Quantitation of malondialdehyde in gingival crevicular fluid by a high-performance liquid chromatography-based method

Lipid peroxidation (LPO) has been associated with periodontal disease, and the evaluation of malondialdehyde (MDA) in the gingival crevicular fluid (GCF), an inflammatory exudate from the surrounding tissue of the periodontium, may be useful to clarify the role of LPO in the pathogenesis of periodontal disease. We describe the validation of a method to measure MDA in the GCF using high-performance liquid chromatography. MDA calibration curves were prepared with phosphate-buffered solution spiked with increasing known concentrations of MDA. Healthy and diseased GCF samples were collected from the same patient to avoid interindividual variability. MDA response was linear in the range measured, and excellent agreement was observed between added and detected concentrations of MDA. Samples' intra- and interday coefficients of variation were below 6.3% and 12.4%, respectively. The limit of quantitation (signal/noise = 5) was 0.03 mu M. When the validated method was applied to the GCF, excellent agreement was observed in the MDA quantitation from healthy and diseased sites, and diseased sites presented more MDA than healthy sites (P < 0.05). In this study, a validated method for MDA quantitation in GCF was established with satisfactory sensitivity, precision, and accuracy. (C) 2012 Elsevier Inc. All rights reserved.

DEVELOPMENT AND VALIDATION OF AN ANALYTICAL METHOD FOR QUANTITATION OF THE DRUG BEVACIZUMAB BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

DEVELOPMENT AND VALIDATION OF AN ANALYTICAL METHOD FOR QUANTITATION OF THE DRUG BEVACIZUMAB BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY. In this study, an analytical method was developed and validated for quantitation of the drug bevacizumab (Avastin (R)) by high performance liquid chromatography (HPLC). The HPLC column was a BioSuite 250 (R) HR SEC, 300 x 7.8 mm x 5 mu m (Waters, USA). The mobile phase consisted of phosphate buffered saline (PBS). The results revealed that the method was specific, precise. accurate, robust and linear (r(2) = 0.998) from 5 to 75 mu g mL(-1). Therefore, this method can be used in drug release studies or in quality control ampoules of the drug.

Evaluation of Solid-Phase Microextraction using a Polythiophene Film and Liquid Chromatography with Spectrophotometric Detection for the Determination of Antidepressants in Plasma Samples

Polythiophene (PTh) phase electropolymerized on the stainless steel wire was evaluated as solid-phase microextraction (SPME), and analysis by liquid chromatography with spectrophotometric detection (LC-UV) for determination of new-generation antidepressants, selective serotonin reuptake inhibitors (SSRIs) (citalopram, paroxetine, fluoxetine and sertraline), in plasma samples. The influence of electropolymerization variables (scan rate, potential range and scan cycles) was evaluated on SPME performance. The SPME variables (extraction time, temperature, matrix pH, ionic strength and desorption procedure), as well as the influence of plasma proteins on sorption mechanisms were also evaluated. The SPME/LC-UV method developed for determination of antidepressants in plasma sample presented a linear range between the limit of quantification (LOQ, 200-250 ng mL(-1)) to 4000 ng mL(-1), and interday precision with coefficient of variation (CV) ranged from 11 to 15%. The proposed method can be a useful tool for the determination of antidepressants in human plasma samples in urgent toxicological analysis after the accidental or suicidal intake of higher doses of medications.

High-Performance Liquid Chromatography Under Partially Denaturing Conditions (dHPLC) is a Fast and Cost-Effective Method for Screening Molecular Defects: Four Novel Mutations Found in X-Linked Chronic Granulomatous Disease

Implementing precise techniques in routine diagnosis of chronic granulomatous disease (CGD), which expedite the screening of molecular defects, may be critical for a quick assumption of patient prognosis. This study compared the efficacy of single-strand conformation polymorphism analysis (SSCP) and high-performance liquid chromatography under partially denaturing conditions (dHPLC) for screening mutations in CGD patients. We selected 10 male CGD patients with a clinical history of severe recurrent infections and abnormal respiratory burst function. gDNA, mRNA and cDNA samples were prepared by standard methods. CYBB exons were amplified by PCR and screened by SSCP or dHPLC. Abnormal DNA fragments were sequenced to reveal the nature of the mutations. The SSCP and dHPLC methods showed DNA abnormalities, respectively, in 55% and 100% of the cases. Sequencing of the abnormal DNA samples confirmed mutations in all cases. Four novel mutations in CYBB were identified which were picked up only by the dHPLC screening (c.904 insC, c.141+5 g>t, c.553 T>C, and c.665 A>T). This work highlights the relevance of dHPLC, a sensitive, fast, reliable and cost-effective method for screening mutations in CGD, which in combination with functional assays assessing the phagocyte respiratory burst will contribute to expedite the definitive diagnosis of X-linked CGD, direct treatment, genetic counselling and to have a clear assumption of the prognosis. This strategy is especially suitable for developing countries.

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.

Development and validation of a high-performance liquid chromatography method for quantification of egonol and homoegonol in Styrax species

Styrax camporum Pohl, known in Brazil as estoraque do campo or cuia de brejo, has been used in the treatment of gastrointestinal diseases. The therapeutic action of S. camporum has been attributed to the ethyl acetate fraction, although the chemical composition of this fraction has not yet been analyzed. In this study, a high-performance liquid chromatography photodiode array detection (HPLC-PAD) method for analysis of Brazilian Styrax species has been developed. The compounds egonol (1) and homoegonol (2) were found to be present in all the samples investigated by HPLC. These compounds were isolated by open column chromatography followed by preparative TLC, and were identified by 1H NMR. Compounds 1 and 2 were thus proposed as phytochemical markers for Styrax, owing to their biological properties and presence in other Styrax species. The developed method has been validated and successfully applied for quantification of 1 and 2 in S. camporum dried leaves and crude ethanolic extracts from S. ferrugineus and S. pohlii aerial parts. Copyright (c) 2011 John Wiley & Sons, Ltd.

Analysis of carvedilol enantiomers in human plasma using chiral stationary phase column and liquid chromatography with tandem mass spectrometry

Carvedilol is an antihypertensive drug available as a racemic mixture. (-)-(S)-carvedilol is responsible for the nonselective beta-blocker activity but both enantiomers present similar activity on a1-adrenergic receptor. To our knowledge, this is the first study of carvedilol enantiomers in human plasma using a chiral stationary phase column and liquid chromatography with tandem mass spectrometry. The method involves plasma extraction with diisopropyl ether using metoprolol as internal standard and direct separation of the carvedilol enantiomers on a Chirobiotic T (R) (Teicoplanin) column. Protonated ions [M + H]+ and their respective ion products were monitored at transitions of 407 > 100 for the carvedilol enantiomers and 268 > 116 for the internal standard. The quantification limit was 0.2 ng ml-1 for both enantiomers in plasma. The method was applied to study enantioselectivity in the pharmacokinetics of carvedilol administered as a single dose of 25 mg to a hypertensive patient. The results showed a higher plasma concentration of (+)-(R)-carvedilol (AUC08 205.52 vs. 82.61 (ng h) ml-1), with an enantiomer ratio of 2.48. Chirality, 2012. (C) 2012 Wiley Periodicals, Inc.

SENSITIVE METHOD FOR DETERMINATION OF PIPLARTINE, AN ALKALOID AMIDE FROM Piper SPECIES, IN RAT PLASMA SAMPLES BY LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY

Piplartine (PPTN) is an alkaloid amide found in Piper species that presents different activities. PPTN determination in rat plasma is necessary to better understand its biological effects. The aim of this study was to develop a sensitive LC-MS/MS method for the determination of PPTN in rat plasma. The performance criteria for linearity, sensitivity, precision, accuracy, recovery, and stability have been assessed and were within the recommended guidelines. The validated method proved to be suitable in a pilot study of PPTN kinetic disposition in rat plasma after a single intraperitoneal dose, and represents an appropriate tool to further pharmacokinetic studies.

Study of photorespiration in marine microalgae through the determination of glycolic acid using hydrophilic interaction liquid chromatography

Determination of organic acids in intracellular extracts and in the cultivation media of marine microalgae aid investigations about metabolic routes related to assimilation of atmospheric carbon by these organisms, which are known by their role in the carbon dioxide sink. The separation of these acids was investigated by hydrophilic interaction liquid chromatography (HILIC) using isocratic elution with a mobile phase composed of 70: 30 v/v acetonitrile/20 mmol/L ammonium acetate buffer (pH 6.8) and detection at 220 nm. HILIC allowed the determinations of glycolic acid, the most important metabolite for the evaluation of the photorespiration process in algae, to be made with better selectivity than that achieved by reversed phase liquid chromatography, but with less detectability. The concentration of glycolic acid was determined in the cultivation media and in intracellular extracts of the algae Tetraselmis gracilis and Phaeodactylum tricornutum submitted to different conditions of aeration: (i) without forced aeration, (ii) aeration with atmospheric air, and (iii) bubbling with N(2). The concentration of glycolic acid had a higher increase as the cultures were aerated with nitrogen, showing higher photorespiratory flux than that occurring in the cultures aerated with atmospheric air.

Stability-indicating methods for the enantioselective determination of dihydropyridines by high performance liquid chromatography and capillary electrophoresis

This paper presents simple, rapid, precise and accurate stability-indicating HPLC and CE methods, which were developed and validated for the determination of nitrendipine, nimodipine and nisoldipine. These drugs are calcium channel antagonists of the 1,4-dihydropyridine type which are used in the treatment of cardiovascular diseases. Experimental results showed a good linear correlation between the area and the concentration of drugs covering a relatively large domain of concentration in all cases. The linearity of the analytical procedures was in the range of 2.0-120.0 mu g mL-1 for nitrendipine, 1.0-100.0 mu g mL(-1) for nimodipine and 100.0-600.0 mu g mL(-1) for nisoldipine, the regression determination coefficient being higher than 0.99 in all cases. The proposed methods were found to have good precision and accuracy. The chemical stability of these drugs was determined under various conditions and the methods have shown adequate separation for their enantiomers and degradation products. In addition, degradation products produced as a result of stress studies did not interfere with the detection of the drugs' enantiomers and the assays can thus be considered stability-indicating.

Determination of anticonvulsants in human plasma using SPME in a heated interface coupled online to liquid chromatography (SPME-LC)

A simple and sensitive method using solid phase microextraction (SPME) and liquid chromatography (LC) with heated online desorption (SPME-LC) was developed and validated to analyze anticonvulsants (AEDs) in human plasma samples. A heated lab-made interface chamber was used in the desorption procedure, which allowed the transference of the whole extracted sample. The SPME conditions were optimized by applying an experimental design. Important factors are discussed such as fiber coating types, pH, extraction time and desorption conditions. The drugs were analyzed by LC, using a C18 column (150 mm x 4.6 mm x 5 mm); and 50 mmol L-1, pH 5.50 ammonium acetate buffer : acetonitrile : methanol (55 : 22 : 23 v/v) as the mobile phase with a flow rate of 0.8 mL min(-1). The suggested method presented precision (intra-assay and inter-assay), linearity and limit of quantification (LOQ) all adequate for the therapeutic drug monitoring (TDM) of AEDs in plasma.

Sensitive method for determination of piplartine, an alkaloid amide from Piper species, in rat plasma samples by liquid chromatography-tandem mass spectrometry

Piplartine (PPTN) is an alkaloid amide found in Piper species that presents different activities. PPTN determination in rat plasma is necessary to better understand its biological effects. The aim of this study was to develop a sensitive LC-MS/MS method for the determination of PPTN in rat plasma. The performance criteria for linearity, sensitivity, precision, accuracy, recovery, and stability have been assessed and were within the recommended guidelines. The validated method proved to be suitable in a pilot study of PPTN kinetic disposition in rat plasma after a single intraperitoneal dose, and represents an appropriate tool to further pharmacokinetic studies.

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.

Evaluation of solid-phase microextraction using a polythiophene film and liquid chromatography with spectrophotometric detection for the determination of antidepressants in plasma samples

Polythiophene (PTh) phase electropolymerized on the stainless steel wire was evaluated as solid-phase microextraction (SPME), and analysis by liquid chromatography with spectrophotometric detection (LC-UV) for determination of new-generation antidepressants, selective serotonin reuptake inhibitors (SSRIs) (citalopram, paroxetine, fluoxetine and sertraline), in plasma samples. The influence of electropolymerization variables (scan rate, potential range and scan cycles) was evaluated on SPME performance. The SPME variables (extraction time, temperature, matrix pH, ionic strength and desorption procedure), as well as the influence of plasma proteins on sorption mechanisms were also evaluated. The SPME/LC-UV method developed for determination of antidepressants in plasma sample presented a linear range between the limit of quantification (LOQ, 200-250 ng mL-1) to 4000 ng mL-1, and interday precision with coefficient of variation (CV) ranged from 11 to 15%. The proposed method can be a useful tool for the determination of antidepressants in human plasma samples in urgent toxicological analysis after the accidental or suicidal intake of higher doses of medications.

Hydroxocobalamin quantification in human plasma by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry in a pharmacokinetic study

A rapid, sensitive and specific method for quantifying hydroxocobalamin in human plasma using paracetamol as the internal standard (IS) is described. The analyte and the IS were extracted from plasma by liquid-liquid extraction using an organic solvent (ethanol 100%; -20°C). The extracts were analyzed by high performance liquid chromatography coupled with electrospray tandem mass spectrometry (HPLC-MS-MS). Chromatography was performed on Prevail C8 3 μm, analytical column (2.1×100 mm i.d.). The method had a chromatographic run time of 3.4 min and a linear calibration curve over the range 5-400 ng.mL-1 (r>0.9983). The limit of quantification was 5 ng.mL-1. The method was also validated without the use of the internal standard. The precision in the intra-batch validation with IS was 9.6%, 8.9%, 1.0% and 2.8% whereas without IS was 9.2%, 8.2%, 1.8% and 1.5% for 5, 15, 80 and 320 ng/mL, respectively. The accuracy in intra-batch validation with IS was 108.9%, 99.9%, 98.9% and 99.0% whereas without IS was 101.1%, 99.3%, 97.5% and 92.5% for 5, 15, 80 and 320 ng/mL, respectively. The precision in the inter-batch validation with IS was 9.4%, 6.9%, 4.6% and 5.5% whereas without IS was 10.9%, 6.4%, 5.0% and 6.2% for 5, 15, 80 and 320 ng/mL, respectively. The accuracy in inter-batch validation with IS was 101.9%, 104.1%, 103.2% and 99.7% whereas without IS was 94.4%, 101.2%, 101.6% and 96.0% for 5, 15, 80 and 320 ng/mL, respectively. This HPLC-MS-MS procedure was used to assess the pharmacokinetics of Hydroxo cobalamin following intramuscular injection 5000 μg in healthy volunteers of both sexes (10 males and 10 females). The volunteers had the following clinical characteristics (according to gender and expressed as mean ± SD [range]): males: age: 32.40 ± 8.00 y [23.00-46.00], height: 1.73 ± 0.07 m [1.62-1.85], body weight: 72.48 ± 10.22 Kg [60.20- 88.00]; females: age: 28.60 ± 9.54 y [18.00-44.00], height: 1.60 ± 0.05 m [1.54-1.70], body weight: 58.64 ± 6.09 Kg [51.70- 66.70]. The following pharmacokinetic parameters were obtained from the hydroxocobalamin plasma concentration vs. time curves: AUClast, T1/2, Tmax, Vd, Cl, Cmax and Clast. The pharmacokinetic parameters were 120 (± 25) ng/mL for Cmax, 2044 (± 641) ng.h/mL for AUClast, 8 (± 3.2) ng.mL-1 for Clast, 38 (± 15.8) hr for T1/2 and 2.5 (range 1-6) hr for Tmax. Female volunteers presented significant (p=0.0136) lower AUC (1706 ± 704) ng.h/mL) and larger (p=0.0205) clearance (2.91 ± 1.41 L/hr), as compared to male 2383 ± 343 ng.h/mL and 1.76 ± 0.23 L/hr, respectively. These pharmacokinetic differences could explain the higher prevalence of vitamin B12 deficiency in female patients. The method described validated well without the use of the internal standard and this approach should be investigated in other HPLC-MS-MS methods.