Removal of endocrine disrupting compounds in a lab-scale anaerobic/aerobic sequencing batch reactor unit.

The fate and removal of six selected endocrine disrupting compounds in a lab-scale anaerobic/aerobic (A/O) sequencing batch reactor (SBR), operating at 5 days, solids retention time (SRT) were investigated. A carbamazepine (CBZ), acetaminophen (ATP), diltiazem (DTZ), butyl benzyl phthalate (BBP), estrone and progesterone mix was spiked as model endocrine disrupting compounds (EDC) into domestic wastewater obtained from a nearby sewage treatment plant. The influent, effluent and sludge samples from the SBR unit were analysed by using an LC/MS/MS instrument equipped with electrospray ionization. More than 80% removal was observed for all the EDCs tested. It was found that biodegradation is the most important mechanism for BBP, ATP and progesterone. Biodegradation constants were calculated according to the simplified Monod model for these compounds. The DTZ seemed to have lower rate of biodegradation. The CBZ appeared totally resistant to biodegradation. However, it presented a high rate of sorption onto the sludge and was thereby treated. This contradicts with the literature studies.

New trend in the LC separation analysis of pharmaceuticals--high performance separation by ultra high-performance liquid chromatography (UHPLC) with core-shell particle C18 columns--.

This article presents a mini-review of the recent results in the ultra high-performance liquid chromatography (UHPLC) separation of pharmaceuticals by our group. High performance UHPLC separation employing core-shell particle C18 columns was demonstrated. High performance (high theoretical plate number of approximately 20000/10 cm, low theoretical plate height of 5 µm) was obtained without any specific devices in the conventional HPLC apparatus, only through changing detector sampling times and the inner diameter of the connecting tube. High theoretical plate numbers with low column back pressure obtained by the core-shell particle columns enabled fast separation of the analytes. Methanol, which gives high column pressure drops in the reversed-phase mode HPLC compared with acetonitrile, can be used without any trouble. One analysis of the purity testing of diltiazem hydrochloride was performed within 100 s. One analysis in the photostability testing of mecobalamin (vitamin B12 analogue) was successful within 180 s.

Liquid-liquid microextraction without phase separation in a multicommuted flow system for diltiazem determination in pharmaceuticals.

Liquid-liquid microextraction without phase segmentation was implemented in a multicommuted flow system for determination of the anti-hypertensive diltiazem. The procedure was based on ion pair formation between the drug and the dye bromothymol blue at pH 3.5. The detection was performed without phase separation in a glass tube coupled to a fiber-optics spectrophotometer. The total volume of chloroform was reduced to 50 µL in comparison with 10 mL consumed in batch. A linear response was observed between 9 and 120 µmol L(-1), with a detection limit of 0.9 µmol L(-1) (99.7% confidence level). The coefficient of variation (n=10), sampling rate and extraction efficiency were estimated as 0.6%, 78 determinations per hour and 61%, respectively. About 30 µg of bromothymol blue was consumed and the waste volume was 380 µL per determination. The results for pharmaceutical samples agreed with those obtained by the reference procedure at the 95% confidence level.

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.

Simultaneous enantioseparation of cis-diltiazem hydrochloride and its metabolite cis-desacetyldiltiazem using high-performance liquid chromatography and capillary electrophoresis.

Two alternative methods were developed using high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) for a simultaneous enantioselective determination of Ca(2+) blocking chiral drug cis-diltiazem hydrochloride (DLT) and its degradation product and metabolite cis-desacetyldiltiazem (Desac-DLT). The methods were compared from the viewpoint of analytical performance. The identification of HPLC peaks were performed using off-line electrospray ionization mass spectrometry. In addition, CE method was evaluated for quantitative determination of minor enantiomeric impurities in pharmacologically active (+)-cis-DLT.

Enantioseparation using selected polysaccharides as chiral buffer additives in capillary electrophoresis.

Selected water-soluble, native polysaccharides--such as amylose, laminaran, pullulan--and derivatized polysaccharides--methyl cellulose, hydroxypropyl cellulose, and carboxymethyl amylose sodium salt (CM-Am)--were investigated as chiral selectors in capillary electrophoresis. Effects of degree of polymerization and concentration of amylose on the separation of enantiomers of 1,1'-binaphthyl-2,2'-diyl hydrogen phosphate (BDHP) and a chiral cardiovascular drug cis-diltiazem were also studied. Pullulan and amyloses used in this study showed the same migration order for enantiomers of BDHP. In contrast, the migration order of BDHP enantiomers for cellulose derivatives and luminaran as well as with beta-cyclodextrin was opposite to that for amylose and pullulan. The enantioseparation of several chiral drugs was also performed using high-molecular-mass amylose (Am-4900) and CM-Am.

Optical resolution of a 1,5-benzothiazepine derivative, a synthetic intermediate of diltiazem, by preferential crystallization and diastereomeric salt formation.

Practical preparation methods of an optically active intermediate of diltiazem, (+)-(2S,3S)-5-[2-(dimethylamino)ethyl]-2,3-dihydro-3-hydroxy- 2-(4-methoxyphenyl)-1,5-benzothiazepin-4(5H)-one [(+)-7], have been developed by the use of physicochemical and chemical resolutions. 1) The salt of (+/-)-7 with 3-amino-4-hydroxy-benzenesulfonic acid (AHS), was found to exist as a conglomerate and could be reproducibly resolved into (+)-7.AHS and (-)-7.AHS of 94-98% ee by a preferential crystallization procedure. 2) (+)-(1R)-3-Bromocamphor-9-sulfonic acid [(+)-BCS] was found to be an efficient resolving agent for (+/-)-7 and the diastereomeric resolution provided (+)-7.(+)-BCS.2H2O salt in > 43% yield and > 97% ee by fractional crystallization. It is presumed that the crystal water of (+)-7.(+)-BCS.2H2O plays an important role in the selective crystallization during this efficient resolution.

Enantiomer separation of basic drugs by capillary electrophoresis using ionic and neutral polysaccharides as chiral selectors.

The enantiomer separation of basic drugs by capillary electrophoresis was investigated employing two types of polysaccharides as chiral selectors. One type consists of electrically neutral polysaccharides such as dextran and dextrin, in which the separation mode is capillary zone electrophoresis (CZE), and ionic drugs are suitable for this mode. The other consists of ionic polysaccharides such as chondroitin sulfate C. Chondroitin sulfates are known as mucopolysaccharides and natural components, being charged, linear, sulfated polysaccharides of high mass. Therefore, the latter approach can be classified as affinity electrokinetic chromatography (AEKC). Both ionic and neutral drugs can be separated by AEKC owing to its separation principle. The separation of enantiomers of some basic drugs such as diltiazem and trimetoquinol was investigated with both CZE and AEKC employing polysaccharides as mentioned above and the enantioselectivity was compared. A brief mechanism of enantiorecognition by polysaccharides is also described.

Chromatographic (TLC) analysis of diltiazem in pharmaceutical form in presence of other antiarrhythmics.

Diltiazem was identified by TLC method on silica gel by ascending technique, in presence of five another antiarrhythmic drugs (disopyramide, flecainide, lidocaine, lorcainide, procainamide). Suitable conditions for separation of diltiazem were established using ethanol-benzene-dioxane-ammonia (2:20:16:3) as the mobile phase. When using acidified iodoplatinate solution or Dragendorff reagent as indicators, the amount of diltiazem as small as 100 ng can be identified. The TLC method is simple and sensitive and it was used to identify diltiazem in tablets.

Enantiomeric separation of diltiazem, clentiazem, and its related compounds by capillary electrophoresis using polysaccharides.

The direct separation of the enantiomers of diltiazem and its three chloro derivatives, including clentiazem, was investigated by capillary zone electrophoresis (CZE) and electrokinetic chromatography (EKC) using polysaccharides. An electrically neutral beta-cyclodextrin polymer, dextran sulfate, and mucopolysaccharides, such as heparin and chondroitin sulfate C, were employed as chiral selectors. Among the tested solutes, 8-chlorodiltiazem (clentiazem) was well enantiorecognized by the methods. Affinity electrokinetic chromatography (AEKC) using chondroitin sulfate C was the most successful; all tested solutes were completely enantioseparated by this mode.

Investigation and optimisation of the use of micellar electrokinetic chromatography for the analysis of six cardiovascular drugs.

A micellar electrokinetic chromatography method was optimised for the separation of the six cardiovascular drugs atenolol, nicardipine, nifedipine, diltiazem, verapamil, and amlodipine by investigating the effects of pH, sodium dodecyl sulphate (SDS) concentration, selection and concentration of organic modifier. An electrophoresis buffer of 100 mM borate pH 8.1 containing 50 mM SDS and 15% (v/v) acetone was found to provide the optimum separation with respect to resolution and migration time.

Automatic determination of diltiazem and desacetyldiltiazem in human plasma using liquid-solid extraction on disposable cartridges coupled to HPLC--Part I: Optimization of the HPLC system and method validation.

A sensitive and automated method for the analysis of diltiazem and desacetyldiltiazem in plasma has been developed using liquid-solid extraction (LSE) on disposable extraction cartridges (DECs) in combination with HPLC. After isolation from plasma, the analytes are separated on a highly deactivated octyl silica column with a mobile phase of methanol-0.05 M phosphate buffer (pH 7.4) (62:38, v/v). The analytes are monitored photometrically at 238 nm. The complete preparation of the plasma sample as well as the injection of the final extract on to the analytical column are performed automatically by means of a sample processor equipped with a robotic arm to which is attached a needle dispensing the different liquids. The internal standard solution is first added to the plasma sample. The DEC is then conditioned successively with methanol and phosphate buffer (pH 7.4). A 1.0-ml volume of sample containing the internal standard solution is applied on an extraction cartridge filled with cyanopropyl silica (50 mg). After the DEC has been washed with the same buffer, the analytes are eluted with 0.16 ml of methanol. A 0.14-ml volume of buffer is then passed through the DEC and 0.25 ml of the final extract is injected onto the HPLC column. The absolute recoveries of the drugs are about 90% and the limit of detection for diltiazem is 0.8 ng ml-1. Relative standard deviations of 2.6% (within-day) and 3.7% (between-day) have been obtained for this compound at a plasma concentration of 50 ng ml-1.

Automatic determination of diltiazem and desacetyldiltiazem in human plasma using liquid-solid extraction on disposable cartridges coupled to HPLC--Part II: Optimization of liquid-solid extraction.

An automatic liquid-solid extraction (LSE) procedure to be coupled to HPLC for the determination of diltiazem and desacetyldiltiazem in plasma has been developed. The LSE operations are performed on disposable extraction cartridges (DECs) by means of a sample processor equipped with a robotic arm holding a needle through which the different liquids are dispensed. The operating parameters of LSE have been optimized with respect to recovery, detectability and reproducibility by using, whenever possible, aqueous solutions of the analytes. Different kinds of DECs have been tested. For the compounds studied, DECs filled with 50 mg of cyanopropyl silica have been selected. The influence of the pH of the buffer used in the washing step has been studied, leading finally to the selection of the same phosphate buffer (pH 7.4) as in the HPLC mobile phase. The minimum volume of methanol which still gives a nearly complete elution of the analytes from the extraction cartridges has been determined. Under these conditions, a high sensitivity can be obtained without an evaporation step. Moreover, the volume of buffer to be added to the methanolic eluate before injection into the HPLC system has been optimized in such a way that a focusing effect is obtained at the top of the analytical column while the dilution of the extract is minimized.