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.

DETERMINATION OF CHLORFENVINPHOS, FIPRONIL, AND CYPERMETHRIN RESIDUES IN MEAT AND BOVINE FAT USING QUECHERS METHOD AND GAS CHROMATOGRAPHY-MASS SPECTROMETRY

Analytic methods were applied and validated to measure residues of chlorfenvinphos, fipronil, and cypermethrin in meat and bovine fat, using the QuEChERS method and gas chromatography-mass spectrometry. For the meat, 2 g of sample, 4mL of acetonitrile, 1.6 g of MgSO4, and 0.4 g of NaCl were used in the liquid-liquid partition, while 80 mg of C18, 80 mg of primary and secondary amine and 150 mg of MgSO4 were employed in the dispersive solid-phase extraction. For the fat, 1 g of sample, 5 mL of hexane, 10 mL of water, 10 mL of acetonitrile, 4 g of MgSO4, and 0.5 g of NaCl were used in the liquid-liquid partition and 50 mg of primary and secondary amine and 150 mg of MgSO4 were used in the dispersive solid-phase extraction. The recovery percentages obtained for the pesticides in meat at different concentrations ranged from 81 to 129% with relative standard deviation below 27%. The corresponding results from the fat ranged from 70 to 123% with relative standard deviation below 25%. The methods showed sensitivity, precision, and accuracy according to EPA standards and quantification limits below the maximum residue limit established by European Union, except for chlorfenvinphos in the fat.

Study of the osteoconductive capacity of hydroxyapatite implanted into the femur of ovariectomized rats

Osteoporosis is a global public health that affects postmenopausal women due to the deficiency of estrogen, a hormone that plays an important role in the microarchitecture of bone tissue. Osteoporosis predisposes to pathological bone fracture that can be repaired by conventional methods. However, depending on the severity and quantity of bone loss, the use of autogenous grafts or biomaterials such as hydroxyapatite might be necessary. The latter has received increasing attention in the medical field because of its good biological properties such as osteoconductivity and biocompatibility with bone tissue. The objective of this study was to evaluate using histologic and radiographic analyses, the osteogenic capacity of hydroxyapatite implanted into the femur of rats with ovariectomy-induced osteoporosis. Eighteen rats were divided into three groups with six animals in each: group nonovariectomized, bilaterally ovariectomized not receiving estrogen replacement therapy, and bilaterally ovariectomized submitted to estrogen replacement therapy. Defects were created experimentally in the distal epiphysis of the femur with a surgical drill and filled with porous hydroxyapatite granules. The animals were sacrificed 8 weeks after surgery. The volume of newly formed bone in the implant area was quantified by morphometrical methods. The results were analyzed by ANOVA followed by the Tukey test (P < 0.05). The hydroxyapatite granules showed good radiopacity. Histological analysis revealed less quantity of newly formed bone in the ovariectomized group not submitted to hormone replacement therapy. In conclusion, bone neoformation can be expected even in bones compromised by estrogen deficiency, but the quantity and velocity of bone formation are lower. Microsc. Res. Tech., 2011. (c) 2011 Wiley Periodicals, Inc.

Hollow-fiber liquid-phase microextraction and gas chromatography-mass spectrometry of barbiturates in whole blood samples

Here, we present a method for measuring barbiturates (butalbital, secobarbital, pentobarbital, and phenobarbital) in whole blood samples. To accomplish these measurements, analytes were extracted by means of hollow-fiber liquid-phase microextraction in the three-phase mode. Hollow-fiber pores were filled with decanol, and a solution of sodium hydroxide (pH 13) was introduced into the lumen of the fiber (acceptor phase). The fiber was submersed in the acidified blood sample, and the system was subjected to an ultrasonic bath. After a 5 min extraction, the acceptor phase was withdrawn from the fiber and dried under a nitrogen stream. The residue was reconstituted with ethyl acetate and trimethylanilinium hydroxide. An aliquot of 1.0 mu L of this solution was injected into the gas chromatograph/mass spectrometer, with the derivatization reaction occurring in the hot injector port (flash methylation). The method proved to be simple and rapid, and only a small amount of organic solvent (decanol) was needed for extraction. The detection limit was 0.5 mu g/mL for all the analyzed barbiturates. The calibration curves were linear over the specified range (1.0 to 10.0 mu g/mL). This method was successfully applied to postmortem samples (heart blood and femoral blood) collected from three deceased persons previously exposed to barbiturates.

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.

Characterization of volatile composition of Laurencia dendroidea by gas chromatography-mass spectrometry

In this study we report the characterization of the volatile compounds of Laurencia dendroidea. Solvent extracts (dichloromethane and methanol), hydrodistillation extracts and headspace solid-phase microextraction samples were obtained and analyzed by GC-MS. Forty-six volatile components were identified in L. dendroidea, among them hydrocarbons, alcohols, phenols, aldehydes, ketones, acids, esters and terpenes.

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.

XRD, AFM, IR and TGA study of nanostructured hydroxyapatite

In this work, the synthetic hydroxyapatite (HAP) was studied using different preparation routes to decrease the crystal size and to study the temperature effect on the HAP nano-sized hydroxyapatite crystallization. X-ray diffraction (XRD) analysis indicated that all samples were composed by crystalline and amorphous phases . The sample with greater quantity of amorphous phase (40% of total mass) was studied. The nano-sized hydroxyapatite powder was heated and studied at 300, 500, 700, 900 and 1150 °C. All samples were characterized by XRD and their XRD patterns refined using the Rietveld method. The crystallites presented an anisotropic form, being larger in the [001] direction. It was observed that the crystallite size increased continuously with the heating temperature and the eccentricity of the ellipsoidal shape changed from 2.75 at 300 °C to 1.94, 1.43, 1.04 and 1.00 respectively at 500, 700, 900 and 1150 °C. In order to better characterize the morphology of the HAP the samples were also examined using atomic force microscopy (AFM), infrared spectrometry (IR) and thermogravimetric analysis (TGA).

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.

Stopping power and depth dose profile of H+ and He+ ion beams in hydroxyapatite thin films.

Hadron therapy is a promising technique to treat deep-seated tumors. For an accurate treatment planning, the energy deposition in the soft and hard human tissue must be well known. Water has been usually employed as a phantom of soft tissues, but other biomaterials, such as hydroxyapatite (HAp), used as bone substitute, are also relevant as a phantom for hard tissues. The stopping power of HAp for H+ and He+ beams has been studied experimentally and theoretically. The measurements have been done using the Rutherford backscattering technique in an energy range of 450-2000 keV for H+ and of 400-5000 keV for He+ projectiles. The theoretical calculations are based in the dielectric formulation together with the MELF-GOS (Mermin Energy-Loss Function – Generalized Oscillator Strengths) method [1] to describe the target excitation spectrum. A quite good agreement between the experimental data and the theoretical results has been found. The depth dose profile of H+ and He+ ion beams in HAp has been simulated by the SEICS (Simulation of Energetic Ions and Clusters through Solids) code [2], which incorporates the electronic stopping force due to the energy loss by collisions with the target electrons, including fluctuations due to the energy-loss straggling, the multiple elastic scattering with the target nuclei, with their corresponding nuclear energy loss, and the dynamical charge-exchange processes in the projectile charge state. The energy deposition by H+ and He+ as a function of the depth are compared, at several projectile energies, for HAp and liquid water, showing important differences.