Determination of diclofenac from paediatric urine samples by stir bar sorptive extraction (SBSE)-HPLC-UV technique

A novel stir bar sorptive extraction (SBSE) method coupled with high performance liquid chromatography (HPLC) and UV detection for the extraction of diclofenac (DIC) from paediatric urine samples has been developed and validated. Selectivity and sensitivity being the prime objectives of the bioanalytical method for clinical samples, an optimised SBSE protocol was developed that selectively extracted DIC from various concurrently administered drugs. The validated assay was found to be linear (r=0.9999) over a concentration range of 100-2000 ng mL(-1). SBSE showed consistent recoveries (similar to 70%) of DIC across the validated linearity range. Overall, the method exhibited excellent accuracy and precision across all QC concentrations, tested over three days. Calculated LOD and LOQ were found to be 12.03 ng mL(-1) and 36.37 ng mL(-1), respectively, however, for the experimental purposes, 100 ngmL(-1) was considered as the validated LOQ(accuracy and precision at this LQC was

Depletion of four nitrofuran antibiotics and their tissue-bound metabolites in porcine tissues and determination using LC-MS/MS and HPLC-UV

Depletion of the nitrofuran antibiotics furazolidone, furaltadone, nitrofurantoin and nitrofurazone and their tissue-bound metabolites AOZ, AMOZ, AHD and SEM from pig muscle, liver and kidney tissues is described. Groups of pigs were given feed medicated with one of the nitrofuran drugs at a therapeutic concentration (400 mg kg(-1)) for ten days. Animals were slaughtered at intervals and tissue samples collected for analysis for six weeks following withdrawal of medicated feed. These samples were analysed both for parent nitrofurans (using LC-MS/MS and HPLC-UV), and for tissue-bound metabolites (using LC-MS/MS). The parent drugs were detectable only sporadically and only in pigs subjected to no withdrawal period whatsoever. This confirms the instability of the four major nitrofuran antibiotics in edible tissues. In contrast, the metabolites accumulated to high concentrations in tissues (ppm levels) and had depletion half lives of between 5.5 and 15.5 days. The metabolites of all four drugs were still readily detectable in tissues six weeks after cessation of treatment. This emphasizes the benefits of monitoring for the stable metabolites of the nitrofurans.

Investigation into mercury bound to biothiols:structural identification using ESI-ion-trap MS and introduction of a method for their HPLC separation with simultaneous detection by ICP-MS and ESI-MS

Mercury in plants or animal tissue is supposed to occur in the form of complexes formed with biologically relevant thiols (biothiols), rather than as free cation. We describe a technique for the separation and molecular identification of mercury and methylmercury complexes derived from their reactions with cysteine (Cys) and glutathione (GS): Hg(Cys)(2), Hg(GS)(2), MeHgCys, MeHgGS. Complexes were characterised by electrospray mass spectrometry (MS) equipped with an ion trap and the fragmentation pattern of MeHgCys was explained by using MP2 and B3LYP calculations, showing the importance of mercury-amine interactions in the gas phase. Chromatographic baseline separation was performed within 10 min with formic acid as the mobile phase on a reversed-phase column. Detection was done by online simultaneous coupling of ES-MS and inductively coupled plasma MS. When the mercury complexes were spiked in real samples (plant extracts), no perturbation of the separation and detection conditions was observed, suggesting that this method is capable of detecting mercury biothiol complexes in plants.

Human aflatoxin albumin adducts quantitatively compared by ELISA, HPLC with fluorescence detection, and HPLC with isotope dilution mass spectrometry

Essential to the conduct of epidemiologic studies examining aflatoxin exposure and the risk of heptocellular carcinoma, impaired growth, and acute toxicity has been the development of quantitative biomarkers of exposure to aflatoxins, particularly aflatoxin B-1. In this study, identical serum sample sets were analyzed for aflatoxin-albumin adducts by ELISA, high-performance liquid chromatography (HPLC) with fluorescence detection (HPLC-f), and HPLC with isotope dilution mass spectrometry (IDMS). The human samples analyzed were from an acute aflatoxicosis outbreak in Kenya in 2004 (n = 102) and the measured values ranged from 0.018 to 67.0, nondetectable to 13.6, and 0.002 to 17.7 ng/mg albumin for the respective methods. The Deming regression slopes for the HPLC-f and ELISA concentrations as a function of the IDMS concentrations were 0.71 (r(2) = 0.95) and 3.3 (r(2) = 0.96), respectively. When the samples were classified as cases or controls, based on clinical diagnosis, all methods were predictive of outcome (P < 0.01). Further, to evaluate assay precision, duplicate samples were prepared at three levels by dilution of an exposed human sample and were analyzed on three separate days. Excluding one assay value by ELISA and one assay by HPLC-f, the overall relative SD were 8.7%, 10.5%, and 9.4% for IDMS, HPLC-f, and ELISA, respectively. IDMS was the most sensitive technique and HPLC-f was the least sensitive method. Overall, this study shows an excellent correlation between three independent methodologies conducted in different laboratories and supports the validation of these technologies for assessment of human exposure to this environmental toxin and carcinogen.

A rapid HPLC protocol for detection of polyols and trehalose

A procedure was developed to extract polyols and trehalose (protectants against stress) from fungal conidia. Conidia were sonicated (120 s) and immersed in a boiling water bath (5.5 min) to optimize extraction of polyols and trehalose, respectively. A rapid method was developed to separate and detect low-molecular-weight polyols and trehalose using high-performance liquid chromatography (HPLC). An ion exchange column designed for standard carbohydrate analysis was used in preference to one designed for sugar alcohol separation. This resulted in rapid elution (less than 5 min), without sacrificing peak resolution. The use of a pulsed electrochemical detector (gold electrode) resulted in limits of reliable quantification as low as 1.6 μg ml-1 for polyols and 2.8 μg ml-1 for trehalose. This is very sensitive and rapid method by which these protectants can be analysed. It avoids polyol derivatization that characterizes analysis by gas chromatography and the long run times (up to 45 min) that typify HPLC analysis using sugar alcohol columns.

Arsenic-glutathione complexes - Their stability in solution and during separation by different HPLC modes

Complexes of arsenic compounds and glutathione are believed to play an essential part in the metabolism and transport of inorganic arsenic and its methylated species. Up to now, the evidence of their presence is mostly indirect. We studied the stability and Chromatographic behaviour of glutathione complexes with trivalent arsenic: i.e. As^III(GS)₃, MA ^III(GS)₂ and DMA^III(GS) under different conditions. Standard ion chromatography using PRP X-100 and carbonate or formic acid buffer disintegrated the complexes, while all three complexes are stable and separable by reversed phase chromatography (0.1% formic acid/acetonitrile gradient). As^III(GS)₃ and MA^III(GS)₂ were more stable than DMA^III(GS), which even under optimal conditions tended to degrade on the column at 25 °C. Chromatography at 6 °C can retain the integrity of the samples. These results shed more light on the interpretation of a vast number of previously published arsenic speciation studies, which have used Chromatographic separation techniques with the assumption that the integrity of the arsenic species is guaranteed. © The Royal Society of Chemistry 2004.

Otimização e validação de um método de cromatografia líquida de alta resolução (HPLC) para a determinação do edulcorante ciclamato: ocorrência em adoçantes de mesa

Trabalho Final de Mestrado para obtenção do grau de Mestre em Engenharia Química e Biológica

An HPLC method for simultaneous determination of residual benomyl and MBC on apple foliage without cleanup

A simple High Performance Liquid Chromatograph (HPLC) method has been developed to identify benamyl (methyl 1- (butylcarbamoyl)-2-benzimidazole carbamate) and MBC (methyl 2-benzimidazole carbamat~ residues on apple leaves without cleanup. Sample leaves are freeze dried in a Mason jar and residues are then extracted by tumbling them in chloroform containing 5,000 microgram per milliliter of n-propyl isocyanate (PIC) at 10 C. To the extract, n-butyl isocyanate (BIC) was added at 5,000 microgram per milliliter and 20 microliter of this mixture injected onto the HPLC system. Separation is accomplished by the use of a Brownlee LiChrosorb silica gel column with a guard column and' operated with a mixed mobile phase consisting of chloroform and hexane (4:1) saturated with water. MBC, a degradation product of benomyl is identified if present as methyl l-(npropyl carbamoyl)-2-benzimidazole carbamate (MBC-n-PIC). Both benomyl and MBC-n-PIC can be detected with aKUltraviolet (UV) detector (280nm) at a concentration as low as 0.2 microgram per milliliter in apple leaves. The fate of benomyl on apple foliage after spray application of benomyl (Ben late 50 per cent wettable powder) was investigated by the method thus described. Benomyl quickly dissipated during the first 3-7 days, but the dissipatio'n sltowed down thereafter. In contrast, the concentration of MBC in leaves gradually increased after repeated applications of Benlate.

Effects of sample solvent composition and injection volume on chromatographic peak profiles of methyl w- benzimidazolecarbamate and 3-butyl1-2,4-dioxo[1,2-a]-s- triazinobenzimidazole in RP-HPLC

The effects of sample solvent composition and the injection volume, on the chromatographic peak profiles of two carbamate derivatives, methyl 2-benzimidazolecarbamate (MBC) and 3-butyl-2,4-dioxo[1,2-a]-s-triazinobenzimidazole (STB), were studied using reverse phase high performance liquid chromatograph. The study examined the effects of acetonitrile percentage in the sample solvent from 5 to 50%, effects of methanol percentage from 5 to 50%, effects of pH increase from 4.42 to 9.10, and effect of increasing buffer concentration from ° to 0.12M. The effects were studied at constant and increasing injection mass and at four injection volumes of 10, 50, 100 and 200 uL. The study demonstrated that the amount and the type of the organic solvents, the pH, and the buffer strength of the sample solution can have a pronounced effect on the peak heights, peak widths, and retention times of compounds analysed. MBC, which is capable of intramolecular hydrogen bonding and has no tendency to ionize, showed a predictable increase .in band broadening and a decrease in retention times at higher eluting strengths of the sample solvent. STB, which has a tendency to ionize or to strongly interact with the sample solvent, was influenced in various ways by the changes in ths sample solvent composition. The sample solvent effects became more pronounced as the injection volume increased and as the percentage of organic solvent in the sample solution became greater. The peak height increases for STB at increasing buffer concentrations became much more pronounced at higher analyte concentrations. It was shown that the widely accepted procedure of dissolving samples in the mobile phase does not yield the most efficient chromatograms. For that reason samples should be dissolved in the solutions with higher aqueous content than that of the mobile phase whenever possible. The results strongly recommend that all the samples and standards, regardless whether the standards are external or internal, be analysed at a constant sample composition and a constant injection volume.

Determinación de la actividad estabilizadora de microtúbulos de fracciones ricas en diterpenoides obtenidas mediante HPLC de extractos de hojas, tallos y corteza de Taxus globosa.

Tesis (Maestría en Ciencias con Especialidad en Química de Productos Naturales) UANL

Validierung von HPLC-Methoden zur Bestimmung von Polyphenolen und Carotinoiden in Möhren, Weizen und Mais unterschiedlicher Herkunft

In den vorliegenden Untersuchungen wurde der Gehalt von Carotinoiden in Weizen, Mais und Möhren sowie der Polyphenolgehalt in Möhren mit analytischen Methoden zum Nachweis dieser Substanzen gemessen. Der Gehalt der Carotinoide in Mais und der Gehalt der phenolischen Bestandteile in Möhren wurde mit Messungen mittels HPLC-Analytik gemessen. Die Methoden wurden aus literaturbekannten Verfahren abgeleitet und an die Anforderungen der untersuchten Probenmatrices angepasst und validiert. Dem Verfahren lag die Frage zugrunde, ob es möglich ist, Kulturpflanzen aus verschiedenen Anbausystemen auf der Basis des Gehaltes bestimmter sekundärer Pflanzeninhaltsstoffe zu differenzieren und aufgrund von Unterschieden im Gehalt der sekundären Pflanzeninhaltsstoffe zu klassifizieren. Die Gesamtverfahren wurden dabei gemäß der ISO 17025 validiert. Für die Messungen standen Proben aus definierten Langzeitversuchen und Erzeugerproben ausgesuchter ökologisch bzw. konventionell arbeitender Anbaubetriebe zur Verfügung. Als Grundlage für eine valide Methodeneinschätzung wurden die Messungen an codierten Proben vorgenommen. Eine Decodierung der Proben erfolgte erst nach der Vorlage der Messergebnisse in den genannten Projekten. Die Messung und Auswertung des Carotinoidgehaltes in Weizen, Mais und Möhren vor dem Hintergrund der Differenzierung und Klassifizierung erfolgte in Proben eines Erntejahres. Die Messung des Gehaltes phenolischer Substanzen in Möhren erfolgte in Möhren aus 3 Erntejahren. Die verwendeten HPLC-Verfahren konnten in Bezug auf den analytischen Teil der Messungen in den einzelnen Verfahrensschritten Linearität, Spezifität, Präzision und Robustheit erfolgreich überprüft werden. Darüber hinaus wurden wichtige Einflussgrößen auf die Messungen bestimmt. Für die Verfahren zur photometrischen Bestimmung der Gesamtcarotinoide konnte eine Grundkalibrierung der Parameter Präzision und Linearität des Verfahrens erfolgreich durchgeführt werden. Während der Anwendung der HPLC-Methoden an codierten Proben konnten in allen untersuchten Probenmatrices quantitativ bedeutende Inhaltsstoffe nachgewiesen und identifiziert werden. Eine vollständige Identifizierung aller dargestellten Peaks konnte in den Untersuchungen der Polyphenole in Möhren und der Carotinoide in Mais nicht erfolgen. Im Hinblick auf die Frage nach der Differenzierung und Klassifizierung ergab sich in den verschiedenen Proben ein unterschiedliches Bild. Sowohl durch den Carotinoid- als auch den Polyphenolgehalt konnten einzelne Proben statistisch signifikant differenziert werden. Die Trennleistung hing dabei sowohl von den jeweiligen Komponenten als auch von der untersuchten Probenmatrix ab. Ein durchgängig höherer Gehalt sekundärer Pflanzeninhaltsstoffe in Proben aus ökologischem Anbau konnte nicht bestätigt werden. Für die Klassifizierung der Proben verschiedener Anbauvarianten und konnten multivariate statistische Methoden, wie lineare Diskriminantenanalyse (LDA) und Classification and Regression Tree (CART), erfolgreich angewandt werden. Eine Klassifizierung mit unterschiedlichen statistischen Verfahren erbrachte dabei unterschiedliche Ergebnisse. In der Klassifizierung der decodierten Proben mittels LDA wirkten sich die Faktoren Sorte und Standort stärker auf das Klassifizierungsergebnis aus als der Faktor Anbausystem. Eine Klassifizierung der decodierten Proben nach dem Anbausystem wurde mit dem CART-Verfahren durchgeführt. Auf dieser Basis wurden für die Polyphenole in Möhren 97 % der Proben richtig klassifiziert. Durch die Messwerte des Carotinoidgehaltes und des Luteingehaltes in Weizen konnte der größere Teil der Proben (90 %) korrekt klassifiziert werden. Auf der Basis des Carotinoidgehaltes in Mais wurde der Großteil der Proben (95 %) korrekt nach dem Anbausystem klassifiziert. Auf der Basis des mittels HPLC gemessenen Carotinoidgehaltes in Möhren konnten die Proben 97 % korrekt klassifiziert werden (97 %). Insgesamt erscheint der Grundgedanke der Klassifizierung durch den Gehalt sekundärer Pflanzeninhaltsstoffe vielversprechend. Durch die vielfältigen Einflussgrößen auf den Sekundärstoffwechsel von Pflanzen müssten Veränderungen, die durch Sorte und Standort auftreten, über mehrere Jahre erhoben und systematisiert werden.

Identification of lipophilic flavones and flavonols by comparative HPLC, TLC and UV spectral analysis

The identification of lipophilic flavones and flavonols using a combination of high performance liquid chromatography, thin layer chromatography and UV spectral analysis is discussed. Data are provided for the flavones, apigenin, luteolin and tricetin and twelve of their methyl ethers, 8-hydroxyluteolin, 6-hydroxyluteolin and scutellarein and fourteen of their methyl ethers, and some 6,8-dihydroxyapigenin and 6,8-dihydroxyluteolin derivatives. Data for some forty two flavonols with extra 6- and/or 8-hydroxylation, mostly 6-hydroxykaempferol and quercetagetin derivatives, are also presented. The remaining compounds analysed include fourteen 5-deoxyflavones, four 5-methoxyflavones and five 5-deoxyflavonols plus further 5-hydroxylated flavones and flavonols without B-ring oxidation or with 2-, 5- or 6-hydroxylation. Copyright © 2003 John Wiley & Sons, Ltd.