Determination of the antileukemic drug mitoguazone and seven other closely related bis(amidinohydrazones) in human blood serum by high-performance liquid chromatography.

A reversed-phase (C18) HPLC method with diode-array detection was developed for the separation and determination of methylglyoxal bis(amidinohydrazone) (mitoguazone) and seven closely related aliphatic analogs thereof, namely the bis(amidinohydrazones) of glyoxal, dimethylglyoxal, ethylmethylglyoxal, methylpropylglyoxal, butylmethylglyoxal, diethylglyoxal and dipropylglyoxal. The mobile phase consisted of a non-linear binary gradient of methanol and 0.03 M aqueous sodium acetate buffer (pH 4.3). Good separation of the eight congeners was achieved. On increasing the methanol content of the eluent, the bis(amidinohydrazones) eluted in the order of increasing number of carbon atoms in the side-chains. The method was also applied to the quantitative analysis of the compounds in aqueous solution and, combined with ultrafiltration, for the separation of the eight congeners in spiked human blood serum. A separate simplified method for the quantitative determination of each of the compounds in spiked human blood serum samples was also developed. The methods developed made for the first time possible the simultaneous HPLC analysis of more than one bis(amidinohydrazones). The results obtained indicate that the bis(amidinohydrazones) studied obviously have a distinct tendency to form ion associates with acetate ions and probably also other carboxylate ions in aqueous solution. This aspect may be of biochemical significance, especially concerning the intracellular binding of the compounds. Each one of the compounds studied invariably gave rise to one peak only, this result supporting the theory that the conventional synthesis of each of the compounds gives rise to one geometrical isomer only. This result is completely in agreement with the results of previous proton and carbon NMR spectroscopic as well as X-ray diffraction studies.

Ion-pair chromatography and micellar electrokinetic capillary chromatography in analyzing beta-adrenergic blocking agents from human biological fluids.

Ion-pair chromatography (IPC) and micellar electrokinetic capillary chromatography (MECC) were used for the separation and determination of parent beta-blockers from human biological fluids. In both these techniques, N-cetyl-N,N,N-trimethylammonium bromide (CTAB) was used as a buffer additive. In IPC, CTAB was an ion-pair former, and in MECC it was a micelle-forming surfactant. The effectiveness of the IPC method using methanol-gradient elution and that of MECC were compared for drug-spiked serum and urine samples. Detection was performed with a diode-array detector in the IPC method and with a 214-nm filter in the MECC technique. In both methods a phosphate buffer (pH 7.0) was used. In MECC the buffer solution contained 10 mM CTAB, while in IPC the CTAB concentration was decreased from 7 to 4 mM during the separation when a methanol gradient was used. The study showed that the IPC technique performed better for bioanalyses than the high-performance MECC technique, since in MECC UV detection presented a problem because of the low sample concentration. However, in MECC sample preparation was less time-consuming, using hydrolyzation and protein precipitation and, unlike the IPC technique, it did not require any liquid-liquid extraction step.

Determination of nine beta-blockers in serum by micellar electrokinetic capillary chromatography.

beta-Adrenergic blocking agents are used for the treatment of angina pectoris, cardiac arrhythmia, hypertension, anxiety attacks, thyrotoxicosis, migraine and glaucoma. Owing to their sedative effect, they are also used as doping agents in sport. All beta-blockers have an alkanol amine side chain terminating in a secondary amino group in their structure. The pKa values vary from 9.2 to 9.8. Because some beta-blockers are hydrophilic and some lipophilic, simultaneous determination is difficult. In this work, a method based on micellar electrokinetic capillary chromatography (MECC) was developed for the separation and determination of beta-blockers in serum. The phosphate buffer 0.08 M (pH 6.7) solution contained 15 mM N-cetyl-N,N,N-trimethylammonium bromide. Nine parent beta-blockers could be separated in a single run and the concentrations determined by internal standard (ephedrine) method. The simple clean-up procedure consisted of enzyme hydrolysis (Helix pomatia), protein precipitation, and filtration through 0.5-microns PTFE membranes. The MECC method exhibited good repeatability and a linear range of 75-300 micrograms/ml. The method was successfully applied after concentration to the determination of propranolol in real samples.

Effects of organic mobile phase modifiers on elution and separation of beta-blockers in micellar electrokinetic capillary chromatography.

A study was made of the effect of organic modifiers (acetone, acetonitrile, ethanol, ethylene glycol, methanol and 2-propanol) in phosphate buffer (0.08 M) containing 15 mM cetyltrimethylammonium bromide as surfactant on the elution and separation of eleven common beta-adrenergic blocking agents. The amount of the modifier was varied from 0.1 to 10.0% (v/v). At maximum addition, the organic solvents increased the viscosity of the buffer solution as follows: acetone 16%, acetonitrile 9%, ethanol 26%, ethylene glycol 27%, methanol 20% and 2-propanol 29%. In contrast to the migration time of the other beta-blockers, that of labetalol was not increased by the addition of organic solvent to the buffer solution. Rather, labetalol eluted more quickly with increase in the amount of modifier, and thereby effected changes in the elution order of the beta-blockers. The addition of modifiers also affected the resolution, and the best resolution values were achieved with the following amounts of organic solvent in MEKC buffer: acetone 0.1%, acetonitrile 0.1-0.5%, ethanol 5.0-7.5%, ethylene glycol 1.0-2.5%, methanol 5.0% and 2-propanol 1.0-2.5% (v/v). No significant relationship was found between the elution order and separation and the structure of the beta-blockers in micellar electrokinetic capillary chromatography with an organic modifier in buffer solutions.

Effect of buffer solution pH on the elution and separation of beta-blockers by micellar electrokinetic capillary chromatography.

Study was made of the effect of the pH of phosphate buffer (0.08 M) containing 15 mM cetyltrimethylammonium bromide as surfactant on the elution order of eleven widely used beta-adrenergic blocking agents. In the pH range 6.0-7.8 the elution order of six of the beta-blockers remained the same, while the order of five of them changed. Sotalol eluted as the sixth compound at pH 6.8 and migrated more quickly with increasing pH. Below pH 7.0 labetalol eluted before propranolol and above pH 7.0 afterwards. Likewise, the order of elution of atenolol and timolol was reversed at pH 7.0. The pH also affected the resolution; the best resolution values were achieved between pH 6.6 and 7.0 and between pH 7.4 and 7.8. The relationship between the structure of the beta-blockers described by molecular and molecular connectivity indices and the elution order and separation of the beta-blockers in micellar electrokinetic capillary chromatography at varying pH of the buffer solution is discussed.

Determination of ten beta-blockers in urine by micellar electrokinetic capillary chromatography.

beta-Adrenergic blocking agents are of therapeutic value in the treatment of migraine and various cardiovascular disorders (angina pectoris, cardiac arrhythmia, hypertension). Owing to their sedative effect, they are also used as doping agents in sport. A characteristic feature of beta-blockers is the alkanolamine side-chain terminating in a secondary amino group. The pKa values vary from 9.2 to 9.8. Because some beta-blockers are hydrophilic and some lipophilic, simultaneous determination is difficult. In this work, a method based on micellar electrokinetic capillary chromatography (MECC) was developed for the separation and determination of beta-blockers. The 0.08 M phosphate buffer (pH 7.0) solution contained 10 mM N-cetyl-N,N,N-trimethylammonium bromide (CTAB). Ten parent beta-blockers in human urine could be separated in a single run and determined quantitatively by the internal standard (2,6-dimethylphenol) method. Neither endogenous compounds in urine nor caffeine and its metabolites interfered with the analysis. The clean-up procedure for urine consisted of a simple filtration through 0.5-microns PTFE membranes. The MECC method exhibited good repeatability and a linear range of 25-150 micrograms/ml. The method was applied to determination of oxprenolol in real samples.

Screening of beta-blockers in human serum by ion-pair chromatography and their identification as methyl or acetyl derivatives by gas chromatography-mass spectrometry.

A simultaneous screening method for atenolol, acebutolol, metoprolol, oxprenolol, alprenolol and propranolol by ion-pair chromatography with a column-switching technique was developed. The serum samples were purified using either liquid-liquid extraction or solid-phase extraction methods. The pretreatment of the samples consisted of hydrolysis and protein precipitation. The drug separation was on either octadecylsilica or polymer-based alkyl column material. Binary eluent mixtures containing methanol and a buffer solution with a quaternary ammonium salt as an ion-pair former were used. Detection of the compounds in liquid chromatographic analysis was based on ultraviolet spectra. The effects of methanol, two buffers and the ion-pair former on the retention of the compounds were studied. The determination limits ranged from nanograms to micrograms in the ion-pair chromatographic method, depending on the drug studied. Identification was based on the mass spectra or, if necessary, on selected-ion monitoring spectra of either the methylated or the acetylated compounds obtained by means of gas chromatography-electron impact or negative chemical ionization mass spectrometry. The detection limits for the identified compounds were in the picogram range. The matrix effect was strong, and this resulted in determination limits in the nanogram range with the scan method.

Determination of bis(amidinohydrazones) by micellar electrokinetic capillary chromatography.

A micellar electrokinetic capillary chromatography method was developed for the separation and determination of aliphatic congeners of bis(amidinohydrazones) in standard solution. Eight bis(amidinohydrazones) could be determined in less than 15 min at an applied voltage of 22 kV, using 0.05 M sodium phosphate as buffer (pH 7.0) together with 1 mM N-cetyl-N,N,N-trimethylammonium bromide. Hydrostatic sample injection was employed. The method exhibited good repeatability and a linear range of 2.5-100 micrograms ml-1. A detection limit of 1 micrograms ml-1 was achieved. The method also allows the determination of bis(amidinohydrazones) in human serum samples.