Chiral separation of four phenothiazines by nonaqueous capillary electrophoresis and quality by design-based method development for quantification of dextromepromazine as chiral impurity of levomepromazine.

The separation of the enantiomers of mepromazine, promethazine, thioridazine and alimemazine was studied by nonaqueous capillary electrophoresis in the presence of cyclodextrins using 1 M acetic acid and 50 mM ammonium acetate in methanol as background electrolyte. Heptakis(2,3-di-O-acetyl-6-O-sulfo)-ß-cyclodextrin, heptakis(2,3-di-O-methyl-6-O-sulfo)-ß-cyclodextrin (HDMS-ß-CD) and octakis(2,3-di-O-methyl-6-O-sulfo)-γ-cyclodextrin were the most effective chiral selectors for mepromazine, promethazine and alimemazine. Subsequently, a method for the determination of dextromepromazine as chiral impurity of levomepromazine was developed employing quality by design principles. Using HDMS-ß-CD as selector, a fractional factorial resolution V+ design was employed for evaluating the knowledge space, while a central composite face centered design provided further method optimization and the basis for the computation of the design space by Monte Carlo simulations. The final experimental conditions included a 30/40.2 cm fused-silica capillary with 75 µm inner diameter and a background electrolyte composed of 0.75 M acetic acid and 55 mM ammonium acetate in methanol containing 27.5 mg/mL HDMS-ß-CD. The applied voltage was 22 kV and the capillary temperature was 15°C. Following method robustness testing via a Plackett-Burman design, the method was validated for dextromepromazine in the range of 0.01 to 3.0 % relative to a concentration of 0.74 mg/mL levomepromazine and applied to the analysis of reference standards of the European Pharmacopoeia and commercial tablets. The assay also allowed the detection of levomepromazine sulfoxide although the quantitation of the compound was hampered by the poor peak shape of the late migrating diastereomer.

Uncyclized xanthommatin is a key ommochrome intermediate in invertebrate coloration.

Ommochromes are widespread pigments that mediate multiple functions in invertebrates. The two main families of ommochromes are ommatins and ommins, which both originate from the kynurenine pathway but differ in their backbone, thereby in their coloration and function. Despite its broad significance, how the structural diversity of ommochromes arises in vivo has remained an open question since their first description. In this study, we combined organic synthesis, analytical chemistry and organelle purification to address this issue. From a set of synthesized ommatins, we derived a fragmentation pattern that helped elucidating the structure of new ommochromes. We identified uncyclized xanthommatin as the elusive biological intermediate that links the kynurenine pathway to the ommatin pathway within ommochromasomes, the ommochrome-producing organelles. Due to its unique structure, we propose that uncyclized xanthommatin functions as a key branching metabolite in the biosynthesis and structural diversification of ommatins and ommins, from insects to cephalopods.

Antioxidative Properties of Melanins and Ommochromes from Black Soldier Fly Hermetia illucens.

A comparative study of melanin and ommochrome-containing samples, isolated from the black soldier fly (BSF) by enzymatic hydrolysis, alkaline and acid alcohol extraction or by acid hydrolysis, was carried out. Melanin was isolated both as a melanin-chitin complex and as a water-soluble melanin. Acid hydrolysis followed by delipidization yielded a more concentrated melanin sample, the electron spin resonance (ESR) signal of which was 2.6 × 1018 spin/g. The ommochromes were extracted from the BSF eyes with acid methanol. The antiradical activity of BSF melanins and ommochromes was determined by the method of quenching of luminol chemiluminescence. It has been shown that delipidization of water-soluble melanin increases its antioxidant properties. A comparison of the antioxidant activity of BSF melanins and ommochromes in relation to photoinduced lipid peroxidation was carried out. The ESR characteristics of native and oxidized melanins and ommochromes were studied. It is assumed that H. illucens adult flies can be a useful source of natural pigments with antioxidant properties.

Enantioseparation of phenothiazines through capillary electrophoresis with solid phase extraction and polymer based stacking.

This study developed a sensitive method involving capillary electrophoresis (CE) coupled with ultraviolet absorption for the simultaneous separation of chiral phenothiazine drugs at nanomolar concentration levels. The method consists of hydroxypropyl-γ-cyclodextrin (Hp-γ-CD) as a chiral selector and poly (diallyldimethylammonium chloride) (PDDAC)-based CE. Five pairs of d,l-phenothiazines were baseline separated using a background electrolyte containing 0.9% PDDAC, 5 mM Hp-γ-CD, and 100 mM tris(hydroxymethyl)aminomethane (Tris)-formate (pH 3.0). The five pairs were successfully stacked on the basis of the difference in viscosity between the PDDAC-containing background electrolyte and the sample solution, with almost no loss of resolution. The combination of a solid-phase extraction and PDDAC-mediated CE can efficiently improve the sensitivity of the phenothiazine enantiomers. Under optimal conditions, calibration graphs displayed the linear range between 6 and 1500 nM, with relative standard deviation values lower than 3.5% (n = 5). Detection limit ranged from 2.1 to 6.3 nM for target analytes, and 607- to 1555-fold enhancement was achieved. The practicality of using the proposed method to determine five pairs of d,l-phenothiazines in urine is also validated, in which recoveries between recoveries of all phenothiazines from urine ranged from 89% to 101%.

Combination of poly(diallyldimethylammonium chloride) and hydroxypropyl-γ-cyclodextrin for high-speed enantioseparation of phenothiazines by capillary electrophoresis.

High-speed capillary electrophoresis (CE) enables the simple, rapid, and inexpensive analysis of large sets of chiral samples in the pharmaceutical industry. Hence, we developed a novel method for separating enantiomers of d,L-phenothiazines simply and rapidly, based on using poly(diallyldimethylammonium chloride) (PDDAC) as an additive and hydroxypropyl-γ-cyclodextrin (Hp-γ-CD) as a chiral selector in capillary electrophoresis. Adding 0.9% PDDAC to the background electrolyte generated a stable, high, and reversed electroosmotic flow (EOF). Hp-γ-CD not only worked as a complexing agent to increase the chiral resolution between d,L-phenothiazines but also decreased the effective electrophoretic mobility of these drugs. Combining PDDAC and Hp-γ-CD as buffer additives enabled CE to achieve a high-speed enantioseparation of five pairs of d,L-phenothiazines. A decrease in capillary length and an increase in the intensity of the electric field further shortened the separation time. When the background electrolyte contained 0.9% PDDAC, 5mM Hp-γ-CD, and 75 mM formic acid (pH 3.0), enantioseparation of the d,L-phenothiazines was attained within 230 s by applying a capillary length of 32.5 cm and an electric field of 292 V cm(-1). The limit of detection (LOD) of the d,L-phenothiazines at a signal-to-noise ratio of 3 ranged from 2 to 8 µM. We demonstrated the feasibility of this method by detecting the five pairs of d,L-phenothiazines in urine samples.

Enantioseparation of phenothiazines in CD-modified CZE using single isomer sulfated CD as a chiral selector.

Enantioseparations of five chiral phenothiazines in CD-modified CZE using the single isomer sulfate-substituted beta-CD (heptakis(2,3-dihydroxy-6-O-sulfo)-beta-CD, SI-S-beta-CD) and dual CD systems consisting of SI-S-beta-CD and a neutral CD as chiral selectors in a citrate buffer at pH 3.0 were investigated. The results indicate that SI-S-beta-CD is an excellent chiral selector for enantioseparation of promethazine. The enantiomers of trimeprazine were well separated, while those of ethopropazine could also be baseline-resolved with SI-S-beta-CD. With dual CD systems, especially with hydroxypropyl-beta-CD (HP-beta-CD) as neutral CD, the enantioselectivity of thioridazine and ethopropazine was considerably enhanced. Effective enantioseparation of phenothiazines, except for methotrimeprazine, could thus be favorably and simultaneously achieved. Moreover, reversal of the enantiomer migration order of ethopropazine and thioridazine occurred by varying the concentration of gamma-CD in the presence of SI-S-beta-CD. These phenomena may be attributable to the opposite effects of sulfated beta-CD and gamma-CD on the mobility of the enantiomers of ethopropazine and of thioridazine. Comparative studies on the enantioseparations of phenothiazines with single CD and dual CD systems containing SI-S-beta-CD and randomly sulfate-substituted beta-CD (MI-S-beta-CD) were made.

Enantioseparation of phenotiazines by affinity electrokinetic chromatography using human serum albumin as chiral selector: application to enantiomeric quality control in pharmaceutical formulations.

Nowadays, there is a special interest within the pharmaceutical laboratories to develop single enantiomer formulations and consequently a need for analytical methods to determine the enantiomeric purity of drugs. The present paper deals with the enantiomeric separation of promethazine and trimeprazine enantiomers by affinity electrokinetic chromatography (AEKC)-partial filling technique using human serum albumin (HSA) as chiral selector. A multivariate optimization of the most critical experimental variables in enantioresolution, running pH, HSA concentration and plug length, is carried out to obtain enantioresolution of promethazine and trimeprazine. The estimated maximum and optimum resolution of trimeprazine and prometazine enantiomers (Rs=1.74 and 2.01, respectively) corresponded to the following experimental conditions: pH 7.5; [HSA] 170 microM and plug length 190 s and pH 7.6; [HSA] 170 microM and plug length 170 s, for trimeprazine and prometazine, respectively. The developed methodologies were applied for the enantiomeric quality control of promethazine and trimeprazine enantiomers in commercially available pharmaceutical formulations. Resolution, accuracy, reproducibility, cost and sample throughput of the proposed methodologies make it suitable for quality control of the enantiomeric composition of promethazine and trimeprazine in pharmaceutical preparations.

Enantioseparation of phenothiazines in cyclodextrin-modified capillary zone electrophoresis using sulfated cyclodextrins as chiral selectors.

In this study, enantioseparations of five phenothiazines, including promethazine, ethopropazine, trimeprazine, methotrimeprazine, and thioridazine, in CD-modified CZE using dual CD systems consisting of randomly sulfate-substituted CD (MI-S-beta-CD) and a neutral CD as chiral selectors in a citrate buffer (100 mM) at pH 3.0 were investigated. The results indicate that MI-S-beta-CD is an excellent chiral selector for enantioseparation of ethopropazine. The enantiomers of promethazine can also be baseline-resolved with MI-S-beta-CD at concentrations in the range of 0.5-1.0% w/v. On the other hand, thioridazine and trimeprazine interact strongly with neutral CDs. As a result, the enantioselectivity of these two phenothiazines is remarkably and synergistically enhanced with increasing the concentration of neutral CDs in the presence of MI-S-beta-CD and simultaneous enantioseparations of these phenothiazines, except for methotrimeprazine, could favorably be achieved with the use of dual CD systems. Moreover, by varying the concentration of beta-CD or gamma-CD at a fixed concentration of MI-S-beta-CD (0.75% w/v) reversal of the enantiomer migration order of promethazine occurred. This may be attributable to the opposite effects of charged and neutral CDs on the mobility of the enantiomers of promethazine.

Development and validation of a capillary electrophoresis method for the determination of phenothiazines in human urine in the low nanogram per milliliter concentration range using field-amplified sample injection.

A capillary zone electrophoresis (CZE) method with ultraviolet-visible detection has been established and validated for the determination of five phenothiazines: thiazinamium methylsulfate, promazine hydrochloride, chlorpromazine hydrochloride, thioridazine hydrochloride, and promethazine hydrochloride in human urine. Optimum separation was obtained on a 64.5 cm x 75 microm bubble cell capillary using a buffer containing 150 mM tris(hydroxymethyl)aminomethane and 25% acetonitrile at pH 8.2, with temperature and voltage of 25 degrees C and 20 kV, respectively. Naphazoline hydrochloride was used as an internal standard. Field-amplified sample injection (FASI) has been applied to improve the sensitivity of the detection. Considering the influence of parameters affecting the on-line preconcentration (nature of preinjection plug, sample solvent composition, injection times, and injection voltage) and due to the significant interactions among them, in this paper we propose for the first time the application of a multivariate approach to carry out the study. The optimized conditions were as follows: preinjection plug of water for 7 s at 50 mbar, electrokinetic injection for 40 s at 6.2 kV, and 32 microm of H3PO4 in the sample solvent. Also, a solid-phase extraction (SPE) procedure is developed to obtain low detection limits and an adequate selectivity for urine samples. The combination of SPE and FASI-CZE-UV allows adequate linearities and recoveries, low detection limits (from 2 to 5 ng/mL), and satisfactory precisions (3.0-7.2% for an intermediate RSD %).

Electrophoretic separations of twelve phenothiazines and N-demethyl derivatives by using capillary zone electrophoresis and micellar electrokinetic chromatography with non ionic surfactant.

We focused our work on the separation of phenothiazines that are important drugs used for the treatment of psychic diseases. For a better understanding of the metabolism of these solutes, we wanted to separate not only a mixture of 12 phenothiazines but also a mixture containing phenothiazines and their N-demethyl metabolites by capillary electrophoresis. Separations in capillary zone electrophoresis were performed using 3 x 10(-2) mol/L H3PO4 (pH 2.5) but the obtained resolutions were not entirely satisfactory especially with regard to phenothiazine -N-demethyl derivative pairs. To improve the obtained results, we have performed separations by using micellar electrokinetic chromatography. In this approach, we used a running electrolyte containing 3 x 10(-2) mol/L H3PO4 electrolyte (pH 2.5) and octaethylene glycol monododecyl ether (C12E8) as neutral surfactant. By introducing 2 x 10(-3) mol/L C12E8 in the electrolyte, 11 out of 12 phenothiazines have been baseline separated. With respect to the separation of a mixture containing 3 phenothiazines and their 3 demethyl derivatives, we obtained an excellent separation by using a running electrolyte prepared with 7.5 x 10(-4) mol/L C12E8 and 3 x 10(-2) mol/L H3PO4.

Enantioseparation of phenothiazines in cyclodextrin-modified capillary zone electrophoresis: reversal of migration order.

Enantioseparations of phenothiazines with gamma-cyclodextrin (gamma-CD) as a chiral selector were investigated using citrate and phosphate buffer electrolytes at pH 3.0. Reversal of the enantiomer migration order of promethazine, ethopropazine, and trimeprazine was observed by varying gamma-CD concentration in the range of 5-9 mM, 2.5-4.5 mM and 1.5-2.8 mM, respectively, using 100 mM citrate buffer at pH 3.0. As in the case of beta-CD, the (+)-enantiomers of phenothiazines possess greater binding strength to gamma-CD than the (-)-enantiomers. The evaluation of the binding constants and limiting mobility of the complexes formed between the enantiomers of phenothiazines and gamma-CD reveals that the binding strength of phenothiazines to gamma-CD and the differences in the binding constants and limiting mobility of the complexes are responsible for the enantiomer migration reversal. Both the binding constants and limiting mobility of the complexes between the (+)-enantiomers of phenothiazine and gamma-CD are greater than those of the corresponding (-)-enantiomers in a citrate buffer, while the binding constants of the complexes primarily determined the migration order of the enantiomers in a phosphate buffer. Compared with the results obtained using a phosphate buffer, we may conclude that citrate buffer which involves competitive complexation with chiral selector plays a significant role in the enantiomer migration reversal.

Separation and migration behavior of structurally related phenothiazines in cyclodextrin-modified capillary zone electrophoresis.

The influences of buffer pH and the concentration of beta-cyclodextrins (beta-CDs) on the separation and migration behavior of 13 structurally related phenothiazines in CD-modified capillary zone electrophoresis (CD-CZE) using a phosphate background electrolyte at low pH were investigated. We focused on the separation of these phenothiazines, including the enantiomers of chiral analytes, with the use of beta-CD and hydroxypropyl-beta-CD (HP-beta-CD) as electrolyte modifiers or chiral selectors at concentrations less than 8 mM. The results indicate that the interactions of phenothiazines with beta-CDs are very strong and that effective separations of 13 analytes can be achieved with addition of 0.3 mM beta-CD or 0.5 mM HP-beta-CD in a phosphate buffer at pH 3.0. Binding constants of phenothiazines to beta-CDs were evaluated for a better understanding of the interactions of phenothiazines with beta-CDs.

Enantioseparation of phenothiazines in cyclodextrin-modified micellar electrokinetic chromatography.

In this study, enantioseparations of five phenothiazines in cyclodextrin (CD)-modified micellar electrokinetic chromatography (MEKC) were investigated using a citrate buffer containing tetradecyltrimethylammonium bromide (TTAB) as a cationic surfactant at low pH. Beta-cyclodextrin (beta-CD) and hydroxylpropyl-beta-CD (HP-beta-CD) were selected as chiral selectors. The results indicate that the separation window is greatly enlarged by beta-CD concentration and that the separability and selectivity of phenothiazines are remarkably influenced by the concentrations of both beta-CD and TTAB, as well as buffer pH. The interaction of thioridazine with beta-CDs is considerably reduced in the presence of TTAB micelles due to competitive complexation of thioridazine with TTAB micelles, which is pH-dependent. As a result, effective enantioseparation of thioridazine is simultaneously achievable with that of trimeprazine and promethazine or ethopropazine in MEKC with addition of either beta-CD or HP-beta-CD, respectively, to a micellar citrate buffer containing TTAB at pH 3.5. Better enantioresolution of thioridazine in MEKC than in capillary zone electrophoresis can be obtained.

Separation, conformation in solution and absolute configuration of ethopropazine enantiomers.

Enantiomers of ethopropazine x HCl (10-(2-diethylaminopropyl)phenothiazine hydrochloride) were prepared by fractional crystallization of diastereomeric dibenzoyltartaric acid salts, and their optical purity (enantiomeric excess, ee) determined by HPLC on Chiralcel OJ column. With a solvent mixture n-hexane/t-butanol/triethylamine (100:3:0.5) as eluent a very good enantioseparation (alpha = 1.68) for racemic ethopropazine was obtained. Enantiomeric purity for (-)-enantiomer was 99.1% and for (+)-enantiomer 97.9%. Combined data from NMR and CD spectra of both enantiomers, along with previously reported X-ray structure analyses of racemic ethopropazine, revealed skewed conformation of tricyclic system in solution, and (S)-configuration on the stereogenic center for (-)-enantiomer, and (R)-configuration for (+)-enantiomer.

Extractive-spectrophotometric determination of some phenothiazines with dipicrylamine and picric acid.

Dipicrylamine and picric acid have been tested as reagents for the determination of promethazine and perphenazine. They react in neutral media with these drugs forming the coloured compounds. The compounds are sparingly soluble in water and quantitatively extracted into organic solvents. The extracts are intensely coloured and very stable. These properties have been exploited for the extractive spectrophotometric determination of promethazine and perphenazine in pure solutions and pharmaceuticals. Linear calibration graphs were obtained in the concentration range 4-40, 3-30 microg ml(-1) of promethazine and 4-80, 8-60 microg ml(-1) of perphenazine for picric acid and dipicrylamine, respectively. The relative standard deviation (RSD) is less than 0.8%.

Tuning of the selectivity in capillary electrophoresis by cyclodextrins illustrated by the separation of some structurally related phenothiazines.

Cyclodextrins were used to affect the selectivity of the capillary electrophoresis system in the separation of 10 widely used phenothiazines. It was shown that the addition of cyclodextrins substantially improved the selectivity. The effect of temperature and cyclodextrin concentration was studied on the resolution between the screened phenothiazines. The best results were obtained with 8 mM hydroxypropyl-beta-cyclodextrin at 15.5 degrees C. Under these conditions, a resolution of at least 1.5 between all phenothiazines could be obtained. In addition, the chiral separation of the enantiomers of trimeprazine could be accomplished. Structure separability relations between the phenothiazines showed that a change in the side chain at the R10 position had the greatest effect on the migration.

Effect of organic modifiers on retention and enantiomeric separations by capillary electrophoresis with human serum albumin as a chiral selector in solution.

We have investigated the effect of methanol, ethanol, 1-propanol, 2-propanol and acetonitrile on the retention and enantiomeric separations of benzoin and propiomazine by capillary electrophoresis, using human serum albumin as a chiral selector. The effects of these modifiers on mobilities of analytes are rather difficult to interpret. Calculation of capacity factors reveals the underlying analyte-protein interactions; pitfalls in making such calculations are pointed out. In the case of benzoin and propiomazine binding to human serum albumin, capacity factors were observed to always decrease upon addition of organic modifiers, although the effects of 1- and 2-propanol suggest a possible specific interaction or modification of the protein conformation.

Development of a melanin-based high-performance liquid chromatography stationary phase and its use in the study of drug-melanin binding interactions.

Drug-melanin interactions were studied using a melanin-based HPLC column. Two approaches were chosen for the preparation of the stationary phases: covalent coupling of synthetic L-dopa melanin and in situ polymerization of L-dopa. Retention of a series of phenothiazines on melanin-based stationary phases was attributed to binding to melanin. Frontal affinity chromatography experiments on one melanin-based column allowed us to calculate the affinity and binding capacity of chlorpromazine and promethazine. A competition was observed between chlorpromazine and haloperidol which was qualitatively consistent with previously described results. Data indicated that the interaction was not a simple competition at one site.

Identification of ommin in the integument of the silkworm, Bombyx mori.

An ommin was isolated from the integument of the quail mutant of the silkworm, Bombyx mori, using column chromatography on SP-Sephadex and cellulose powder. As a standard, ommin was isolated from diapause eggs of the normal silkworm. The red pigment from the larval integument of quail mutants was identical to the standard compound with respect to absorption spectrum, infrared spectrum and RF values in thin-layer chromatography (TLC). After acid hydrolysis of the pigment, 3-hydroxykynurenine was detected by TLC. This is the first report of an ommin in a lepidopteran larval integument.

Purification and properties of an ommochrome-binding protein from the hemolymph of the tobacco hornworm, Manduca sexta.

A yellow-colored protein (YCP) was isolated from the hemolymph (i.e. blood) of fifth instar wandering stage larvae of Manduca sexta. The molecular mass of YCP was 31 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration chromatography suggested that native YCP was a monomer. The absorbance spectrum of YCP revealed maxima at 278 and 405 nm. Chromophore was released from YCP through denaturation of the protein with methanol and chloroform. In neutral solution and in acid, the released chromophore showed the absorbance characteristics of an ommochrome: ommatin D. In addition, the chromophore was sensitive to treatment with arylsulfatase as would be expected for ommatin D. The amino acid composition and the N-terminal sequence of YCP were determined. The YCP polypeptide chain was found to be glycosylated. Carbohydrate analysis suggested that Man and GlcNAc were present in a 3:1 ratio. Circular dichroism indicated that YCP consisted of 68% beta-pleated sheet with no alpha-helices being detected. An in vitro incubation of larval fat body in the presence of [35S]methionine indicated that this organ was the site of synthesis. Ommochromes arise in insects as end products of the metabolism of tryptophan. It is well-documented that ommochromes occur in both the tissues and the excreta of insects. We propose that in M. sexta, one such tryptophan metabolite is found in the hemolymph associated with a specific protein.