Quantification of tryptophan in plasma by high performance liquid chromatography

A simple, rapid and selective method using high-performance liquid chromatography with ultraviolet detection (267 nm) was applied for the determination of tryptophan in plasma. Separation was carried out on a C18 column (150 x 4.6 mm internal diameter) in 6 min. The mobile phase consisted of 5 mM the sodium acetate and acetonitrile (92:8, v/v). The method was shown to be precise and accurate, and good recovery of analyte was achieved, characterizing the method as efficient and reliable for use in laboratory analysis.

Simultaneous determination of non-steroidal anti-inflammatory drugs in pharmaceutical formulations and human serum by reversed phase high performance liquid chromatography

A rapid and sensitive method using high performance liquid chromatography has been developed and validated for the simultaneous determination of non-steroidal anti-inflammatory drugs (NSAIDs) in pharmaceutical formulations and human serum. Six NSAIDs including: naproxen sodium, diclofenac sodium, meloxicam, flurbiprofen, tiaprofenic and mefenamic acid were analyzed simultaneously in presence of ibuprofen as internal standard on Mediterranea C18 (5 µm, 250 x 0.46 mm) column. Mobile phase comprised of methanol: acetonitrile: H2O (60:20:20, v/v; pH 3.35) and pumped at a flow rate of 1 mL min-1 using 265 nm UV detection. The method was linear over a concentration range of 0.25-50 µg mL-1 (r² = 0.9999).

Specificity and selectivity improvement in doping analysis using comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry

Comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry was used for the identification of forty doping agents. The improvement in the specificity was remarkable, allowing the resolution of analytes that could not be done by one-dimensional chromatographic systems. The sensitivity observed for different classes of prohibited substances was clearly below the value required by the World Anti-Doping Agency. In addition time-of-flight mass spectrometry gives full spectrum for all analytes without any interference from the matrix, resulting in selectivity improvements. These results could support the implementation of an exhaustive monitoring approach for hundreds of doping agents in a single injection.

Multi-component quantitation of loratadine, pseudoephedrine and paracetamol in plasma and pharmaceutical formulations with liquid chromatography-tandem mass spectrometry utilizing a monolithic column

The purpose of this study was to develop a rapid, simple and sensitive quantitation method for pseudoephedrine (PSE), paracetamol (PAR) and loratadine (LOR) in plasma and pharmaceuticals using liquid chromatography-tandem mass spectrometry with a monolithic column. Separation was achieved using a gradient composition of methanol-0.1% formic acid at a flow rate of 1.0 mL min-1. Mass spectral transitions were recorded in SRM mode. System validation was evaluated for precision, specificity and linearity. Limit of detection for pseudoephedrine, paracetamol, and loratadine were determined to be 3.14, 1.86 and 1.44 ng mL-1, respectively, allowing easy determination in plasma with % recovery of 93.12 to 101.56%.

Determination of fipronil in bovine plasma by solid-phase extraction and liquid chromatography with ultraviolet detection

A fast and efficient method has been developed and validated for the determination of fipronil in bovine plasma. Samples were subjected to solid-phase extraction (SPE) followed by reversed phase liquid chromatography (LC) separation, using acetonitrile/water (60:40 v/v) as the mobile phase with a flow rate of 1.0 mL/min and ultraviolet (UV) detection at 210 nm. Ethiprole was used as the internal standard (IS). The method was found to be linear over the range 5-500 ng/mL (r = 0.999). The limit of quantitation (LOQ) was validated at 5 ng/mL. The method was successfully applied to monitor plasma concentrations following subcutaneous administration of fipronil in cattle.

Determination of levodopa in human plasma by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS): application to a bioequivalence study

A sensitive, accurate and simple method using HPLC-MS/MS was developed and validated for levodopa quantitation in human plasma. Analysis was achieved on a pursuit® C18 analytical column (5 µm; 150 x 4.6 mm i.d.) using a mobile phase (methanol and water , 90:10, v/v) containing formic acid 0.5% v/v, after extracting the samples using a simple protein plasma precipitation with perchloric acid. The developed method was validated in accordance with ANVISA guidelines and was successfully applied to a bioequivalence study in 60 healthy volunteers demonstrating the feasibility and reliability of the proposed method.

Preparative separation and purification of bufadienolides from ChanSu by high-speed counter-current chromatography combined with preparative HPLC

Eight bufadienolides were successfully isolated and purified from ChanSu by high-speed counter-current chromatography (HSCCC) combined with preparative HPLC (prep-HPLC). First, a stepwise elution mode of HSCCC with the solvent system composed of petroleum ether - ethyl acetate - methanol - water (4:6:4:6, 4:6:5:5, v/v) was employed and four bufadienolides, two partially purified fractions were obtained from 200 mg of crude extract. The partially purified fractions III and VI were then further separated by prep-HPLC, respectively, and another four bufadienolides were recovered. Their structures were confirmed by ESI-MS and ¹H-NMR spectra.

Preparative separation of polyphenols from artichoke by polyamide column chromatography and high-speed counter-current chromatography

An efficient method for the rapid separation and purification of polyphenols from artichoke by polyamide column chromatography in combination with high-speed counter-current chromatography (HSCCC) was successfully built. The crude ethanol extracts from dry artichoke were first pre-separated by polyamide column chromatography and divided in two parts as sample 1 and sample 2. Then, the samples were further separated by HSCCC and yielded 7.8 mg of chlorogenic acid (compound I), 24.5 mg of luteolin-7-O-β-D-rutinoside (compound II), 18.4 mg of luteolin-7-O-β-D-glucoside (compound III), and 33.4 mg of cynarin (compound IV) with purity levels of 92.0%, 98.2%, 98.5%, and 98.0%, respectively, as determined by high-performance liquid chromatography (HPLC) method. The chemical structures of these compounds were identified by electrospray ionization-mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR).

Quantification of beta-lactam antibiotics in veterinary drugs: amoxicillin and ampicillin determination by high performance liquid chromatography

This work focused on the development and validation of an RP-HPLC-UV method for quantification of beta-lactam antibiotics in three pharmaceutical samples. Active principles analyzed were amoxicillin and ampicillin, in 3 veterinary drugs. Mobile phase comprised 5 mmol L-1 phosphoric acid solution at pH 2.00, acetonitrile with gradient elution mode and detection wavelength at 220 nm. The method was validated according to the Brazilian National Health Surveillance regulation, where linear range and linearity, selectivity, precision, accuracy and ruggedness were evaluated. Inter day precision and accuracy for pharmaceutical samples 1, 2 and 3 were: 1.43 and 1.43%; 4.71 and 3.74%; 2.72 and 1.72%, respectively, while regression coefficients for analytical curves exceeded 0.99. The method had acceptable merit figure values, indicating reliable quantification. Analyzed samples had active principle concentrations varying from -12 to +21% compared to manufacturer label claims, rendering the medicine unsafe for administration to animals.

Preparative separation of C19-diterpenoid alkaloids from Aconitum carmichaelii Debx by pH‑zone-refining counter-current chromatography

The technique of pH-zone-refining counter-current chromatography was successfully applied to preparatively separate three C19-diterpenoid alkaloids from the crude extracts of Aconitum carmichaelii for the first time using a two-phase solvent system of petroleum ether-ethyl acetate-methanol-water (5:5:1:9, v/v/v/v). Mesaconitine (I), hypaconitine (II), and deoxyaconitine (III) were obtained from 2.5 g of the crude alkaloids in a one-step separation; the yields were 4.16%, 16.96%, and 5.05%, respectively. The purities of compounds I, II, and III were 93.0%, 95%, and 96%, respectively, as determined by HPLC. The chemical structures of the three compounds were identified by electrospray ionization mass spectrometry (ESI-MS) and NMR.

Super/subcritical fluid chromatography with packed columns: state of the art and applications

Separations using supercritical fluid chromatography (SFC) with packed columns have been re-discovered and explored in recent years. SFC enables fast and efficient separations and, in some cases, gives better results than high performance liquid chromatography (HPLC). This paper provides an overview of recent advances in SFC separations using packed columns for both achiral and chiral separations. The most important types of stationary phases used in SFC are discussed as well as the most critical parameters involved in the separations and some recent applications.

Separation and purification of three stilbenes from the radix of Polygonum cillinerve (Nakai) Ohwl by macroporous resin column chromatography combined with high-speed counter-current chromatography

An effective method for the rapid separation and purification of three stilbenes from the radix of Polygonum cillinerve (Nakai) Ohwl by macroporous resin column chromatography combined with high-speed counter-current chromatography (HSCCC) was successfully established. In the present study, a two-phase solvent system composed of chloroform-n-butanol-methanol-water (4:1:4:2, v/v/v/v) was used for HSCCC separation. A one-step separation in 4 h from 150 mg of crude extract produced 26.3 mg of trans-resveratrol-3-O-glucoside, 42.0 mg of pieceid-2"-O-gallate, and 17.9 mg of trans-resveratrol with purities of 99.1%, 97.8%, and 99.4%, respectively, as determined by high-performance liquid chromatography (HPLC). The chemical structures of these compounds were identified by nuclear magnetic resonance (NMR) spectroscopy.

DETECTION AND QUANTIFICATION OF PHYTOCHEMICAL MARKERS OF Ilex paraguariensis BY LIQUID CHROMATOGRAPHY

Ilex paraguariensis (yerba-mate) is used as a beverage, and its extract requires adequate quality control methods in order to guarantee quality and safe use. Strategies to develop and optimize a chromatographic method to quantify theobromine, caffeine, and chlorogenic acid in I. paraguariensis extracts were evaluated by applying a quality by design (QbD) model and ultra high-performance liquid chromatography (UHPLC). The presence of these three phytochemical markers in the extracts was evaluated using UHPLC-MS and was confirmed by the chromatographic bands in the total ion current traces (m/z of 181.1 [M+H]+, 195.0 [M+H]+, and 353.0 [M−H]−, respectively). The developed method was then transferred to a high-performance liquid chromatography (HPLC) platform, and the three phytochemical markers were used as external standards in the validation of a method for analyses of these compounds in extracts using a diode array detector (DAD). The validated method was applied to quantify the chlorogenic acid, caffeine, and theobromine in the samples. HPLC-DAD chromatographic fingerprinting was also used in a multivariate approach to process the entire data and to separate the I. paraguariensis extracts into two groups. The developed method is very useful for qualifying and quantifying I. paraguariensis extracts.

Electron transfer by singlet excited state of porphyn and electron acceptor affinity in rigid medium: photoacoustic phase angle analysis

Photoacoustic spectroscopy provides information about both amplitude and phase of the response of a system to an optical excitation process. This paper presents the studies of the phase in the electron transfer process between octaethylporphyn (OEP) and quinone molecules dispersed in a polymeric matrix. It was observed a tendency in the phase behavior to small values only in the spectral region near to 620 nm, while for shorter wavelength did not show any tendency. These measurements suggested that the electron transfer to acceptor occurred with the participation of octaethylporphyn singlet excited state.

Isolation of lignans glycosides from Alibertia sessilis (Vell.) K. Schum. (Rubiaceae) by preparative high-performance liquid chromatography

Enantiomeric aglycone lignans contained in a mixture were separated from a fraction of the extract of the stems of Alibertia sessilis (Vell.) K. Schum. (Rubiaceae) by preparative high-performance liquid chromatography. An efficient and fast separation can be achieved with methanol-water (30:70, v/v). Their structures were identified as (+)-lyoniresinol 3alpha-O-beta-glucopyranoside and (-)-lyoniresinol 3alpha-O-beta-glucopyranoside, being reported for the first time in Rubiaceae.