p-coumaroyl)-serotonin (1) and N-(trans-feruloyl)-serotonin (maoschamine, 2), isolated from the methanol extract of the seeds of Centaurea nigra, have been assessed for antibacterial and free radical scavenging activities. The general toxicity of 1 and 2 has also been determined by the brine shrimp lethality bioassay.

Eremostachiin: a new furanolabdane diterpene glycoside from Eremostachys glabra

The reversed-phase preparative HPLC analysis of the methanol extract of the rhizomes of Eremostachys glabra (Lamiaceae) led to the isolation of two furanolabdane diterpene glycosides, phlomisoside II, and β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl ester of phlomisoic acid (named eremostachiin), the latter being a novel natural product. The structure of the new compound has been elucidated unambiguously by HRMS and a series of 1D- and 2D-NMR spectroscopic techniques. The free radical scavenging activity of these compounds was assessed using the DPPH assay.

Determination of antibacterial quaternary ammonium compounds in lozenges by capillary electrophoresis

A method for the specific determination of three quaternary ammonium compounds, benzalkonium chloride, cetylpyridinium chloride and dequalinium chloride, used as antibacterial agents in candy-based lozenges, is described based on capillary zone electrophoresis. It is shown that, following optimisation of buffer composition with respect to organic modifier concentration. pH and buffer concentration together with the inclusion of sodium dodecylsulphate as an ion-pairing agent in the case of dequalinium chloride, these analytes migrate in less than 5 min. The resultant electrophoretic peaks are sharp and readily quantified. The individual alkyl components of benzalkonium chloride can be resolved as can related impurities in dequalinium chloride lozenges. The quantitative characteristics of the assay method, based on peak areas normalised with respect to migration times, are reported and the method is compared with a previously published method based on liquid chromatography.

Evaluation of quality control strategies in Scutellaria herbal medicines

The statutory regulation of herbal medicines is under review within the United Kingdom (UK) and by 2011 all herbal medicines will require either a Product Licence or a Traditional Herbal Registration. The species Scutellaria baicalensis has been shown to possess anti-inflammatory, anti-viral and anti-tumor properties and is one of the most widely used Chinese herbal extracts in Eastern and Western medicines. The bioactivity of this herbal medicine is due to the radical scavenging activities of the flavone components of which there are more than 60. This research has characterised 5 key flavones in 18 extracts of Scutellaria using a combination of HPLC with DAD and MS detection. Employing an internal standard approach, the validated HPLC method afforded good sensitivity and excellent assay precision. Assays for the ferric reducing antioxidant power (FRAP) and total phenol determinations enabled determination of the antioxidant coefficient (PAC) of each Scutellaria extract. The potential usefulness of employing multivariate statistical analysis using a combination of the key parameters collected namely, FRAP activity, total phenol content, levels of 5 flavone biomarkers and the PAC as a means of quality evaluation of the Scutellaria herbal extracts was investigated. The PAC value was predicted by soft independent modelling of class analogy (SIMCA) as being the most discriminatory parameter and applying this ranking the herbal extracts were grouped into 3 clusters. The second most influential parameter in determining the clustering of the samples was the level of baicalin in each extract. It is proposed that the PAC value alone or in combination with a chromatographic fingerprint of key biomarkers [e.g. baicalin or (baicalin+baicalein)] may be useful indicators to adopt for the quality control of S. baicalensis.

Analysis of proguanil and its metabolites by application of the sweeping technique in micellar electrokinetic chromatography

The method of applying large sample volumes in micellar electrokinetic chromatography termed sweeping is applied to determine the conservative limits of detection of some basic drugs in plasma and urine. The biguanides proguanil, 4-chlorophenylbiguanide and cycloguanil are used as models of basic drugs and the limits of detection obtained compared with those previously reported for capillary zone electrophoresis using field-amplified sample injection (FASI) and also by LC using off-line preconcentration. It is found that the sweeping method can be applied to extracts of such biological matrices. The limits of detection obtained by sweeping are improved over FASI for plasma but not for urine and the limits of detection are higher than those reported for LC, for these compounds.

Analysis of basic antimalarial drugs by CZE; Part 2. Validation and application to bioanalysis

This report describes some of the quantitative aspects of the CZE separation of proguanil, chloroquine and their respective metabolites, the separations of which, by CE and MEKC, were reported in Part 1. Results obtained on the precision of migration time and peak areas using the alternative injection methods of vacuum and electrokinetic are described and discussed. The increase in concentration sensitivity using electrokinetic injection with an organic injection solvent reported in Part 1 is confirmed and the resultant limits of detection in urine reported. An assay method for these compounds in urine is described which incorporates a pretreatment stage of solid phase extraction and the main analytical parameters used in the validation of such an assay are reported. The limitation of the sample pretreatment used when applied to matrices of plasma and saliva are reported and discussed in the context of the electrokinetic injection method used.