LC UV Research Articles

Development and Application of an Off-Line SPE–LC–UV Methodology for the Determination of Penoxsulam in Aquatic Systems Adjacent to Rice Fields

An accurate, simple, reproducible and sensitive off-line SPE–LC–UV method was developed and validated for the determination of penoxsulam, a new triazolopyrimidine sulphonamide herbicide widely used in rice culture. The method was validated in terms of precision, linearity, detection limit, quantification limit and accuracy. The repeatability and intermediate precision of LC–UV conditions chosen were evaluated, with an RSD always bellow 10%. A limit of detection of 0.07 μg L−1 was achieved after SPE concentration. Recovery studies, performed in two different matrixes (run-off water from rice fields and brackish water), ranged from 93 to 105%. The methodology was successfully used to detect penoxsulam in aquatic systems, during an application period in rice fields.

Simultaneous RP-LC Analysis of Diltiazem and Non-Steroidal Anti-Inflammatory Drugs in Pharmaceutical Formulations and Human Serum

A simple, precise, accurate, selective, and sensitive reversed-phase LC–UV method has been developed for simultaneous analysis of diltiazem and non-steroidal anti-inflammatory drugs (NSAIDs) in the bulk drug, tablet dosage forms, and human serum. Chromatographic separation of the drugs was performed at ambient temperature on a C18 stationary phase with 80:20 (v/v) methanol–water, pH 3.1 ± 0.02, as isocratic mobile phase. The mobile phase flow rate was initially 0.5 mL min−1 then increased to 1 mL min−1. All the NSAIDs were well separated from each other and from diltiazem. Total run time was 10 min. The assay was successfully applied to pharmaceutical formulations and serum and there was no chromatographic interference from tablet excipients. The method was linear in the range 1.25–50 μg mL−1 both for diltiazem and the NSAIDs. The suitability of this HPLC method for quantitative analysis of the drugs was proved by validation in accordance with International Conference on Harmonization (ICH) guidelines. The validation results, and results from statistical analysis of the data, demonstrated the method was reliable.

Flavonoid glycosides of the black locust tree, Robinia pseudoacacia (Leguminosae)

Four flavone glycosides isolated from extracts of the leaves of Robinia pseudoacacia (Leguminosae) were characterised by spectroscopic and chemical methods as the 7- O-β- d-glucuronopyranosyl-(1 → 2)[α- l-rhamnopyranosyl-(1 → 6)]-β- d-glucopyranosides of acacetin (5,7-dihydroxy-4′-methoxyflavone), apigenin (5,7,4′-trihydroxyflavone), diosmetin (5,7,3′-trihydroxy-4′-methoxyflavone) and luteolin (5,7,3′,4′-tetrahydroxyflavone). Assignment of glycosidic 1H and 13C resonances in their NMR spectra was facilitated by 2 J HC correlations detected using the H2BC (heteronuclear two-bond correlation) pulse sequence. Spectroscopic analysis of two known triglycosides, acacetin 7- O-β- d-glucopyranosyl-(1 → 2)[α- l-rhamnopyranosyl-(1 → 6)]-β- d-glucopyranoside (previously unrecorded from this species) and acacetin 7- O-β- d-xylopyranosyl-(1 → 2)[α- l-rhamnopyranosyl-(1 → 6)]-β- d-glucopyranoside (‘acacetin trioside’), enabled inconsistencies in the literature relating to these structures to be resolved. Comparison of the flavonoid chemistry of leaves and flowers of R. pseudoacacia using LC–UV and LC–MS showed that flavone 7- O-glycosides, particularly of acacetin, predominated in the former, whereas the latter comprised mainly flavonol 3,7-di- O-glycosides, including several examples new to this species. Tissue dependent differences in flavonoid chemistry were also evident from the glycosylation patterns of the compounds.

Kinetics and initial photocatalytic pathway of tryptophan, important constituent of microorganisms

The adsorption equilibrium in the dark and under UV light, the kinetic of photocatalytic oxidation and the main initial pathways of tryptophan photocatalytic degradation has been investigated in aerated TiO2 Degussa P-25 aqueous suspensions illuminated at λ>340nm. Tryptophan has been chosen as a model of organic compounds of amino acids, elemental constituents of DNA, microorganisms, e.g. bacteria, fungi, virus among others to better understand their photocatalytic degradation.Langmuir and Langmuir–Hinshelwood models have been used to determine the adsorption, the rate constants and the surface coverage of TiO2. Despite a low coverage of the TiO2 surface in the dark a high photocatalytic degradability and initial mineralization occurred. However, the surface coverage of TiO2 was about doubled under UV light suggesting the formation of active sites under irradiation.LC–MS, LC–UV with or without derivatization analyses allowed us to identify, mono-, di- and tri-hydroxylated compounds, deamination of heterocycle, the formation of six organic acid, oxalic, oxamique, lactic, formic, acetic and propanoic acids and of three linear amino acids, serine, aspartic acid and glycine. A degradation mechanism is proposed to the formation of oxamic acid.The study of the mineralization of carbon and nitrogen showed the presence of trace of organic molecules mineralizable with difficulty. Nitrogen atoms were predominantly photoconverted into NH4+. After more than 95% of carbon mineralization, NH4+/NO3− ratio was 4.The identification of the photocatalytic products, their evolution and the TOC evolution indicated three initial competitive pathways: about 50% of hydroxylation with 6–7% of the hydroxylation of carbon bearing NH2 and COO−, about 30% of decarboxylation and less than 20% of nitrogen–carbon (N–C) cleavage.

Reversed-Phase Liquid Chromatographic Determination of Two Manufacturing Intermediates in D&C Red No. 34 and Its Lakes

A reversed-phase LC method was developed to determine two manufacturing intermediates in the monosulfo monoazo color additive D&C Red No. 34 and its lakes. The analytes are 2-amino-1-naphthalenesulfonic acid (Tobias acid) and 3-hydroxy-2-naphthalenecarboxylic acid (3-hydroxy-2-naphthoic acid). This method can be used for batch certification of the color additives by the U.S. Food and Drug Administration to ensure that each lot meets published specifications for coloring drugs and cosmetics. The new method uses lithium oxalate in methanol-water to dissolve the color additives for analysis. The analytes were identified by comparison of their LC retention times and UV absorption spectra with those of standards. Peak area calibrations were generally linear (R > 0.999) and recoveries were 105% for Tobias acid and 103% for 3-hydroxy-2-naphthoic acid. The limits of determination (LOD) were 0.01% for Tobias acid and 0.03% for 3-hydroxy-2-naphthoic acid. The RSDs at the specification levels were 0.9% for Tobias acid and 3.2% for 3-hydroxy-2-naphthoic acid. Survey analyses of 14 samples of certified D&C Red No. 34 straight colors and lakes from six domestic and foreign manufacturers yielded results for Tobias acid that generally agreed with results previously obtained by using a gravity elution column chromatographic method. Nine of the results for 3-hydroxy-2-naphthoic acid were 2 to 5 times higher than the results obtained using the column chromatographic method. We attribute the lower accuracy of the column chromatographic method to incomplete solubility of the samples using the method conditions and difficulty with interpreting the UV spectrophotometric results.

FTRIFS biosensor based on double layer porous silicon as a LC detector for target molecule screening from complex samples

Post-column identification of target compounds in complex samples is one of the major tasks in drug screening and discovery. In this work, we demonstrated that double layer porous silicon (PSi) attached with affinity ligand could serve as a sensing element for post-column detection of target molecule by Fourier transformed reflectometric interference spectroscopy (FTRIFS), in which trypsin and its inhibitor were used as the model probe–target system. The double layer porous silicon was prepared by electrical etching with a current density of 500 mA/cm 2, followed by 167 mA/cm 2. Optical measurements indicated that trypsin could infiltrate into the outer porous layer (porosity 83.6%), but was excluded by the bottom layer (porosity 52%). The outer layer, attached with trypsin by standard amino-silane and glutaraldehyde chemistry, could specifically bind with the trypsin inhibitor, acting as a sample channel, while the bottom layer served as a reference signal channel. The binding event between the attached trypsin and trypsin inhibitor samples could be detected by FTRIFS in real-time through monitoring the optical thickness change of the porous silicon layer. The baseline drift caused by sample matrix variation could be effectively eliminated by a signal correction method. Optical signals had a linear relationship with the concentration of trypsin inhibitor in the range of 10–200 ng mL −1. The FTRIFS biosensor based on double layer porous silicon could be combined with a UV detector for screening the target molecule from complex component mixtures separated by a LC column. Using an LC–UV–FTRIFS system, a fraction containing a trypsin inhibitor could be separated from a soybean extract sample and identified in real-time.

Comparison of LC–UV, LC–ELSD and LC–MS Methods for the Determination of Sesquiterpenoids in Various Species of Artemisia

Simple and specific analytical methods for the quantitative determination of sesquiterpenoids from various species of Artemisia plant samples were developed. By LC–UV, LC–ELSD, the separation was achieved by reversed-phase chromatography on a C18 column with water and acetonitrile both containing 0.025% trifluoroacetic acid as the mobile phase. In the LC–MS system, trifluoroacetic acid was replaced by 0.1% formic acid. The wavelength used for quantification of sesquiterpenoids with a diode array detector was 205 nm. The limits of detection by LC–MS was found to be 5, 10, 25, 50, 50 ng mL−1. The limits of detection by LC–UV and LC–ELSD were found to be 5.0, 3.0, 100, 100, 7.5 μg mL−1, by LC–UV and 50, 25, 30, 100 and 75 μg mL−1 by LC–ELSD. LC–mass spectrometry coupled with electrospray ionization (ESI) interface is described for the identification and quantification of sesquiterpenoids in various plant samples. This method involved the use of the [M + H]+ ions of sesquiterpenoids in the positive ion mode with extractive ion monitoring.

Simultaneous identification of glucosinolates and phenolic compounds in a representative collection of vegetable Brassica rapa

Brassica rapa rapa group is widely distributed and consumed in northwestern Spain. The consumption of Brassica vegetables has been related to human health due to their phytochemicals, such as glucosinolates and phenolic compounds that induce a variety of physiological functions including antioxidant activity, enzymes regulation and apoptosis control and the cell cycle. For first time in Brassica crops, intact glucosinolates and phenolic compounds were simultaneously identified and characterized. Twelve intact glucosinolates, belonging to the three chemical classes, and more than 30 phenolic compounds were found in B. rapa leaves and young shoots (turnip greens and turnip tops) by LC–UV photodiode array detection (PAD)–electrospray ionization (ESI). The main naturally occurring phenolic compounds identified were flavonoids and derivatives of hydroxycinnamic acids. The majority of the flavonoids were kaempferol, quercetin and isorhamnetin glycosylated and acylated with different hydroxycinnamic acids. Quantification of the main compounds by HPLC-PAD showed significant differences for most of compounds between plant organs. Total glucosinolate content value was 26.84 μmol g −1 dw for turnip greens and 29.11 μmol g −1 dw for turnip tops; gluconapin being the predominant glucosinolate (23.2 μmol g −1 dw). Phenolic compounds were higher in turnip greens 51.71 μmol g −1 dw than in turnip tops 38.99 μmol g −1 dw, in which flavonols were always the major compounds.

Oxidized Multiwalled Carbon Nanotubes as an SPME Fiber Coating for Rapid LC–UV Analysis of Benzimidazole Fungicides in Water

The potential of oxidized multiwalled carbon nanotubes as a fiber coating for solid-phase microextraction of carbendazim, thiabendazole, and thiophanate-methyl from aqueous solution has been investigated, and the mechanism of adsorption has been explored. When an analytical method combining SPME with high-performance liquid chromatography was applied to real environmental water samples, recoveries of the spiked compounds was 75.7–104.5%, the linear range was 5–500 ng mL−1, the squared correlation coefficients (r 2) were from 0.9978 to 0.9984, LOD were from 0.3 to 1.5 ng mL−1, and RSD were from 2.3 to 10.5%.

LC–UV and LC–MS evaluation of stress degradation behaviour of tenatoprazole

In the present study, comprehensive stress testing of tenatoprazole was carried out according to ICH guideline Q1A (R2). Tenatoprazole was subjected to stress conditions of hydrolysis, oxidation, photolysis and neutral decomposition. Additionally, the solid drug was subjected to 50 °C for 60 days in dry-bath, and to the combined effect of temperature and humidity at 40 °C/75% RH. Extensive degradation was found to occur in acidic, neutral and oxidative conditions. Mild degradation was observed in basic conditions. The drug is relatively stable in the solid-state. The products formed under different stress conditions were investigated by LC and LC–MS. Successful separation of drug from degradation products formed under stress conditions was achieved on a Chemito ODS-3 column [C 18 (5 μm, 250 mm × 4.6 mm, i.d.)] using methanol: 0.01 M acetate buffer pH 4.5 adjusted with glacial acetic acid (55:45) as the mobile phase at flow rate of 1 mL/min and the peak was detected using a UV detector set at 306 nm. The LC–MS m/ z values and fragmentation patterns of degradation products formed under different stress conditions were studied and characterized through LC–MS fragmentation. Based on the results, degradation pathway for drug has been proposed.

Effect of simulated gastrointestinal conditions and epithelial transport on extracts of green tea and sage

Few in vitro screening studies on the biological activities of plant extracts that are intended for oral administration consider the effect of the gastrointestinal system. This study investigated this aspect on extracts of Camellia sinensis (green tea) and Salvia officinalis (sage) using antimicrobial activity as a model for demonstration. Both the crude extracts and their products after exposure to simulated gastric fluid (SGF) as well as simulated intestinal fluid (SIF) were screened for antimicrobial activity. The chromatographic profiles of the crude plant extracts and their SGF as well as SIF products were recorded and compared qualitatively by means of high performance liquid chromatography coupled to mass spectrometry. The effect of epithelial transport on the crude plant extracts was determined by applying them to an in vitro intestinal epithelial model (Caco-2). The crude extracts for both plants exhibited reduced antimicrobial activity after exposure to SGF, while no antimicrobial activity was detected after exposure to SIF. These results suggested chemical modification or degradation of the antimicrobial compounds when exposed to gastrointestinal conditions. This was confirmed by a reduction of the peak areas on the LC–UV–MS chromatograms. From the chromatographic profiles obtained during the transport study, it is evident that some compounds in the crude plant extracts were either not transported across the cell monolayer or they were metabolised during passage through the cells. It can be deduced that the gastrointestinal environment and epithelial transport process can dramatically affect the chromatographic profiles and biological activity of orally ingested natural products.

On-line high-performance liquid chromatography–ultraviolet–nuclear magnetic resonance method of the markers of nerve agents for verification of the Chemical Weapons Convention

This paper details an on-flow liquid chromatography–ultraviolet–nuclear magnetic resonance (LC–UV–NMR) method for the retrospective detection and identification of alkyl alkylphosphonic acids (AAPAs) and alkylphosphonic acids (APAs), the markers of the toxic nerve agents for verification of the Chemical Weapons Convention (CWC). Initially, the LC–UV–NMR parameters were optimized for benzyl derivatives of the APAs and AAPAs. The optimized parameters include stationary phase C 18, mobile phase methanol:water 78:22 (v/v), UV detection at 268 nm and 1H NMR acquisition conditions. The protocol described herein allowed the detection of analytes through acquisition of high quality NMR spectra from the aqueous solution of the APAs and AAPAs with high concentrations of interfering background chemicals which have been removed by preceding sample preparation. The reported standard deviation for the quantification is related to the UV detector which showed relative standard deviations (RSDs) for quantification within ±1.1%, while lower limit of detection upto 16 μg (in μg absolute) for the NMR detector. Finally the developed LC–UV–NMR method was applied to identify the APAs and AAPAs in real water samples, consequent to solid phase extraction and derivatization. The method is fast (total experiment time ∼2 h), sensitive, rugged and efficient.

Development and Validation of an RP–LC–UV Method for the Determination of Ondansetron: Application to Pharmaceutical Dosage Forms

A new, simple, rapid, sensitive and specific isocratic RP–LC–UV method was developed and validated for the determination of ondansetron in pharmaceutical dosage forms of orally disintegrating tablets, oral solution and injection. The LC separation was achieved on a Hypersil C4 column (250 × 4.6 mm, 5 μm) using a mobile phase of 50 mM potassium dihydrogen phosphate anhydrous adjusted to pH 3.5 with orthophosphoric acid and acetonitrile (30:70, v/v) at a flow rate of 1.0 mL min−1 and UV detection at 310 nm. The method was validated for specificity, linearity, precision, accuracy, limit of quantification, limit of detection, robustness and solution stability. The calibration curve was linear over a concentration range of 100–1,000 ng mL−1 (r 2 = 0.9996) with limit of detection and limit of quantification 50 and 100 ng mL−1, respectively. The intra-day and inter-day precision and accuracy were between 0.79 and 2.37% and −0.64 and 1.65%, respectively. The method was successfully applied for analysis of ondansetron in the presence of excipients in commercially available pharmaceutical dosage forms.

LC Determination of Isosorbide-5-Mononitrate in Human Plasma

A simple, sensitive, selective and cost effective LC–UV method was developed for determination of isosorbide mononitrate in human plasma using guaifenesin as an internal standard. Isosorbide mononitrate in plasma was extracted by a single step liquid extraction using tert-butyl methyl ether and chromatographed on a C18 column using water and acetonitrile (80:20 v/v) as mobile phase. The method was validated and exhibited a linear range from 51.6 to 2064.4 ng mL−1. The inter- and intra-assay accuracy ranged from 97.2–102.7 to 94.2–105.5%, respectively, with precision less than 10% in both the cases. The LLQ was 51.6 ng mL−1. The validated method was applied to the quantitation of isosorbide mononitrate from plasma samples in a pharmacokinetic study.

Simultaneous Determination of Four Active Components in Tobacco Wastes by LC.

A liquid chromatographic method was developed for the simultaneous quantification of four major active components in tobacco (Nicotiana tobaccum L.) wastes. Samples were extracted with 70% v/v aqueous methanol, four compounds including chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid and caffeic acid were identified and determined by using LC coupled to electrospray tandem mass spectrometry and LC–UV method, respectively. Separation in LC–UV was on an Alltima C18 column (250 mm × 4.6 mm i.d.; 5 μm) with a mobile phase consisting acetonitrile: ammonium acetate buffer (pH 4.5) (5:95 v/v), at a flow rate of 1.0 mL min−1, detected at 327 nm. Four regression equations showed good linear relationships (r 2 > 0.999) between the peak area of each marker and concentration. The method has good repeatability and precision, the intra-day and inter-day RSD for both retention time and peak area was less than 1.0%. The recoveries, measured at three concentration levels, varied from 96.33 to 101.10%. The LOD (S/N = 3) and LOQ (S/N = 6) were less than 0.010 and 0.795 μg·mL−1, respectively. This assay was successfully applied to the determination of four active compounds in ten samples. The results indicated that the developed assay method was rapid, accurate, reliable and could be readily utilized as a quantitative analysis method for various of tobacco wastes.

LC–UV Methodology for Simultaneous Determination of Lamivudine and Zidovudine in Plasma by Liquid–Liquid Extraction

This study describes an accurate, sensitive, and specific chromatographic method for the simultaneous quantitative determination of lamivudine and zidovudine in human blood plasma, using stavudine as an internal standard. The chromatographic separation was performed using a C8 column (150 × 4.6 mm, 5 μm), and ultraviolet absorbency detection at 270 nm with gradient elution. Two mobile phases were used. Phase A contained 10 mM potassium phosphate and 3% acetonitrile, whereas Phase B contained methanol. A linear gradient was used with a variability of A-B phase proportion from 98–2% to 72–28%, respectively. The drug extraction was performed with two 4 mL aliquots of ethyl acetate.

On-line solid phase extraction coupled to capillary LC-ESI-MS for determination of fluoxetine in human blood plasma

An instrumental setup including on-line solid phased extraction coupled to capillary liquid chromatography-electrospray ionization-mass spectrometry (SPE-capLC-ESI-MS) has been constructed to improve the sensitivity for quantification of fluoxetine hydrochloride in human plasma. Prior to injection, 0.5 mL of plasma spiked with metronidazole (internal standard) was mixed with ammonium formate buffer for effective chloroform liquid–liquid extraction. The method was validated in the range 5–60 ng mL −1 fluoxetine, yielding a correlation coefficient of 0.999 (r 2). The within-assay and between-assay precisions were between (8.5 and 11%) and (6.6 and 7.5%), respectively. The method was used to determine the amount of fluoxetine in a healthy male 14 h after an intake of one capsule of the antidepressant and anorectic Flutin ®, which contains 20 mg fluoxetine per each capsule. Fluoxetine was detected, and the concentration was calculated to 9.0 ng mL −1 plasma. In the preliminary experiments, conventional LC–UV instrumentation was employed. However, it was found that employing a capillary column with an inner diameter of (0.3 mm I.D. × 50 mm, Zorbax C 18) increased the sensitivity by a factor of ∼100, when injecting the same mass of analyte. Incorporating an easily automated C 18 reversed phase column switching system with SPE (1.0 mm I.D. × 5.0 mm, 5 μm) made it possible to inject up to 100 μL of solution, and the total analysis time was 5.5 min.

Determination of Shikimic Acid in Fruits of Illicium Species and Various Other Plant Samples by LC–UV and LC–ESI–MS

A simple and specific analytical method for the quantitative determination of shikimic acid from the methanol extract of the fruits of Illicium species and from various plant samples was developed. The LC–UV separation was achieved by reversed-phase chromatography on a C18 column with potassium dihydrogen phosphate and methanol as the mobile phase. In the LC–MS method, the separation was achieved by a C12 column using water and acetonitrile, both containing 0.1% acetic acid as the mobile phase. The methods were successfully used to study the percentage compositions of shikimic acid present in nine species of Illicium and various other plant samples. The detector response was linear with concentrations of shikimic acid in the range from 1.0–500.0 μg mL−1 by LC–UV and 100–1000 ng mL−1 by LC–MS. Mass spectrometry coupled with electrospray ionization interface is described for the identification of shikimic acid in various plant samples. This method involved the use of the [M-H]− ions of shikimic acid at m/z 173.0455 (calculated mass) in the negative ion mode with extractive ion monitoring.

Simultaneous Nonchiral Determination of Artemisinin and Arteannuin B in Artemisia annua Using Circular Dichroism Detection

A new method was performed using on-line coupling of nonchiral reversed-phase liquid chromatography (RP-LC) to circular dichroism (CD) spectroscopy for simultaneous determination of artemisinin and arteannuin B in crude plant extracts of Artemisia annua. Analysis was carried out on an LC–CD system equipped with an Agilent TC-C18 column (4.6 mm I.D. × 200 mm L, 5 μm) using gradient of acetonitrile. The method was validated to be practicable and reliable at alterable wavelength in the ranges of 220–420 nm as desired. LOD and LOQ of artemisinin and arteannuin B were 0.08, 0.26 and 0.31, 1.02 μg mL−1, respectively. It was more sensitive than conventional LC–UV and comparatively cheaper than LC–MS in analysis of TCM.

Structural elucidation of a PDE-5 inhibitor detected as an adulterant in a health supplement

A PDE-5 inhibitor was detected and isolated from a health supplement claimed to be a preparation of fresh oyster extracts and be able to promote and support healthy sexual function and endurance, etc. The structure of this PDE-5 inhibitor was elucidated using LC–UV, LC–TOF-MS, MS–MS, IR spectroscopy, and 2D NMR. It was characterized as 8-(2-(4-(hydroxymethyl)piperidin-1-yl)benzylamino)-3-ethyl-1H-imidazo[4,5- g]quinazoline-2(3H)-thione, a compound reported to be a PDE-5 inhibitor.