Analysis of diltiazem and its related substances by HPLC and HPLC/MS.

Diltiazem (DTZ) is an optically active calcium channel blocker having a benzodiazepine structure. The drug used in therapy is (+)-cis-diltiazem with configuration (2S,3S). To describe the analytical profile of DTZ different stationary phases (RP-18, RP-8, monolithic support) were tested. The best separation of DTZ from A, B, E and F was obtained using as stationary phase a RP-8 or a monolithic RP-18. The characterization of impurities was carried out using two analytical systems, HPLC and HPLC/MS.

Determination of the binding of a beta 2-blocker drug, frusemide and ceftriaxone to serum proteins by capillary zone electrophoresis.

A modified Hummel-Dreyer method was used to study the binding of drugs with serum proteins by high performance capillary electrophoresis. The study was carried out to check the possible interaction between serum proteins and a highly selective beta 2-blocker, ICI 118551 (ICI). To prove the suitability of the method the protein binding of frusemide and ceftriaxone, drugs previously investigated, was also studied. The analyses were carried out by injecting a solution of s alpha(1)-acidic glycoprotein (alpha(1)-AGP) or human serum albumin in 70 mM NaH2PO4/Na2HPO4(pH 7.4) buffer into an uncoated fused silica capillary filled with the same buffer. In the capillary, maintained at a working temperature of 35 degrees C, a known amount of the ICI, frusemide or ceftriaxone was added. The method allows the bound drug to be determined directly.

Determination of MPTP, a toxic impurity of pethidine.

Pethidine, predominantly a mu-receptor agonist, is a phenyl-piperidinic synthetic drug. It is used in the management of moderate to several pain. A possible hydrolytic degradation of an ester group can generate a very toxic compound, the N-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) which contaminates the drug. Because of the toxicity of MPTP a suitable method for its determination must be selective and sensitive. Afterwards we propose simple methods to determine pethidine and MPTP by capillary electrophoresis (CE), MECK and RP-high performance liquid chromatography (HPLC) looking at the limit of detection obtained using these three techniques. CE was carried out using as running buffer ammonium acetate (pH 8.3). MECK was performed with a borate buffer (pH 8.3) containing sodium dodecylsulphate and trimethyl-beta-cyclodextrins. RP-HPLC was carried out on a RP18 stationary phase, using as mobile phase a mixture of phosphate buffer (pH 7) containing acetonitrile and 1% of diethylamine.

Enantiomeric separation of substituted flavanoids by LC-DAD.

A variety of racemic flavanoids with anti-rhinovirus activity have been resolved for the first time by HPLC, using a chiral stationary phase. Baseline separation was easily obtained for racemic 4',6-dichloroisoflavan (V). The absolute configurations of two enantiomers (Va and Vb) were established by comparing their circular dichroism curves with those of other known isoflavans. Both the isomers were tested against human rhinovirus serotype 1B infection in vitro; the S form was approximately four times more effective than the R form.

Human alpha1-glycoprotein acid as chiral selector in the enantioseparation of midodrine and deglymidodrine racemates by HPLC.

Human alpha1-acid glycoprotein (alpha1-AGP) has been used as a chiral stationary phase (CSP) for the enantioseparation of midodrine and deglymidodrine racemates in the same HPLC run. The imobilized AGP resulted as the best chiral selector for the enantioresolution of two compounds. Due to the modification of alpha1-AGP characters as a result of changing the composition of the mobile phase, an attempt study of the watery mobile phase (ionic strength and pH of the buffer, nature and concentration of the organic modifier) allowed for an increase in the enantioselectivity of the chromatographic system and an optimization of the resolution base-line of both enantiomeric pairs.

Determination of silymarine in the extract from the dried silybum marianum fruits by high performance liquid chromatography and capillary electrophoresis.

Silybine (SBN), isosilybine (ISBN), silycristine (SCN), silydianine (SDN), and taxifoline (TXF) are the main active flavanoids, generally found in the dried fruits of silybum marianum. The concentrations of these compounds, excepted TXF, are all together usually expressed as silymarine content. In this paper the determination of the silymarine titre was made by high performance liquid chromatography (HPLC), and high performance capillary electrophoresis (HPCE). Two reversed stationary phases, RP-18 and RP-8, were observed comparing the resolutions of all considered flavanoids with each stationary phases. The HPCE was carried out considering the possible improvement in the resolution of SBN, CN, SDN and TXF using, 8-cyclodextrines or organic modifier. The qualitative and quantitative data obtained by HPLC and HPCE were compared.

Analysis of ticlopidine and related impurities by capillary electrophoresis.

Capillary electrophoresis has been used to analyse ticlopidine and its main impurities in bulk material. The minimum detectable amount of impurities was determined and the electrophoretic conditions used were discussed. The proposed method is also suitable to analyse the drug in plasma.

Analysis of non-benzodiazepinic anxiolytic agents by capillary zone electrophoresis.

A simple capillary electrophoretic method was developed for the analysis of a new generation of serotonergic anxiolytics and their related substances: zalospirone, gepirone, ipsapirone and buspirone. All compounds run in a Tris/phosphate buffer at pH 3 as cations and the experimental conditions allowed good resolution of four drugs and their principal impurities. The analyses were made using two different kinds of capillary. The suitability of CZE and HPLC methods for the analysis of these non-benzodiazepinic anxiolytic agents and their impurities was compared.

The indirect UV detection in the analysis of ursodeoxycholic acid and related compounds by HPCE.

A high-performance capillary zone electrophoretic (HPCE) assay has been developed for the determination of ursodeoxycholic acid (UDCA) and its usual impurities. Considering the low molecular absorptivity of UDCA and its related compounds indirect UV detection was used. The electrophoretic capillary was filled with a background electrolyte (BGE) containing an UV absorbing ion: benzoic acid (BA) or 5,5-diethylbarbituric acid (DBA). To enhance the selectivity of the assay diimethyl-beta-cyclodextrines (D-beta-CDs) or trimethyl-beta-cyclodextrines (T-beta-CDs) have been added to the running buffer together with methylcellulose or urea. All considered impurities were well resolved with two buffers studied, with the exception of methylursodehoxycholate, a neutral compound.

LTB4 as marker of 5-LO inhibitory activity of two new N-omega-ethoxycarbonyl-4-quinolones.

The supposed 5-LO inhibitory activity of two N-omega-ethoxycarbonyl-4-quinolones was tested determining leukotriene B4 (LTB4) in RBL-1 cell cultures, pretreated with the two compounds of interest. LTB4, obtained by solid-phase extraction (SPE) from cell cultures supernatants, was determined by micellar electrokinetic chromatography (MEKC). The analysis was performed using an uncoated capillary, filled with borate buffer at pH 8.3, containing 12.5 mM SDS as micelles generator. Therefore, following the decreasing of LTB4 it was possible to verify the 5-LO inhibitory activity of two quinolone derivatives. To asses the suitability of the use of LTB4 as marker of the activity of the new compounds, the analysis was repeated using quercetin, a well known 5-LO inhibitor.

Determination of optical purity by nonenantioselective LC using CD detection.

The use of a circular dichroism (CD) based HPLC detection system was recently described by some authors and proposed for a nonenantioselective HPLC enantiomeric purity determination. Indeed the system, measuring both CD and UV signals simultaneously, allows the evaluation of the g anisotropy factor. In order to experimentally support such an analytical procedure as an alternative to the enantioselective chromatographic method currently found in some pharmacopoeial monographs, we have studied its application to the analysis of dexchlorpheniramine maleate, an active substance which exhibits a poor CD signal in the 250-270 nm spectral region with a g value of the order of 10(-4). The results reported indicate that the suitability of the studied procedure for the enantiomeric purity determination is obtained only when the CD-detector reaches high stability; indeed a certain time lag is systematically necessary to obtain stable responses, i.e. adequate precision. The enantioselective HPLC procedure seems to be more precise for enantiomeric purity values < or = 2% than the CD based detection system; such a disadvantage might be counterbalanced by the use of non chiral stationary phases.

Determination of methyl bromide traces in some food products by HS-GC/MS.

A simple and rapid method has been developed for fast selective determination of residues of methyl bromide in food products. The method is based on the use of a GC/MS computer-aided system equipped with a headspace. Analysis of methyl bromide was carried out directly on the food products without preliminary extraction of fumigant. An MSD quadrupole mass spectrometer with data processing allowed rapid identification of the separated volatile compound in the samples. The technique of focusing on characteristic single ion monitoring for quantification of methyl bromide, compared with chromatographic peak area measurement, turned out to have a good degree of accuracy and sensitivity. The use of known amount of added methyl bromide allowed better evaluation of traces of this compound. The proposed method was applied to the analysis of methyl bromide residues in alimentary pastes, white flour, rice, hazelnuts, peanuts and dried mushrooms (boletus).

Chiral separations with coated and uncoated capillaries.

Chiral resolution depends on the time the two enantiomers have for active migration within the capillary. Consequently, the magnitude of the electroosmotic flow (EOF) has a paramount influence on resolution, especially for chiral acidic compounds (counterosmotic migration). In this case, when the mobility of the analytes relative to that of the EOF is known, a rule of thumb can be deduced: Coated capillaries with suppressed EOF should be used when the effective mobility of the analyte is less than 50% of that of the EOF. Uncoated capillaries give better resolution when the effective mobility of the analyte is higher than 50% of that of the EOF because of the longer effective migration distance. The efficiencies achieved are mainly a function of the time the analytes spend in the capillary when solute-wall interactions can be neglected.

Remifentanil for cesarean section under general anesthesia: effects on maternal stress hormone secretion and neonatal well-being: a randomized trial.

BACKGROUND: Remifentanil may attenuate maternal hemodynamic response during cesarean section under general anesthesia, but could cause transient but significant neonatal depression. We investigated the effect of low-dose remifentanil on maternal neuroendocrine response and fetal wellbeing. METHODS: Forty-two ASA I-II parturients undergoing cesarean section at term under general anesthesia were randomized to receive either fentanyl after delivery (n=21, group C) or remifentanil bolus 0.5 microg/kg before induction followed by a continuous infusion at 0.15 microg x kg(-1)min(-1) until peritoneal incision, then restarted after delivery (n=21, group R). Maternal heart rate and blood pressure, and epinephrine, norepinephrine, adrenocorticotropic hormone (ACTH), and growth hormone levels were measured at baseline, uterine incision, and the end of surgery. Remifentanil was measured in maternal and umbilical arterial and venous blood. One- and 5-minute Apgar scores and umbilical arterial and venous pH were recorded. RESULTS: ACTH was significantly higher in group C at uterine incision (P<0.01). No significant differences were observed in hemodynamics, catecholamines or growth hormone. Apgar scores at 1 (P<0.05) and 5 min (P<0.01) were significantly higher in group C. Mean umbilical pH values were within normal range but significantly higher in group C. Three neonates in group R required intubation but recovered at 5 min without naloxone. Mean+/-SD maternal remifentanil concentration was 1.67+/-1.04 ng/mL. CONCLUSIONS: Remifentanil administration before peritoneal incision partially reduced the hormonal stress response. Maternal benefits must be weighed against transitory but significant neonatal respiratory depression. Neonatal resuscitation facilities are mandatory when remifentanil is used.

Determination of the maximal carcinoma/normal skin ratio after HpD or m-THPC administration in Hairless mice (SKH-1) by fluorescence spectroscopy.

The two major steps in our study on the treatment of skin carcinomas by photochemotherapy (PCT) were the development of a skin tumor model in Hairless mice by a chemical carcinogenesis and the use of fluorescence spectroscopy, a semi-quantitative and non-invasive method, in order to determine the time after i.p. injection of photosensitizer when the tumor/normal skin ratio was the highest. A three-step carcinogenesis protocol provided mice bearing carcinomas and these were used to determine the tumor/normal skin ratios of two photosensitizers by fluorescence spectroscopy. Hematoporphyrin derivative (HpD) (5 mg/kg body weight) and m-tetra(hydroxyphenyl) chlorine (m-THPC) (0.3 mg/kg body weight) were injected i.p., and fluorescence was measured at 1, 4, 8, 12, 24, 48, 72 and 96 h after injection. The best carcinoma/normal skin ratio would be 3.2+/-1.4 for HpD and 2.7+/-2.1 for m-THPC, respectively. The delays required to reach these ratios were 72 h for HpD and 24 h for m-THPC. These results have to be considered with caution due to the high SEs and they must be confirmed by organic extraction. Photodynamic therapy with the same doses of HpD and m-THPC used in this pharmacokinetic study has to be carried out in order to compare the toxicities of the two photosensitizers and to determine which one is the best for this type of tumor.

Enatioseparation and anti-rhinovirus activity of 3-benzylchroman-4-ones.

In a series of homo-isoflavonoids, chloro-substituted rac-3-benzylchroman-4-ones (3 d-f) showed an antiviral in vitro activity against selected picornaviruses. In order to study the anti-rhinovirus activity of each stereoisomer, racemic mixtures of 3 d and 3 e were successfully resolved by high-performance liquid chromatography, using a Whelk-O 1 column as chiral stationary phase. The CD spectra confirm that the two eluates of each compound are enantiomers but do not allow the assignment of their absolute configurations. The antiviral activity of the isomers and their racemates was tested in vitro against human rhinovirus serotype 1B and 14 infection, by means of the plaque reduction assay. All homoisoflavonoids tested exhibited an inhibitory effect on rhinovirus replication with an activity depending on virus serotype and compound. The two enantiomers of each compound and the corresponding racemate were equipotent, clearly showing that the configuration of the chiral center in position 3 does not influence the activity against both rhinovirus serotypes.

Determination of the maximal tumor/normal skin ratio after HpD or m-THPC administration in hairless mouse (SKh-1) by fluorescence spectroscopy--a non-invasive method.

Two major steps in our study on the treatment of skin tumors by photochemotherapy (PCT) were the development of a skin tumor model in hairless mice by chemical carcinogenesis and by the use fluorescence spectroscopy, a semi-quantitative and non-invasive method, to determine the time after i.p. injection of photosensitizer when the tumor/normal skin ratio is the highest. Carcinogenesis provided mice bearing many benign papillomas and these were used to determine the tumor/normal skin ratios of two photosensitizers by fluorescence spectroscopy. Hematoporphyrin derivative (HpD) (5 mg/kg body weight) and m-tetra(hydroxyphenyl)-chlorin (m-THPC) (0.3 mg/kg body weight) were injected, and fluorescence measured at 4, 8, 24, 48, 72 and 96 h after injection. The tumor/normal skin ratio was 6.2 for HpD and 5.1 for m-THPC. The times required to reach these ratios were 48 h for HpD and 72 h for m-THPC. Published reports indicate that m-THPC gives a much higher tumor/normal skin ratio than HpD. These results must be confirmed by organic extraction. Photodynamic therapy with the same doses of HpD and m-THPC used in this pharmacokinetic study must also be carried out to compare the toxicities of the two photosensitizers and to determine which is best for this type of tumor.