Buffalo yogurt fermented with commercial starter and Lactobacillus plantarum originating from breast milk lowered blood pressure in pregnant hypertensive rats.

Nutritional therapy, which may have advantages over medication, is being investigated as a novel treatment for pregnancy-induced hypertension. Several studies have shown that probiotic yogurt supplementation during pregnancy has beneficial effects on maternal and fetal health. In this study, fermented buffalo milk was produced with yogurt culture and Lactobacillus plantarum B, a probiotic isolated from healthy breast milk with high angiotensin-converting enzyme inhibitory activity. The fermentation conditions under which the angiotensin-converting enzyme (ACE) inhibitory activity reached 84.51% were optimized by the response surface method as follows: 2 × 106 cfu/mL of L. plantarum B, yogurt culture 2.5 × 105 cfu/mL, and 8 h at 37°C. The distribution of ACE inhibitory peptides from fermented buffalo milk and fermented cow milk were further analyzed by liquid chromatography-mass spectrometry. By searching according to the structural features of ACE inhibitory peptides, 29 and 11 peptides containing ACE inhibitory peptide features were found in fermented buffalo milk and fermented cow milk, respectively. To investigate the in vivo antihypertensive activity of fermented buffalo milk, 18 pregnant rats were divided into 3 groups (n = 6 in each group) and administered 10 mL of normal saline, yogurt (20 mg/kg), or labetalol hydrochloride (4 mg/kg) daily from the beginning of pregnancy to parturition. To induce hypertension, methyl nitrosoarginine (125 mg/kg) was injected subcutaneously every day from d 15 of pregnancy to the day of delivery. Blood pressure was not significantly changed in the yogurt and labetalol groups after induction of hypertension and was lower compared with the normal saline group, but there was no difference between the yogurt and labetalol groups. This implied that the buffalo yogurt had a preventive and antihypertensive effect in the pregnancy-induced hypertensive rat model. Further studies to determine the mechanism of action, as well as a randomized control trial, are warranted.

DETERMINATION OF SOTALOL, OXPRENOLOL AND LABETALOL IN BINARY MIXTURES AND IN SPIKED HUMAN SERUM BY DERIVATIVE SPECTROPHOTOMETRIC METHOD.

The usefulness of derivative spectrophotometry for the determination of labetalol, sotalol and oxprenolol in binary mixtures and in human spiked serum was checked. To this aim a spectrophotometric analysis of samples in the UV range was carried out and the obtained results revealed that derivative spectropho- tometry allows for the fast, accurate and precise determination of the tested substances in spite of their clear interference in the zero-order spectra. For quantitative determinations "zero-crossing" technique was used to establish wavelengths for zeros of specified component. In a mixture of labetalol and oxprenolol the following wavelengths were established: D1 λ = 245.32 nm and 266.03 nm, D2 λ = 243.30 nm and 301.09 nm. respectively. D3 derivative did not show zeros suitable for quantitative analysis. For the analysis of labetalol and sotalol mixture, D3 derivative spectrophotometry was used at the following wavelengths: = 246.03 nm and λ = 249.91 rum, respectively. In this case, the curves of Dl and D2 derivatives showed no zeros that can be used in quantitative analysis. To determine the concentration of the components in a mixture containing oxprenolol and sotalol the following wavelengths were selected: for oxprenolol DI λ = 245.32 nm, D2 λ = 240.18 run, D3 λ = 232.05 nm and for sotalol Dl λ = 230.56 nm, D2 Xλ= 232.65 nm and D3 X = 238.84 tm, respectively. The developed spectrophotometric method was characterized by high sensitivity and accuracy, LOD determined for sotalol was in the range of 0.21-1.88 µg/mL, for labetalol 1.00-3.43 µg/mL and for oxprenolol 0.16-2.06 µg/mL; LOQ determined for sotalol was in the range of 0.65-5.70 µg/mL, for labetalol 3.11-10.39 µg/mL and for oxprenolol 0.47-6.23 µg/mL, depending on the composition of the tested mixture and the order of the deriv- ative. The recovery of the individual components was within the range of 100 ± 5%. The linearity range was wide and estimated for sotalol in the range of 11.00-38.50 µg/mL, for labetalol 12.80-44.80 µg/mL and for oxprenolol 12.60-44.10 µg/mL with correlation coefficients in the range of 0.9977-0.9999.

Development and validation of a bio-analytical method for simultaneous quantification of nebivolol and labetalol in aqueous humor and plasma using LC-MS/MS and its application to ocular pharmacokinetic studies.

Beta blockers is the class of choice of drugs in treatment of open angle Glaucoma. However, many of these drugs suffer from systemic side effects due to their absorption into systemic circulation via nasolachrymal duct. To evaluate the safety and efficacy of nebivolol and labetalol for the treatment of open angle glaucoma, it is important to have a bioanalytical method for measuring the drug concentrations both in aqueous humor and plasma. A simple, sensitive and high throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with protein precipitation technique was developed for simultaneous quantification of nebivolol and labetalol using nebivolol-d4 and metoprolol, respectively, as internal standards in aqueous humor and plasma. Nebivolol and labetalol were monitored in electrospray positive ionization (ESI) mode at transition 406.2/151.1 and 329.2/162.0, respectively. Mobile phase comprised of mixture of aqueous buffer (solvent A) and organic phase (solvent B) (mixture of A:B in the ratio of 30:70, v/v). The aqueous buffer was 5 mM ammonium acetate buffer adjusted to pH 3.5 ±â€¯0.05 with formic acid while the organic phase was a mixture of methanol and acetonitrile in the ratio of 25:75, v/v. Chromatographic separation was achieved using reverse phase Zorbax SB-C18 column (4.6 × 100 mm, 3.5 µm). Method was linear in both the matrices in the concentration range of 0.43-750 ng/mL for nebivolol and 0.39-668 ng/mL for labetalol with r2 > 0.99. Accuracy values, expressed in terms of bias (%), for nebivolol in aqueous humor and plasma were ≤9.6% and ≤11.4% and for labetalol were ≤8.6% and ≤5.9%, respectively. Inter-day and intra-day precision values, expressed in terms of RSD (%), for both the drugs were within 11.4%. No interference was obtained due to matrix components. Mean recovery (%) values in aqueous humor and plasma were 72.4% and 73.0% for nebivolol and 56.7% and 54.4% for labetalol, respectively. No significant degradation was observed in both the drugs in both the matrices when stored at -20 °C for 1 month. Aqueous humor and plasma samples of nebivolol and labetalol on bench top were stable for 18 h and 8 h, respectively. The developed method was applied for determining pharmacokinetic parameters of both drugs in aqueous humor following single dose ocular administration in rabbits.

Chiral separation of beta-blockers by high-performance liquid chromatography and determination of bisoprolol enantiomers in surface waters.

Beta-blockers are chiral compounds with enantiomers that have different bioactivity, which means that while one is active, the other can be inactive or even harmful. Due to their high consumption and incomplete degradation in waste water, they may reach surface waters and affect aquatic organisms. To address this issue we developed a chromatographic method suitable for determining beta-blocker enantiomers in surface waters. It was tested on five beta-blockers (acebutolol, atenolol, bisoprolol, labetalol and metoprolol) and validated on bisoprolol enantiomers. Good enantioseparation of all analysed beta-blockers was achieved on the Chirobiotic V column with the mobile phase composed of methanol/acetic acid/triethylamine (100/0.20/0.15 v/v/v) at a flow rate of 0.5 mL/min and column temperature of 45 °C. Method proved to be linear in the concentration range from 0.075 µg/mL to 5 µg/mL, and showed good recovery. The limits of bisoprolol enantiomer detection were 0.025 µg/mL and 0.026 µg/mL and of quantification 0.075 µg/mL and 0.075 µg/mL. Despite its limitations, it seems to be a promising method for bisoprolol enantiomer analysis in surface water samples. Further research could focus on waste water analysis, where enantiomer concentrations may be high. Furthermore, transferring the method to a more sensitive one such as liquid chromatography coupled with tandem mass spectrometry and using ammonium acetate as the mobile phase additive instead of acetic acid and triethylamine would perhaps yield much lower limits of detection and quantification.

Simultaneous determination of labetalol and furosemide by first-derivative synchronous spectrofluorimetry.

A rapid, simple and highly sensitive first derivative synchronous spectrofluorimetric method was developed for the simultaneous analysis of a binary mixture of labetalol HCl (LBT) and furosemide (FUR) without prior separation. The method was based upon measuring the first derivative of synchronous fluorescence spectra of the two drugs at Deltalambda = 130 nm in aqueous ethanol (55% V/V). The different experimental parameters affecting the synchronous fluorescence of the studied drugs were carefully studied and optimized. The first derivative amplitude-concentration plots were rectilinear over the range of 0.10 to 1.00 microg/mL and 0.05-0.50 microg/mL with lower detection limits of 0.0149 and 7 x 10(-3) microg/mL and quantification limits of 0.045 and 0.021 microg/mL for LBT and FUR, respectively. The proposed method was successfully applied for the determination of the studied drugs in synthetic mixtures. The results obtained were in good agreement with those obtained by the reference methods.

Micellar enhanced spectrofluorometric determination of labetalol through complexation with aluminium(III): application to dosage forms and biological fluids.

Two simple, sensitive, and specific spectrofluorometric procedures have been developed for the determination of labetalol (LBT) in pharmaceuticals and biological fluids. LBT was found to react with Al3+, both in acetate buffer of pH 4.5 (Procedure I) and borate buffer of pH 8.0 (Procedure II), to produce highly fluorescent stable complexes. The fluorescence intensity could be enhanced by the addition of sodium dodecyl sulfate, resulting in 3.5- and 2.7-fold increases in the fluorescence intensity for Procedures I and II, respectively. In both procedures, the fluorescence intensity was measured at 408 nm after excitation at 320 nm. The different experimental parameters affecting the development and stability of the fluorescent products were carefully studied and optimized. The fluorescence intensity-concentration plots were rectilinear over the range of 0.02-0.1 and 0.01-0.05 microg/mL with a detection limit of 0.003 and 0.001 microg/mL for Procedures I and II, respectively. The proposed method was successfully applied to commercial tablets containing LBT. The results were in good agreement with those obtained using a reference spectrofluorometric method. Furthermore, the method was applied for the determination of LBT in spiked human plasma, and the recovery (n = 4) was 93.30 +/- 2.62%. A proposal of the reaction pathway was postulated for Procedures I and II, respectively.

Modeling the electrophoretic mobility of beta-blockers in capillary electrophoresis using artificial neural networks.

Artificial neural networks were used for modeling the mobility of five beta-blockers (i.e., labetalol atenolol, practolol, timolol and propranolol) in running buffer with ternary solvent background electrolyte systems containing 80 mM acetate buffer dissolved in water, methanol, ethanol and their ternary mixtures. The volume fractions of two solvents (f(2), f(3)) and cologarithm of electrophoretic mobilities in pure solvents (i.e., -Lnmu(1), -Lnmu(2) and -Lnmu(3)) were used as inputs and cologarithm of the mobility in mixed solvents was the output of the networks. The number of neurons in hidden layer, learning rate, momentum and the number of epochs were optimized, in which two neurons in hidden layer, 0.2, 0.9 and 20000 were found the optimized values for learning rate, momentum and number of epochs, respectively. Mean percentage deviations (MPD) between calculated and experimental mobilities were computed as an accuracy criterion. To assess the correlative ability of the model, all data points in each set were used as training set and the mobilities were back-calculated by the trained networks, in which the overall MPD (OMPD)+/- standard deviation (SD) for correlative study was 3.1+/- 2.3. To evaluate the prediction capability of the proposed ANN model, the network was trained using 15 data points for each analyte and the remaining data points were predicted. The obtained OMPD (+/-SD) for this analysis was 3.6+/-3.0. To further investigate on the applicability of ANN, a generalized network was trained with 10 data points from each beta-blocker and then the network was employed to predict the mobilities of the analytes in ternary solvent electrolyte systems. The MPDs for predicted mobilities were 3.6%, 3.6%, 3.9%, 3.7% and 2.9% respectively for labetalol, atenolol, practolol, timolol and propranolol.

Spectrofluorometric determination of some beta-blockers in tablets and human plasma using 9,10-dimethoxyanthracene-2-sodium sulfonate.

A simple and sensitive spectrofluorometric method was developed for the quantitative determination of some beta-blockers, namely arotinolol, atenolol and labetalol as hydrochloride salts. The method is based on the reaction of these drugs as n-electron donors with the fluorogenic reagent 9,10-dimethoxy-2-anthracene sulfonate (DMAS) as pi-acceptor in acidic medium. The obtained ion-pairs were extracted into chloroform and measured spectrofluorometrically at 452 nm after excitation at 385 nm. The fluorescence intensity-concentration plots are rectilinear over the ranges of 0.5-5 microg x ml(1), 1.0-11.0 microg x ml(1) and 0.6-6.4 microg x ml(1) for labetalol, atenolol and arotinolol, respectively. The different parameters affecting the reaction pathway were thoroughly studied and optimized. No interference was observed from the common pharmaceutical excipients. The proposed method was successfully applied to the analysis of tablets and the results were statistically compared with those obtained by reference methods. The method was further extended to the in vitro determination of the drugs in spiked human plasma, the % recoveries (n = 3) ranged from 96.98 +/- 1.55 to 98.28 +/- 2.19. A proposal of the reaction pathway was postulated.

Influence of inorganic mobile phase additives on the retention, efficiency and peak symmetry of protonated basic compounds in reversed-phase liquid chromatography.

Inorganic eluent additives affect the retention of protonated basic analytes in reversed-phase HPLC. This influence is attributed to the disruption of the analyte solvation-desolvation equilibria in the mobile phase, also known as "chaotropic effect". With an increase of counteranion concentration analyte retention increases with concomitant decrease in the tailing factor. Different inorganic counteranions at equimolar concentrations affect protonated basic analyte retention and peak symmetry to varying degrees. The effect of the concentrations of four different inorganic mobile phase additives (KPF6, NaClO4, NaBF4, NaH2PO4) on the analyte retention, peak symmetry, and efficiency on a C8-bonded silica column has been studied. The analytes used in this study included phenols, toluene, benzyl amines, beta-blockers and ophthalmic drugs. The following trend in increase of basic analyte retention factor and decrease of tailing factor was found: PF6- > ClO4- approximately BF4- > H2PO4-. With the increase of the counteranion concentration greater analyte loading could be achieved and consequently an increase in the apparent efficiency was observed until the maximum plate number for the column was achieved. At the highest concentration of counteranions, the peak efficiency for most of the basic compounds studied was similar to that of the neutral markers. In contrast, the neutral markers, such as phenols, showed no significant changes in retention, efficiency or loading capacity as counteranion concentration was increased.

Oxidation of beta-blocking agents--VII. Periodate oxidation of labetalol.

The oxidation of labetalol with sodium metaperiodate is described. In spite of the bulky substituent on the amino group of labetalol, glycol cleavage of the molecule occurred. Spectrometric methods verified that the aromatic aldehyde formed was 2-hydroxy-5-formylbenzamide and that the amine was 1-methyl-3-phenylpropylamine. The polarographic behaviour of the aldehyde was examined as well. The half-wave potential in Britton-Robinson buffer pH 5.68 was -1.24 V. Direct linearity (r = 0.9999) was observed between the diffusion current and the concentration of the aldehyde. Quantitation of labetalol was carried out polarographically by measuring the concentration of 2-hydroxy-5-formylbenzamide formed in the oxidation.

Spectrofluorometric determination of labetolol in pharmaceutical preparations and spiked human urine through the formation of coumarin derivative.

A simple, sensitive and specific spectrofluorimetric method has been developed for the determination of labetalol (LBT). The method is based on the reaction between LBT and ethylacetoacetate in the presence of sulphuric acid to give yellow fluorescent product with excitation wavelength of 312 nm and emission wavelength of 432 nm. The reaction conditions were studied and optimized. The fluorescence intensity-concentration plot is rectilinear over the range 1-15 microgram/ml with minimum detectability limit of 0.8 microgram/ml (2.16 x 10(-6) M). The proposed method was successfully applied to commercial tablets containing LBT, the percentage recoveries agreed well with those obtained using the official methods. Hydrochlorothiazide, which is frequently co-formulated with LBT did not interfere with the assay. The method was further extended to the in-vitro determination of LBT in spiked human urine samples. The percentage recovery was 101.50+/-6.18 (n=6). A proposal of the reaction pathway was postulated.

Determination of labetalol in a post mortem case using HPLC.

A case report involving low levels of labetalol is presented. Samples of post mortem stomach contents, liver, kidney, urine, and blood were flocculated with aluminium hydroxide before the labetalol was extracted by using Sep-pakTM C18 cartridges. Quantification of the extracts was performed by HPLC with an ultraviolet detector. The significant of relatively low drug levels found in a patient suffering from a respiratory disease is discussed.

Spectrophotometric determination of labetalol in pharmaceutical preparations and spiked human urine.

Two simple and sensitive spectrophotometric methods were developed for the spectrophotometric determination of labetaolol (LBT). Both methods are based on the phenolic nature of the drug. The first method (Method I) is based on coupling LBT with diazotized benzocaine in presence of trimethylamine. A yellow colour peaking at 410 nm was produced and its absorbance is linear with the concentration over the range 1-10 microg ml(-1) with correlation coefficient (n=5) of 0.9993. The molar absorptivity was 2.633 x 10(4) l mol(-1) cm(-1). The second method (Method II) involves coupling LBT with diazotized p-nitroaniline in presence of sodium carbonate. An orange colour peaking at 456 nm was obtained and its absorbance is linear with concentration over the range 1-10 microg ml(-1) with correlation coefficient (n=5) of 0.99935. The stoichiometry of the reaction in both cases was accomplished adopting the limiting logarithmic method and was found to be 1:1. The developed method could be successfully applied to commercial tablets. The results obtained were in good agreement with those obtained using the official methods. No interference was encountered from co-formulated drugs, such as hydrochlorothiazide. The method was further extended to the in-vitro determination of LBT in spiked human urine. The % recovery (n=4) were 97.7+/-5.75 and 103.27+/-5.42 using the Methods I and II, respectively.

Capillary electrophoresis procedure for the simultaneous analysis and stoichiometry determination of a drug and its counter-ion by using dual-opposite end injection and contactless conductivity detection: application to labetalol hydrochloride.

In this work, a capillary electrophoresis (CE) procedure was developed for the simultaneous determination of a pharmaceutical drug and its counter-ion, namely labetalol hydrochloride. For this purpose, an uncoated fused-silica capillary, a low conductivity background electrolyte (BGE) and a capacitively coupled contactless conductivity detector (C(4)D) were employed. This detection system is highly sensitive and enables detection of inorganic as well as organic ions unlike with direct UV detection. Moreover, to be able to simultaneously analyze the cationic drug (labetalol(+)) and its anionic counter-ion (Cl(-)) in the same electrophoretic run without the need of a coated capillary, a dual-opposite end injection was performed. In this technique, the sample is hydrodynamically injected into both ends of the capillary. This method is simple and easy to perform since the different injection steps are automated by the CE software. This novel CE-C(4)D procedure with dual-opposite end injection has been successfully validated and applied for the analysis of chloride content in an adrenergic antagonist (labetalol hydrochloride). Thus, the hereby developed method has been shown to enable fast (analysis time<10 min), precise (repeatability of migration times<0.7% and of corrected-peak areas < 3.3%; n=6) and rugged analyses for the simultaneous determination of a pharmaceutical drug and its counter-ion.

Fatal intoxication with labetalol (Trandate).

The dead body of a 44-year-old woman, previously known for depression and alcoholism, has been discovered at her place of residence by her husband. A forensic autopsy has been carried out. The results indicated unspecific histological lesions (alveolar oedema, liver steatosis and interstitial nephritis) but did not reveal any apparent cause of death. Several boxes of medicines have been found near the body, justifying a toxicological analysis. This has been performed on peripheral blood and urine samples using liquid chromatography with diode array and mass spectrometric detections, in conjunction with gas chromatography coupled with mass spectrometry. Ethanol has been found (1.24 g/L in blood, 2.63 g/L in urine and 1.33 g/kg in gastric content), as well as therapeutic concentrations of meprobamate (14.1mg/L) and low concentrations of nordazepam (0.12 mg/L) in blood. On the other hand, particularly high levels of labetalol, a widely used beta-blocker, have been found both in blood (1.7 mg/L) and urine (20.2mg/L), which led us to measure labetalol levels in available viscera samples (liver, heart, kidney, and lung) and gastric content. Measured concentrations were 14.2 microg/g, 7.8 microg/g, 5.4 microg/g, 5.2 microg/g and 31.1 microg/g, respectively. We describe here the first report of a fatal intoxication attributed to labetalol that is linked to its acute toxicity, with tissue distribution of this beta-blocker.

Fluorescence optosensing implemented with sequential injection analysis: a novel strategy for the determination of labetalol.

The coupling of sequential injection analysis and optosensing has been developed for the first time. It has been applied to the determination of labetalol in both pharmaceuticals and urine samples, with the analytical signal (native fluorescence) being monitored directly on sensing zone microbeads. The solid support used was the nonionic silica gel C18, using 20% methanol-water (v:v) as a carrier. By using a 1.5-ml sample volume, we achieved a detection limit of 3.3 ng ml-1. This sensitivity allowed the determination of the compound in urine samples. A recovery study was carried out at the labetalol levels usually found in urine after pharmaceuticals administration, and recovery percentages close to 100% were obtained. The relative standard deviation was 3.4% for 100 ng ml-1 labetalol. No pretreatment was needed for urine samples, only an appropriate dilution, therefore minimizing the time required per sample analysis. In addition, the determination of the analyte was also carried out in one pharmaceutical, with a satisfactory result being obtained.

Combined use of an automated sample processor and a polymer-based high-performance liquid chromatographic column to determine the pharmacokinetics of labetalol in man.

An improved high-performance liquid chromatographic (HPLC) assay has been developed for the analysis of labetalol in human plasma. The method is based on the combined use of an automated sample processor, reversed-phase analysis on a microparticulate polymer-based HPLC column and fluorescence detection. The pH stability of the polymeric column packing material allowed the use of a mobile phase adjusted to pH 9.5, which was optimal for the fluorescence of labetalol. Assay validation was undertaken over the labetalol concentration range 2-100 ng/ml. Calibration curves were essentially linear, and the mean coefficient of variation was 5.3%. The assay has been used for the analysis of clinical samples in support of pharmacokinetic studies.

Paired-ion chromatography and high performance liquid chromatography of labetalol in feeds.

A high performance liquid chromatographic (HPLC) method using reverse phase paired-ion chromatography and ultraviolet detection at 280 nm has been developed to determine labetalol, an alpha and beta adrenoceptor blocking agent, in Purina No. 5001 rodent chow. The method is simple and rapid, and demonstrates a separation technique applicable to other acidic and basic drugs. It requires only extraction of the drug with methanol--water--acetic acid (66 + 33 + 1) and separation of insoluble material by filtration before HPLC. Labetalol, is chromatographically separated from soluble feed components by means of a microBondapak C18 column and methanol--water--acetic acid (66 + 33 + 1) mobile phase, 0.005M with respect to sodium dioctylsulfosuccinate paired-ion reagent. Average recovery is 98.7% with a relative standard deviation of +/- 2.3% for the equipment described.

Reversed-phase high-performance liquid chromatographic separation of the stereoisomers of labetalol via derivatization with chiral and non-chiral isothiocyanate reagents.

The antihypertensive agent labetalol is a mixture of two racemates. We report reversed-phase high-performance liquid chromatographic (HPLC) methodology for the separation of the four stereoisomers of labetalol via derivatization with the chiral reagent (4S-cis)-2,2-dimethyl-5-isothiocyanato-4-phenyl-1,3-dioxane. The derivatives were separated on octadecylsilane columns with a methanol-ammonium phosphate buffer mixture as mobile phase. Separations of the diastereomeric forms of labetalol were achieved with the non-chiral derivatizing reagents benzyl isothiocyanate and 1-naphthalenemethyl isothiocyanate. In all cases the derivatives of the R,S/S,R forms eluted before those of the R,R/S,S forms. Isothiocyanates may have general utility in stereoisomer separations of amines by HPLC.