Mass spectrometry of pancuronium bromide and related quaternary ammonium steroids using the moving belt LC/MS interface

Pancuronium and vecuronium are members of a series of quaternary ammonium steroids used as neuromuscular blocking agents in anesthesiology. In this study the mass spectrometric properties of these bromide salts are examined and spectral features which permit their differentiation are evaluated. The relative merits of chemical ionization and fast atom bombardment using the moving belt liquid chromatography/mass spectrometry interface have been investigated. Fragmentation pathways for both ionization methods were determined with deuterium labeling and linked-scan techniques. Cleaner spectra can be obtained via the matrix-free belt introduction system as compared to conventional fast atom bombardment.     

Simple approach to measure metabolic pathways of steroids in living cells.

A simple, rapid approach to the study of conversion rates and metabolic patterns of the steroids testosterone and estradiol is presented. It includes an optimized isocratic high-performance liquid chromatographic procedure in the reversed-phase mode and radioactive on-line detection. The purpose was to estimate the activity of key enzymes of steroid pathways, such as 17 beta-hydroxysteroid dehydrogenase and 5 alpha-reductase, in in vivo conditions. Using this system, we obtained good efficiency and linearity of radio detection, under continuous flow conditions. Sensitivity limits were of the order of 50 and 70 cpm for [3H]estradiol and [14C]estrone, respectively, even though the efficiency was quite dissimilar (17.3% versus 56.2%). The applicability of this approach to studies of steroid metabolic pathways in growing cancer cells in culture is illustrated with examples of the conversion rates of both testosterone and estradiol. The high reproducibility (coefficients of variation of 2.7 and 5.1% for 3H and 14C, respectively) and good extraction efficiency (ranging from 86 to 94%) indicate the feasibility and reliability of this approach.     

Endogenous alkaloids in man. XI. Analysis of glyoxylate-derived 1,3-thiazolidines by ion-pair-assisted reversed-phase chromatography.

Reversed-phase ion-pair chromatography, using cetyltrimethyl ammonium hydrogensulphate as detergent, has been applied to the analysis of the highly polar glyoxylate-derived 1,3-thiazolidines 1, 2 and 3. On the base of this high-performance liquid chromatographic separation of the diastereomeric compounds 1a/b and 3a/b was achieved. Removal of the hydrophobic couterion by precipitation as its insoluble iodide, followed by an extraction with chloroform, seems to be a promising first step to establish ion-pair-assisted chromatography as a preparative high-performance liquid chromatographic method for the isolation of polar compounds.     

Selectivity enhancement for the separation of tocopherols and steroids by integration of highly ordered weak interaction sites along the polymer main chain

A novel alternating copolymer-based organic phase was synthesized using a new N-substituted maleimide monomer for the development of alternating copolymer-grafted silica for high-performance liquid chromatographic applications. This new monomer (DGMI) was copolymerized with octadecyl acrylate (ODA) from 3-mercaptopropyltrimethoxysilane-grafted silica to produce Sil-poly(ODA-alt-DGMI). The organic phase was characterized by the elemental analysis and the diffuse reflectance infrared Fourier transform spectroscopy. Tocopherol isomers and steroids were used as analytes for the evaluation of the chromatographic selectivity profiles of this novel stationary phase. The selectivity of this column was then compared with a polymeric ODS column and previously developed another alternating copolymer-grafted silica (without the glutamide-derived moiety) column, Sil-poly(ODA-alt-N-octadecylmaleimide). The complete baseline separation of tocopherol isomers in an isocratic mode has been achieved within 25 min with the Sil-poly(ODA-alt-DGMI). The separation of eight kinds of estrogenic steroids and corticoids has also been achieved in an isocratic mode with this column. Significant differences in separation selectivity between Sil-poly(ODA-alt-DGMI) and polymeric ODS columns were observed towards the steroids, and compared with the reference columns, a better separation profile for these analytes was obtained with the Sil-poly(ODA-alt-DGMI). The results of this investigation indicated that the enhancement of selectivity of Sil-poly(ODA-alt-DGMI) towards the test analytes arose from the multiple interaction mechanism such as hydrophobic effect, carbonyl-π and hydrogen-bonding interactions, and such integrated interactions originated from the addition of two amide groups in the N-substituted maleimide monomer.     

High-pressure liquid chromatography of steroids.

After a brief discussion of the merits and limitations of high-pressure liquid chromatography (HPLC) relative to other chromatographic methods, special problems in the application to steroids are discussed. Publications on HPLC of steroids are then discussed under the headings of individual classes, arranged generally in the order of increasing polarity.     

Chromatographic retention behaviour, modelling and separation optimisation of the quaternary ammonium salt isometamidium chloride and related compounds on a range of reversed-phase liquid chromatographic stationary phases

This paper describes the reversed-phase liquid chromatographic behaviour of the trypanocidal quaternary ammonium salt isometamidium chloride and its related compounds on a range of liquid chromatographic phases possessing alkyl and phenyl ligands on the same inert silica. In a parallel study with various extended polar selectivity phases which possessed different hydrophobic/silanophilic (hydrogen bonding) activity ratios, the chromatographic retention/selectivities of the quaternary ammonium salts was shown to be due to a co-operative mechanism between hydrophobic and silanophilic interactions. The highly aromatic and planar isometamidium compounds were found to be substantially retained on stationary phases containing aromatic functionality via strong π-π interactions. The chemometric approach of principal component analysis was used to characterise the chromatographic behaviour of the isometamidium compounds on the differing phases and to help identify the dominant retention mechanism(s). Two-dimensional (temperature/gradient) retention modelling was employed to develop and optimise a rapid liquid chromatography method for the separation of the six quaternary ammonium salts within 2.5 min which would be suitable for bioanalysis using liquid chromatography-mass spectrometry. This is the first reported systematic study of the relationship between stationary phase chemistries and retention/selectivity for a group of quaternary ammonium salts.     

High-performance liquid chromatographic analysis of basic drugs on silica columns using non-aqueous ionic eluents. I. Factors influencing retention, peak shape and detector response

The use of silica columns together with non-aqueous ionic eluents provides a stable yet flexible system for the high-performance liquid chromatographic analysis of basic drugs. At constant ionic strength, eluent pH influences retention via ionisation of surface silanols and protonation of basic analytes, pKa values indicating the pH of maximum retention. At constant pH, retention is proportional to the reciprocal of the eluent ionic strength for fully protonated analytes and quaternary ammonium compounds. The addition of water up to 10% (v/v) has little effect on retention if the protonation of the analytes is unaffected. Thus, it is likely that retention is mediated primarily via cation exchange with surface silanols. However, additional factors must play a part with compounds such as morphine which give tailing peaks at acidic or neutral eluent pHs.     

Ion-Pair Reversed-Phase Thin-Layer Chromatography and High-Performance Liquid Chromatography of Benzoic Acids

The separation of benzoic acids by ion-pair reversed-phase thin-layer chromatography and high-performance liquid chromatography was studied. In the optimization of separation conditions, the effect of the nature and concentration of the organic solvent, acidity of the mobile phase, concentration of salts of quaternary ammonium bases, and the length of their hydrocarbon radical on the retention of benzoic acids was studied. Conditions of the separation of a mixture of benzoic, hydroxybenzoic, aminobenzoic, and nitrobenzoic acids on Silufol plates impregnated with cetyltrimethylammonium bromide were selected; the mobile phase was isopropanol–ammonium acetate buffer solution with pH 5–dodecyltrimethylammonium bromide.     

An efficient procedure for extraction and determination of steroids in the tissue of laboratory animals

Summary A high-performance liquid chromatographic method for analysis of steroids in the tissue of laboratory animals is described. A two-stage solid extraction procedure, utilizing Sep-pak C₁₈ and graphitized carbon black, gives a purified product. Steroids in this extract were separated by multistep gradient elution with a reversedphase liquid chromatography system, using acetonitrilewater as solvent. Several columns with different geometry and phase loading were investigated. Optimum resolution was obtained with a medium covered and spherical particle.The applicability of this method was successfully demonstrated in the analysis of the testis and adrenal gland of rat.Dedicated to Prof. Dr. A. Liberti on the occasion of his 70th birthday.     

Analysis of propantheline bromide and related quaternary ammonium drugs by reversed-phase,ion-pair liquid chromatography with two counterions in the eluent

Summary The addition of appropriate concentrations of an organic amine and an alkylsulphonate to the mobile phase in reversed-phase, ion-pair liquid chromatography can introduce unique selectivity in to the chromatographic system allowing separation of complex mixtures of basic, acidic and neutral compounds. As an example, the methodology for a specific stability-indicating determination of propantheline bromide, a quaternary ammonium anticholinergic agent, on several reversedphase stationary phases, was developed. The retention mechanism was studied and it was concluded that both ion-interaction processes and ion-exchange processes were involved in the separation technique developed with two counterions of opposite charge in the eluent.     

Studies on steroids. CCXVI. Separation of bile acid 3-glucuronides by high-performance liquid chromatography

The separation of 3-glucuronides of cholate, chenodeoxycholate, deoxycholate, ursodeoxycholate and lithocholate, and their glyco- and tauro-conjugates, has been carried out by high-performance liquid chromatography on a reversed-phase column. The chromatographic behaviour of bile acid 3-glucuronides was dependent on the type of conjugation. An effect of the pH of the mobile phase on the capacity ratio was observed at higher pH for chenodeoxycholate 3-glucuronide, probably owing to steric interaction of the 7 alpha-hydroxy group with the carboxy group in the glucuronyl moiety. Conversion of the alpha-hydroxy function on the steroid nucleus into an oxo group resulted in a 50% decrease in the capacity ratio. Bile acid 3-glucuronides were efficiently separated on Shodex ODS Pak F-411 using three kinds of ammonium phosphate buffer-acetonitrile systems.     

Separation of steroids using temperature-dependent inclusion chromatography

The influence of temperature on retention and separation of estrogens, progesterone derivatives and beta-cyclodextrin in reversed-phase high-performance liquid chromatography has been studied. Steroids were detected using direct UV detection at 240 and 280 nm. Detection of beta-cyclodextrin was achieved using a post-column indirect photometric method. Chromatographic experiments were performed using an acetonitrile-water mobile phase (30%, v/v) and a wide range of column temperatures from 0 to 80 degrees C with 20 degrees C steps. Linear Van't Hoff plots were observed for steroids and beta-cyclodextrin when an unmodified binary mobile phase was applied. The retention of steroids was strongly influenced by temperature when the mobile phase was modified with beta-cyclodextrin at a concentration of 12 mM. Particularly, for 17beta-estradiol and 20alpha-hydroxyprogesterone a strong deviation from the linear Van't Hoff plots and a remarkable affinity for beta-cyclodextrin was observed. Polynomial regression calculations were performed to fit the set of experimental data points. Using third-order polynomial equations, minimum separation factor values (alphamin) were calculated for temperatures from -10 to + 100 degrees C with 1 degrees C steps. The best chromatographic conditions for separation of multicomponent samples were chosen. A possible retention mechanism for solutes in the presence of macrocyclic additives is discussed. The results presented describe the role of temperature in high-performance liquid chromatography systems in which the mobile phase is modified with an inclusion agent.     

Detection of Quaternary Ammonium Drugs in Equine Urine by Liquid Chromatography-Mass Spectrometry

Quaternary ammonium drugs are anticholinergic agents and some of which have been known to be abused in equine sports. A general screening method for this class of drugs in equine urine by liquid chromatography-mass spectrometry (LC-MS) has not been reported. The paper describes an efficient LC-MS-MS method for the simultaneous detection and confirmation of twenty quaternary ammonium drugs at low ng mL^?1 in equine urine after solid-phase extraction. Quaternary ammonium drugs were extracted from equine urine by solid-phase extraction using ISOLUTE^® CBA SPE Columns and analysed by LC-MS-MS in the positive electrospray ionisation mode. Separation of twenty quaternary ammonium drugs (the quaternary ammonium ions of edrophonium chloride, pyridostigmine bromide, neostigmine bromide, bretylium tosylate, ipratropium bromide, tubocurarine chloride, N-butylscopolammonium bromide, mepenzolate bromide, rocuronium bromide, clidinium bromide, pipenzolate bromide, isopropamide iodide, glycopyrronium bromide, alcuronium chloride, oxyphenonium bromide, propantheline bromide, tridihexethyl chloride, vecuronium bromide, mivacurium chloride and pancuronium bromide) was achieved in a reversed phase column with a mixture of aqueous ammonium formate (pH 3.0, 10 mM) and acetonitrile as the mobile phase. Detection and confirmation of the twenty quaternary ammonium drugs at about 5 ng mL^?1 in equine urine could be achieved within 22 min using product-ion scan MS-MS. The target analytes were detected by examination of extracted-ion chromatograms of their product ions. Drugs spiked in different equine urine (n = 15) were consistently detected. Negative samples (n = 30) of normal post-race equine urine have also been analysed, no matrix interference at the targeted masses and retention times was observed. The method was successfully applied to the analyses of drug-administration samples. Other method validation data including reproducibility and recovery will also be presented. An LC-MS-MS method for the simultaneous detection and confirmation of twenty quaternary ammonium drugs in equine urine was developed. The methodology should be applicable to other biological matrices such as human urine.     

Studies on neurosteroids. IX. Characterization of estrogens in rat brains using gas chromatography-tandem mass spectrometry.

The characterization of the classical estrogens (estrone, estradiol, estriol) and guaiacol estrogens (2-hydroxyestrone 3-methyl ether, 4-hydroxyestrone 3-methyl ether) in rat brains was performed using gas chromatography-tandem mass spectrometry (GC-MS-MS). Estrogens were purified from Wistar strain rat brains by some chromatographic pre-treatments, such as solid-phase extraction, preparative thin-layer chromatography or preparative high-performance liquid chromatography. After the derivatization with O-methylhydroxylamine and/or N,O-bis(trimethylsilyl)trifluoroacetamide, estrogens were identified by comparison of their chromatographic behavior during GC-MS-MS operating in the product ion scan mode and comparison with the product ion MS spectra of an authentic sample. These evidences suggested that estrogens exist in rat brains as neurosteroids or neuroactive steroids.     

A colorimetric assay for the determination of polyhexamethylene biguanide in pool and spa water using nickel-nioxime

A novel nickel-nioxime analytical method to measure polyhexamethylene biguanide (PHMB) in swimming pools and spas was developed. This method utilizes nickel(II) chloride and 1,2-cyclohexanedionedioxime (nioxime) chemistry. In the method, nickel ions bind and neutralize PHMB in the solution. Excess, un-reacted nickel ions react with nioxime and the resulting colored solution is measured at 550 nm using a colorimetric assay. Currently, the colorimetric method to measure PHMB uses bromophenol blue (BPB). However, high levels of quaternary ammonium based algaecides and surfactant based products interfere with this colorimetric method. A time-consuming and expensive high-performance liquid chromatographic (HPLC) analysis can be used for samples with high levels of quaternary ammonium based algaecides or surfactants. The proposed nickel-nioxime detection method achieves comparable PHMB results to HPLC in about 5 min and is a very economical and simple method to perform.     

Determination of 2-mercaptopropionylglycine and its metabolite, 2-mercaptopropionic acid, in plasma by ion-pair reversed-phase high-performance liquid chromatography with post-column derivatization.

A simple and fast high-performance liquid chromatographic method was developed for the simultaneous measurement of 2-mercaptopropionylglycine (Tiopronine) and its metabolite (2-mercaptopropionic acid) in human plasma after the administration of a pharmaceutical dosage form (Acadione). The sample treatment before high-performance liquid chromatographic analysis consisted of the reduction of the corresponding disulphides by tri-n-butylphosphine and protein precipitation with ethanol. Separation was achieved by ion-pair high-performance liquid chromatography on a reversed-phase column (LiChrospher RP 18e) with cetrimonium bromide as counter ion and detection by fluorimetry after post-column derivatization with a selective thiol reagent, i.e. pyrenemaleimide. The high frequency of the analyzed samples and validation results make the method suitable for pharmacokinetic studies, and this was demonstrated by the first results obtained after the administration of an oral dose of 500 mg of Tiopronine to two healthy subjects.     

Selection of high-performance liquid chromatographic methods in pharmaceutical analysis. III. Method validation

The most important steps in the validation of high-performance liquid chromatographic (HPLC) methods are discussed. The establishment of system suitability data and the assessment of peak purity are demonstrated on the example of bisquaternary amino steroids. For the recognition of incomplete resolution of adjacent peak pairs, the absorbance-ratio method in which the ratio of absorbances at two preselected wavelengths are plotted as a function of time in combination with the separation of sample components subjected to various chemical and physico-chemical treatments (stress conditions) is applied. The separation power and performance of the HPLC systems are characterized by the system resolution (SR) and system selectivity (SS). The special demands of stability-indicating methods are summarized.     

Comparison of different high-performance liquid chromatographic systems for the purification of adrenal and gonadal steroids prior to immunoassay

The high-performance liquid chromatography of nineteen hormonal steroids with special respect to its suitability for routine purification of these steroids from crude, organic extracts of biological fluids prior to final quantitation by immunoassay has been studied. In all systems the gradient elution technique was applied. Separation of steroids has been investigated using different stationary phases chemically coated with non-polar, hydroxyl, NO2 and CN groups. Reproducibility of retention times was studied on a stationary phase coated with hydroxyl groups (DIOL column) using different organic eluents. Coefficients of variation range from 0.76 to 8.16%. Reproducibility was shown to be unequivocally better in the gradient part than in the isocratic part of the chromatographic run. In contrast to the other steroids, 18-hydroxylated steroids were more or less unstable in certain systems studied. As to resolution and reproducibility, the DIOL column run with an n-hexane-dioxane gradient has been shown to be superior to the other systems studied.     

Quantitation of quaternary ammonium compounds using electrospray mass spectrometry

A sensitive and specific electrospray mass spectroscopic assay has been developed for the quantitation of the series of quaternary ammonium surfactant compounds dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide, and octadecyltrimethylammonium bromide and neostigmine bromide. Standard solutions were prepared in methanol, with an appropriate quaternary ammonium internal standard used in each case, and injected into a carrier solvent comprising acetonitrile (99%) and methanol (1%). Detection was based upon the positively charged quaternary ammonium ion in each case. Using this new technique, significant increases in sensitivity have been obtained over more traditional gas chromatographic–mass spectroscopic analytical techniques. All of the compounds tested were readily quantifiable at solution concentrations of 50 ppb, with linear correlations (r²>0.995) obtained over the range 50–300 ppb for the alkyltrimethylammonium bromides, and 50–200 ppb for neostigmine bromide. It is envisaged that, using this method, adsorption isotherms for these compounds on highly adsorbing activated carbons should be readily acquired.     

Analysis of Quaternary Ammonium Compounds by Ion-Pair Thin Layer Chromatography on Silica Gel

Abstract

A general approach to the chromatographic analysis of quaternary ammonium compounds on silica gel thin layers is described. The quaternary cations migrate as ion pairs with bromide or iodide as counter ions. Methanol or chloroform-methanol mixtures serve as developing solvents, in which excess amounts of NaBr or NaI are dissolved. If the solvent cannot dissolve sufficient quantities of halide salts, the latter can be sprayed onto the plate as a methanol solution prior to development. The separation patterns of the quaternary ammonium compounds can be influenced by the choice of counter ion and by the choice of the organic solvent components.