Chemical ionization mass spectrometry of doxylamine and related compounds

The chemical ionization mass spectrometric (CIMS) analysis of doxylamine, N,N-dimethyl-2-[1-phenyl-1-(2-pyridinyl)ethoxy]ethanamine, and related compounds, using both ammonia and methane as reagent gases, is discussed. The two reagent gases did not produce the same major fragment ion for doxylamine. Mechanisms for the fragmentation of doxylamine under either ammonia or methane CIMS conditions are proposed. The mechanisms explain the observation of an m/z 182 fragment ion for doxylamine analyzed under methane CIMS conditions and an m/z 184 product ion detected under ammonia CIMS conditions.     

Chromatographic behavior of doxylamine succinate, phenylpropanolamine hydrochloride, chlorpheniramine maleate, dextromethorphan hydrobromide, paracetamol, and guaifenesin in ion pair reverse-phase high performance liquid chromatography

A study of the chromatographic behavior of some cough syrup ingredients has led to the optimum chromatographic separation of four ingredients (doxylamine succinate, phenylpropanolamine hydrochloride, chlorpheniramine maleate, and dextromethorphan hydrobromide). The paracetamol and guaifenesin were overlapping under all chromatographic conditions. The application of 1% chlorotrimethylsilane in methanol to the column (Partisil 5 CCS/C₈) was found to improve the column efficiency significantly. This separation can be applied for the analysis of cough syrup for these ingredients after a study of the interferences due to normal excipients.²     

GC/MS characterization of urinary metabolites of doxylamine succinate: Identification of the aglycones formed from intestinal microflora metabolism of the polar glucuronide metabolites

This study describes the use of high performance liquid chromatography (HPLC) and capillary gas chromatography/mass spectrometry (GC/MS) in the characterization of polar glucuronide conjugates of doxylamine and their subsequent aglycones following deconjugation. Rat urinary extracts which contained doxylamine and both nonconjugated and conjugated doxylamine metabolites, were examined by HPLC before and after incubation with rat intestinal microflora. The subsequent deconjugated urinary metabolites and the nonconjugated products remaining in the urinary extracts were then isolated, acetylated, and assayed by GC/MS. Incubation with the intestinal microflora indicated that anaerobic bacteria were capable of effecting hydrolytic cleavage of these polar O-glucuronide metabolites of doxylamine and its demethylated products to their subsequent aglycones. GC/MS analysis was performed using a fused silica DB-5 GC column and was utilized for the identification of these deconjugated metabolites.     

Optimization using central composite design for antihistamines separation by nonaqueous capillary electrophoresis with electrochemical and electrochemiluminescence detections

The first detailed examination of non-aqueous capillary electrophoresis with electrochemical and electrochemiluminescence detections (NACE-EC/ECL), separation parameters and their interactions via central composite design was presented. This concept was demonstrated by examining the optimization separation conditions of seven antihistamines (chlorpheniramine, cyproheptadine, diphenhydramine, doxylamine, methapyrilene, terfenadine, and triprolidine) by NACE-EC/ECL. To evaluate the NACE separation quality, the chromatography resolution statistic function (CRS(-1) function) with regard to the resolution and migration time was established as the response variable. The influences of three experimental variables (buffer apparent pH value (pH*), buffer (TBAP) concentration, and separation voltage) on the response were investigated. A set of optimal conditions was obtained from central composite design: 9.2 mM TBAP in ACN (pH* 4.0) and voltage (12.7 kV), and under these optimum conditions, the seven antihistamines could be well separated in less than 10 min. The obtained electropherograms indicated that the dual EC/ECL detection system was indispensable since the six antihistamines (except for triprolidine) displayed both EC and ECL response, whereas triprolidine only displayed the EC response. This work is instructive for investigators in simplifying the NACE-EC/ECL development procedures for multi-component analysis.     

Separation of pharmacologically active nitrogen-containing compounds on silica gels modified with 6,10-ionene,dextran sulfate,and gold nanoparticles

New stationary phases for HPLC are obtained via layer-by-layer deposition of polyelectrolytes and studied: (1) silica gel modified layer-by-layer with 6,10-ionene and dextran sulfate (Sorbent 1); (2) silica gel twice subjected to the above modification (Sorbent 2); and (3) silica gel modified with 6,10-ionene, gold nanoparticles, and dextran sulfate (Sorbent 3). The effect the content of the organic solvent in the mobile phase and the concentration and pH of the buffer solution have on the chromatographic behavior of several pharmacologically active nitrogen-containing compounds is studied. The sorbents are stable during the process and allow the effective separation of beta-blockers, calcium channel blockers, alpha-agonists, and antihistamines. A mixture of caffeine, nadolol, tetrahydrozoline, pindolol, orphenadrine, doxylamine, carbinoxamine, and chlorphenamine is separated in 6.5 min on the silica gel modified with 6,10-ionene, gold nanoparticles, and dextran sulfate.

     

Development and validation of a generic RP‐HPLC PDA method for the simultaneous separation and quantification of active ingredients in cold and cough medicines

A novel generic reverse phase high performance liquid chromatography (RP‐HPLC) method is developed and validated for simultaneous determination of seven pharmaceutically active ingredients, namely, acetaminophen, dextromethorphan, doxylamine, phenylephrine, guaifenesin, caffeine and aspirin. All seven ingredients were quantified in soft gel, syrup and tablet formulations of the over‐the‐counter US‐marketed products, as per the guidelines of the International Conference on Harmonization. The separation was achieved in a 16 min run time on an Agilent Zorbax Phenyl column using a gradient method with two mobile phases. Mobile phase A was 0.15% trifluoro acetic acid in purified water and while mobile phase B was a mixture of acetonitrile and methanol (750:250 v/v) with 0.02% trifluoro acetic acid. The flow rate was 1.0 mL min^?1 and injection volume was 10 μL. Detection was performed at 280 nm using a photodiode array detector. As part of the method validation, specificity, linearity, precision and recovery parameters were verified. The concentration and area relationships were linear (R² > 0.999), over the concentration ranges 20–120 μg mL^?1 for acetaminophen, 75–450 μg mL^?1 for dextromethorphan, 31.25–187.5 μg mL^?1 for doxylamine, 25–150 μg mL^?1 for phenylephrine, 25–150 μg mL^?1 for aspirin, 6.5–39 μg mL^?1 for caffeine and 12–72 μg mL^?1 for guaifenesin. The relative standard deviations for precision and intermediate precision were <1.5%. The proposed RP‐HPLC generic method is applicable for routine analysis of cold and cough over‐the‐counter products.     

固相萃取/高效液相色谱法测定化妆品中5种抗组胺药物残留

建立了固相萃取/高效液相色谱(SPE/HPLC)测定化妆品中5种抗组胺药物(多西拉敏、曲吡那敏、溴苯那敏、苯海拉明、氯苯沙明)残留的分析方法。试样经三氯乙酸溶液超声提取,PCX柱净化后,以甲醇-磷酸盐缓冲溶液为流动相,经C18柱分离后进行HPLC检测。5种抗组胺药物在5.0～100 mg/L范围内均呈良好的线性关系,线性系数均大于0.999。在5.0、10.0、25.0 mg/kg 3个加标水平下的平均回收率为92%～108%,相对标准偏差(RSD,n=6)为2.1%～4.2%,检出限为0.5～1.0 mg/L。该方法灵敏度高、重现性好、定量准确。     

Differential protonation and dynamic structure of doxylamine succinate in solution using 1H and 13C NMR

A protonation and dynamic structural study of doxylamine succinate, a 1:1 salt of succinic acid with dimethyl-[2-(1-phenyl-1-pyridin-2-yl-ethoxy)ethyl]amine, in solution using one- and two-dimensional 1H and 13C NMR experiments at variable temperature and concentration is presented. The two acidic protons of the salt doxylamine succinate are in 'intermediate' exchange at room temperature, as evidenced by the appearance of a broad signal. This signal evolves into two distinct signals below about -30 degrees C. A two-dimensional 1H-1H double quantum filtered correlation experiment carried out at -55 degrees C shows protonation of one of the acidic protons to the dimethylamine nitrogen. A two-dimensional rotating frame 1H-1H NOE experiment at the same temperature reveals that the other proton remains with the succinate moiety. Comparison of the 1H and 13C chemical shifts and the 13C T1 relaxation times of the salt with those of the free base further substantiate the findings.     

Determination of diphenylmethane antihistaminic drugs and their analogues in body fluids by gas chromatography with surface ionization detection.

Eleven diphenylmethane antihistaminic drugs and their analogues were tested for their detection by capillary gas chromatography (GC) with surface ionization detection (SID). The GC-SID response was highest for doxylamine, diphenhydramine and orphenadrine and lowest for terodiline, clemastine and pipethanate. The detection limits for drugs with the highest response were 2-5 pg (ca. 6-20 fmol) on-column (100-250 pg/ml of body fluid). The detection limits with GC-SID were 10-100 times higher than those with GC with nitrogen-phosphorus detection. A detailed procedure for the isolation of the antihistaminics from human whole blood and urine by the use of Sep-Pak C18 cartridges, prior to GC-SID, is also presented. The recoveries of the drugs (50 or 500 pmol), which had been added to 1 ml of body fluids, were > 60%. The baselines remained steady as the column temperature was increased and the background was clean, especially for whole blood extracts.     

Separation of cold medicine ingredients using a precise MEKC method at elevated pH

An MEKC method was developed in order to separate a cold medicine formulation containing acetaminophen, ephedrine sulfate, doxylamine succinate, and dextromethorphan hydrobromide as active pharmaceutical ingredients. Because of their similar physical and chemical properties, it was a challenge to separate the basic compounds without sample pretreatment. In addition, the high content of alcohol and sucrose together with the variety of further excipients had to be considered. Thus, the complex matrix required several optimization steps. These included the search for the optimum pH and for a suitable sodium dodecyl sulfate concentration to avoid matrix-capillary wall interaction and to ensure precision. As a second developing step, an internal standard (benzocaine) was chosen to guarantee a high level of quantitative performance. An RSD% value of the peak areas between 1.0 and 2.0 was reached. The employed method development strategy can be generalized to similar separation approaches in the future.     

On-line sample treatment and FT-IR determination of doxylamine succinate in pharmaceuticals

A low solvent consumption method for Fourier transform infrared spectroscopy (FT-IR) determination of doxylamine succinate in pharmaceuticals has been developed. The analyte was continuous and selectively extracted with a 13% (v/v) ethanol:chloroform solvent mixture, recirculating the solvent through the sample and monitoring the process by FT-IR. Doxylamine succinate was determined by on-line standard addition measuring the peak area in the regions 1730–1710 and 1485–1462 cm⁻¹ corrected with a two-point baseline established between 2000 and 1800 cm⁻¹. This new method implies low volumes of chloroformic solvent mixture, only 2.6 mL per sample, in front of classical batch FT-IR methods, improving analytical efficiency and reducing waste generation. The on-line extraction and standard addition determination of doxylamine succinate allowed a throughput of 10 h⁻¹.     

Development and validation of a generic high‐performance liquid chromatography for the simultaneous separation and determination of six cough ingredients: Robustness study on core–shell particles

A generally applicable high‐performance liquid chromatographic method for the qualitative and quantitative determination of pharmaceutical preparations containing phenylephrine hydrochloride, paracetamol, ephedrine hydrochloride, guaifenesin, doxylamine succinate, and dextromethorphan hydrobromide is developed. Optimization of chromatographic conditions was performed for the gradient elution using different buffer pH values, flow rates and two C₁₈ stationary phases. The method was developed using a Kinetex® C₁₈ column as a core–shell stationary phase with a gradient profile using buffer pH 5.0 and acetonitrile at 2.0 mL/min flow rate. Detection was carried out at 220 nm and linear calibrations were obtained for all components within the studied ranges. The method was fully validated in agreement with ICH guidelines. The proposed method is specific, accurate and precise (RSD% < 3%). Limits of detection are lower than 2.0 μg/mL. Qualitative and quantitative responses were evaluated using experimental design to assist the method robustness. The method was proved to be highly robust against 10% change in buffer pH and flow rate (RSD% < 10%), however, the flow rate may significantly influence the quantitative responses of phenylephrine, paracetamol, and doxylamine (RSD% > 10%). Satisfactory results were obtained for commercial combinations analyses. Statistical comparison between the proposed chromatographic and official methods revealed no significant difference.     

Characterization of antihistamine-human serum protein interactions by capillary electrophoresis

An important topic in the drug discovery and development process is the role of drug binding to plasma proteins. In this paper the characterization of the interaction between antihistamines (cationic drugs) towards human serum albumin (HSA) and alpha(1)-acid glycoprotein (AGP) under physiological conditions by capillary electrophoresis-frontal analysis is presented. Furthermore, the binding of these drugs to all plasma proteins is evaluated by using ultrafiltration and capillary electrophoresis. Antihistamines present a wide-ranging behaviour with respect to their affinities towards plasma proteins. Orphenadrine, phenindamine, tripelenamine and tripolidine principally bind to HSA; carbinoxamine, dimetindene and etintidine principally bind to AGP; brompheniramine, chlorpheniramine and ranitidine present an important binding to lipoproteins and/or globulins and finally, chlorcyclizine, cinarizine, cyclizine, doxylamine, hydroxyzine, perphenazine and terfenadine do not bind to lipoproteins and/or globulins but bind to HSA and AGP in different extension. The interaction of antihistamines with HSA is determined by the hydrophobicity (direct relationship) and the polar surface area (indirect relationship) of the compounds. The steric parameters and hydrogen bonding character of compounds seems to be related with the binding of antihistamines to AGP. The antihistamine-HSA affinity constants were evaluated and the K(1) values ranged from 7 x 10(2)M(-1) (for doxylamine) to 4 x 10(4)M(-1) (for phenindamine).     

HPLC-fluorescence detection and MEKC-LIF detection for the study of amino acids and catecholamines labelled with naphthalene-2,3-dicarboxyaldehyde

Naphthalene-2,3-dicarboxyaldehyde (NDA) is commonly used for detection of primary amines in conjunction with their separation with HPLC and CE. The fluorescence of the derivatives can be measured by a conventional fluorometer or via LIF. NDA is a reactive dye, which can replace o-phthaldehyde (OPA) and provides for derivatives which are considerably more stable than OPA derivatives. In addition, NDA can be used to derivatize primary amines at concentrations as low as 100 pM. In this work, HPLC/fluorescence and MEKC/LIF experiments were performed to separate/detect six neuroactive compounds, the amino acids, Gly, Glu, Asp, gamma-aminobutyric acid (GABA) and the catecholamines, dopamine and noradrenaline. The two methods were compared in terms of performance of separation. The amino acids can be separated in HPLC in less than 30 min and an identical separation is obtained in CE using MEKC and lithium salts with greater resolution (the number of theoretical plates was approximately 5000 for HPLC and 200 000 for MEKC). The lowest detected concentration was in the range of 0.1 nM for CE/LIF. The presence of a high salt concentration does not affect the separation of the samples. Examples of the analysis of microdialysate samples as well as amino acids in Ringer's solution are presented.     

Electrofilament deposition and off-column detection of analytes separated by capillary electrophoresis

Capillary electrophoresis interfaced with electrospray is a convenient technique for continuously transferring column effluent from capillary-to-planar format. Conditions are optimized to produce a narrow (approximately 20 microm) liquid filament (electrofilament), which is capable of depositing spatially focused bands with track widths that are routinely 100 microm. A fiber optic-based, laser-induced fluorescence cell is employed to monitor the separation on-column while the separated bands are deposited onto a moving substrate. The photodetection of deposited bands is accomplished by using either a charge-coupled device camera or a photomultiplier tube. Deterioration of on-column separation performance is observed when the electrofilament voltage is applied. Elevating the inlet of the capillary column, to provide hydrodynamic flow, restores separation performance. Substrate temperature and translational rates are optimized with respect to both off-column separation efficiency and signal intensity. Off-column separation efficiencies of 65 000 plates per meter were achieved. A linear dynamic range of 10(3) and a limit of detection of 10(-8) M were obtained for kiton red deposited onto a reversed phase thin-layer chromatography plate. To demonstrate the applicability of this technique to more complex separation solutions, a dye mixture was successfully separated and deposited with sodium dodecyl sulfate in the running buffer.     

Separation and detection of peroxynitrite and its metabolites by capillary electrophoresis with UV detection

A method for the separation and direct detection of peroxynitrite (ONOO(-)) and two of its degradation products, nitrite (NO(2)(-)) and nitrate (NO(3)(-)), using capillary electrophoresis with ultraviolet detection is described. The separation parameters were optimized and included electrokinetic injection, a run buffer consisting of 25 mM K(2)HPO(4) 7.5 mM DTAB, pH 12, and a field strength of -323 V/cm. A diode array UV detector was employed in these studies as it allowed the determination of all three species simultaneously. Nitrate and nitrite provided the maximum response at 214 nm while peroxynitrite generated the best response at 302 nm. All three species could be detected at 214 nm, while simultaneous detection at 214 and 302 nm positively identified each peak.     

Separation and detection of lanthanides by capillary electrophoresis

Due to the importance of application of lanthanides in various industries especially the nuclear ones, and the advantages of capillary electrophoresis method in separation of metal cations, this research was carried out in order to investigate the separation potential of lanthanides using capillary electrophoresis via simulation method at laboratory scale. Since the properties of various types of lanthanides are very similar, the separation of lanthanides using the usual approaches was not possible. Thus, the separation of lanthanides was devised upon partial, competing complexation in order to differentiate their properties. Salicylic acid was firstly used as the primary UV-absorbing ligand, whereas formic, acetic, lactic, tartaric and citric acids, which showed no absorption in UV-spectrum and had weaker complexes in comparison to salicylic acid, were used as auxiliary ligands. Upon the results of spectrometry, the wave length of 210 nm was selected for detecting lanthanides. The properties and stability of lanthanides were examined and furthermore acetic and citric acids were selected as auxiliary ligands. The simulation was carried out with respect to the transport phenomena in the unsteady state. The ion species dissociation was found to be directly dependent upon the concentration, and was also used in complexation. The results of simulation showed that the diffusion control of H⁺ and homogenizing electrical field promoted separation quality. The separation conditions were optimized by using the simulation results as well as the tests obtained. In order to optimize the experimental conditions, variable factors such as voltage, injection time, pH, temperature and ionic strength were examined. Also, methanol was used as dissolving modifier as well as noise reducer on the base line. Sodium nitrate was used as ionic strength controller and sucrose for increasing viscosity which optimized separation quality.     

Parameters influencing separation and detection of anions by capillary electrophoresis

Electrolyte composition is critical in optimizing separation and detection of ions by capillary electrophoresis. The parameters which must be considered when designing an electrolyte system for capillary electrophoresis include electrophoretic mobility of electrolyte constituents and analytes, detection mode, and compatibility of electrolyte constituents with one another. An electrolyte system based on pyromellitic acid is well suited for use with indirect photometric detection, and provides excellent separations of anions. The ability to modify the electrophoretic mobility of pyromellitic acid as a function of ph provides flexibility in matching electrophoretic mobilities of analytes. Additionally, the use of alkyl amines as electroosmotic flow modifiers allows the rapid separation of anions by reversing the direction of electroosmotic flow in a fused-silica capillary. The optimization of a capillary electrophoresis electrolyte for anion analysis is also discussed in terms of pH, ionic strength and applied voltage. The effect of organic solvent on separation selectivity is also discussed.     

Microchip capillary electrophoresis with amperometric detection for rapid separation and detection of phenolic acids

A microchip capillary-electrophoresis protocol for rapid and effective measurements of food-related phenolic acids (including chlorogenic, gentisic, ferulic, and vanillic acids) is described. Relevant parameters of the chip separation and amperometric detection are examined and optimized. Under optimum conditions, the analytes could be separated and detected in a 15 mM borate buffer (pH 9.5, with 10% of methanol) within 300 s using a separation voltage of 2000 V and a detection voltage of +1.0 V. Linear calibration plots are observed for micromolar concentrations of the phenolic acid compounds. The negligible sample volumes used in the microchip procedure obviates surface fouling common to amperometric measurements of phenolic compounds. The new microchip protocol offers great promise for a wide range of food applications requiring fast measurements and negligible sample consumption. An application on a commercial red wine was performed with minimal sample preparation and promising results.     

On-column conductivity detection in capillary-chip electrophoresis

On-column conductivity detection in capillary-chip electrophoresis was achieved by actively coupling the high electric field with two sensing electrodes connected to the main capillary channel through two side detection channels. The principle of this concept was demonstrated by using a glass chip with a separation channel incorporating two double-Ts. One double-T was used for sample introduction, and the other for detection. The two electrophoresis electrodes apply the high voltage and provide the current, and the two sensing electrodes connected to the separation channel through the second double-T and probe a potential difference. This potential difference is directly related to the local resistance or the conductivity of the solution defined by the two side channels on the main separation channel. A detection limit of 15 microM (600 ppb or 900 fg) was achieved for potassium ion in a 2 mM Tris-HCl buffer (pH 8.7) with a linear range of 2 orders of magnitude without any stacking. The proposed detection method avoids integrating the sensing electrodes directly within the separation channel and prevents any direct contact of the electrodes with the sample. The baseline signal can also be used for online monitoring of the electric field strength and electroosmosis mobility characterization in the separation channel.