Liquid chromatographic assay with fluorescence detection to determine ajmaline in serum from patients with suspected Brugada syndrome.

Ajmaline is a sodium channel blocking, class 1A anti-arrhythmic drug. It has gained renewed interest in the field of cardiology as a diagnostic agent to reveal the electrocardiographic characteristics in patients with suspected Brugada syndrome. We developed a simple and precise high-performance liquid chromatographic assay to determine ajmaline in serum of patients. The samples were pre-treated using protein precipitation with perchloric acid and the extract was injected into the chromatographic system. The system consisted of an end-capped octadecyl silica column with isocratic elution using perchloric acid in a water-acetonitrile mixture. Ajmaline was detected by fluorescence at 290 and 355 nm for excitation and emission, respectively. The assay was validated in a 21-5300 ng/ml concentration range, the lower limit of quantification was 25 ng/ml. Within day precisions were 1.3-3.9%, between day precisions 2-7% and accuracies were between 95 and 99% for the whole calibration range. The drug was shown to be chemically stable under all relevant conditions. This assay has been successfully applied to pharmacokinetic-pharmacodynamic evaluations of intravenous ajmaline administration to patients with suspected Brugada syndrome.

Sensitive method for the determination of the antiarrhythmic drug detajmium in serum by solid-phase extraction and high-performance liquid chromatography with fluorimetric detection.

The objective of this study was to develop a very sensitive and selective method for the determination of detajmium (4-3-diethylamino-2-hydroxypropyl-ajmaline), a sodium-channel-blocking drug with antiarrhythmic properties, in serum. A high-performance liquid chromatography (HPLC) method with solid-phase extraction and fluorimetric detection has been applied. Serum samples were diluted with phosphate buffer (pH 3.5) and the extraction of detajmium and ajmaline, which was used as an internal standard, was carried out with Oasis cartridges (Waters). The chromatographic separation was performed on a RP18 column. The limit of quantification for serum samples of detajmium was 1 ng/ml with good reproducibility (R.S.D. < 15%) and a linear response from 1 to 200 ng/ml. The described method is highly sensitive and specific for the determination of detajmium in serum of patients and volunteers.

Fatal poisoning with detajmium: identification of detajmium and its metabolites and artifacts by gas chromatography-mass spectrometry and quantification by high-performance liquid chromatography.

After ingestion of an unknown dose of detajmium, a 14-year-old female collapsed with asystolia. Resuscitation efforts were not successful. A medicolegal autopsy was carried out, and blood, liver and gastric content were extracted and analyzed by gas chromatography-mass spectrometry (GC-MS). After derivatization with acetic anhydride, detajmium and twelve of its derivatives and metabolites were identified. The main metabolic pathways include hydroxylation and subsequent O-methylation of the indol ring, and oxidation as well as reduction of the C-21 hydroxyl function. Cleavage of the N-alkyl side-chain is a further, possibly non-enzymatic degradation pathway. Artifact formation induced by acetylation included dehydratation of the hydroxyl function of C-21 and the N-alkyl side-chain. The detajmium concentration in blood of the decreased was determined by high-performance liquid chromatography with fluorimetric detection (12 micrograms/ml).

Monitoring of ajmaline in plasma with high-performance liquid chromatography.

A rapid, reliable and sensitive assay for routine determination of ajmaline in plasma by high-performance liquid chromatography with fluorimetric detection is presented. A low limit of detection in plasma (less than 1 ng/ml ajmaline) could be achieved by the extraction of plasma samples and the use of fluorimetric detection. Deproteinization of the plasma sample instead of extraction, or the use of an ultraviolet detector, yielded a higher limit of detection (less than 50 ng/ml). Two different eluents were studied. Eluent 1 allowed clear separation of ajmaline from isoajmaline and sandwicine, but did not separate isoajamaline from sandwicine. With eluent 2, separation of isoajmaline and sandwicine was achieved, but separation of ajmaline from sandwicine was less optimal than with eluent 1. Therefore, eluent 1 was used for further clinical studies. No interference was observed from therapeutic doses of other commonly co-administered drugs, such as acetylsalicylic acid, digoxin, digitoxin, ranitidine, dopamine, dobutamine, furosemide, captopril or glycerol trinitrate. In addition, the chemical stability of ajmaline and a possible rearrangement of ajmaline to its stereoisomers isoajmaline and sandwicine was studied in vivo and in vitro. Ajmaline proved to be unusually stable under both in vivo and in vitro conditions.

Mechanisms of pharmacokinetic interaction between ajmaline and quinidine in rats.

In order to elucidate the mechanism of ajmaline-quinidine interaction previously observed in humans, the effects of quinidine on pharmacokinetics of ajmaline were investigated in rats. Concurrent oral administration of 10 mg/kg of quinidine markedly increased the plasma concentration of ajmaline at a dose of 2 mg/kg. On the other hand, it did not affect the pharmacokinetics of ajmaline after intravenous dose. The availability of ajmaline after oral dose showed an increase from 13% to nearly 100% by the presence of quinidine, which suggests a change in the presystemic clearance of ajmaline. In fact, when ajmaline was administered into the intestinal loop, its concentration in mesenteric venous plasma increased approximately 5-fold by the combination with quinidine. Furthermore, quinidine delayed the elimination rate of ajmaline from the perfused rat liver. These results indicate that quinidine prevents presystemic elimination of ajmaline in the intestine and liver, and increases the systemic availability of ajmaline.

Quinidine-induced rise in ajmaline plasma concentration.

A high-performance liquid chromatographic method is described for the simultaneous determination of ajmaline and quinidine in human plasma. With 0.5 ml plasma sample of a ajmaline and quinidine, concentrations as low as 0.001 and 0.01 micrograms ml-1, respectively, could be detected and the technique could be used to investigate the effect of quinidine on the pharmacokinetics of ajmaline. Four healthy subjects were given oral ajmaline (50 mg) alone or in combination with quinidine sulphate (200 mg) on separate occasions. When ajmaline was administered alone, its plasma concentrations were less than 0.03 micrograms ml-1. Quinidine induced a marked increase to give a mean peak concentration of ajmaline which increased from 0.018 micrograms ml-1 after a single administration to 0.141 micrograms ml-1 in combination with quinidine. the area under the ajmaline concentration-time curves was increased 10 to 30-fold by the concurrent administration of quinidine. According to the one compartment open model, the absorption rate constant of ajmaline did not change appreciably, but the elimination rate constant was reduced to approximately 50% of the value in the absence of quinidine. The results indicate the existence of a significant interaction between oral ajmaline and quinidine.

[Acute ajmaline poisoning. Study of 7 cases]. / Intoxication aiguë par l'ajmaline. Etude de 7 observations.

Seven cases of acute ajmaline overdose admitted over a 3 year period to a polyvalent intensive care unit are reported. The severity of this condition is related to the membrane stabilising and depolarising effects of ajmaline on the myocardium. The dose ingested varied from 10 to 40 mg/kg. The delay between ingestion and hospital admission ranged from 3 to 6 1/2 hours. The first cardiac disturbances can appear one hour after ingestion. Three cardiac arrests and one hypovolemic shock occurred. Three atrioventricular blocks, six intraventricular blocks, three ventricular tachycardias, and six prolongations of the QT interval were observed. Serum ajmaline levels varied from 0,8 to 6 mg/l. Symptomatic therapy was mainly based on sodium, temporary cardiac pacing, external DC shock, sympathomimetics and external cardiac massage with assisted ventilation. Cardiac bypass should be a part of the therapeutic arsenal. Elimination of the drug is assisted by a complete digestive evacuation. Renal or extrarenal dialysis is not indicated. One of the seven patients died. Prophylaxis is based on the non-prescription of ajmaline for benign cardiac disturbances.

Acute intoxication with ajmaline.

A 57-year-old man ingested 1,000 mg of ajmaline with suicidal intent. He was unconscious, hypotensive and showed serious disturbances in cardiac conduction on admission. High serum and urine levels of ajmaline were found. Although only 4% of the ingested dose was excreted following forced diuresis, all evidence of toxicity disappeared within 21 h.

Pharmacokinetic studies of the antiarrhythmic drugs ajmaline and N-propylajmaline bitartrate in dogs.

A rapid, sensitive fluorometric method for the determination of ajmaline and N-propylajmaline (NPA) was used to follow serum concentrations after an i.v. administration of ajmaline, after an oral administration of ajmaline, and an oral dose of NPA bitartrate (NPAB) in beagle dogs. Ajmaline was eliminated from serum with an apparent half-life of approximately 1 h and NPA with one of approximately 4 h. 1 h after i.v. and oral administration of 50 mg of ajmaline the serum levels were of the same order of magnitude. The bioavailability of oral ajmaline, calculated from the area under the serum concentration/time curves, was about 90%. The peak serum concentrations of ajmaline and NPA after a single oral dose of 50 mg of ajmaline and NPAB were about 1.4 microgram/ml and 0.6 microgram/ml, respectively, the latter calculated as its bitartrate, with about the same peak time: 1 h.

A spectrofluorometric method for the determination of ajmaline in plasma.

1 Ajmaline was found to have maximum fluorescence at neutral pH with 300 nm excitation and 365 nm emission wavelengths (corrected). 2 The fluorescence intensity had a linear relationship to concentration up to 50 microgram ml-1 and the recovery of ajmaline after extraction from plasma was 92.5 +/- 3%. 3 Extraction of drug-free plasma and of samples containing known concentrations of ajmaline showed that drug levels in the range found clinically could be measured accurately by fluorimetry. 4 Serial plasma ajmaline concentrations were measured in a subject after intravenous injection of ajmaline (50 mg). The rates of plasma clearance of the drug were found to be similar to those obtained in previous studies.

The pharmacokinetics and organ distribution of ajmaline and quindine in the mouse.

After i.v. infusion into mice (lasting 10 s) the time courses of ajmaline and quinidine concentrations in blood, heart, lung, liver, and brain were studied. The drugs were assayed by a spectrofluorophotometric procedure. Blood concentration data obtained were fitted graphically and calculations were performed in accordance with an open two compartment model. Blood kinetic data were very similar for both alkaloids. A rapid distribution phase with a t0.5alpha of 3.0 min for ajmaline and 2.5 min for quindine was followed by a disposition phase with a t0.5beta of 16 min for ajmaline and 20 min for quinidine. High tissue accumulation of both alkaloids was found in lung, liver, and heart and this is also reflected by the volume of distribution Vdbeta, which was 136 ml for ajmaline and 116 ml for quinidine (body weight of the mice = 31 g). With equilibrium dialysis a 62% binding of ajmaline and a 77% binding of quinidine to mouse blood constituents was found. Both drugs were highly metabolized since only 5% of a given dose was excreted unchanged in the urine.

A gas chromatography-mass fragmentographic method for the assay of ajmaline and its monochloroacetyl ester.

A gas chromatograph-mass fragmentography method for simultaneous assay of 17-monochloroacetyl-ajmaline (MCAA) and its hydrolysis product, ajmaline, is described. Recovery of both compounds from whole blood averaged 71%. About 5% of MCAA was hydrolyzed during the assay procedure. The method was accurate and precise to within a few percent. It was suitable for assays of blood levels in the dog after an i.v. dose of 2 mg/kg or an oral dose of 5 mg/kg.

In vitro and in vivo observations on the erythrocyte uptake of 17-monochloroacetylajmaline.

The in vitro and in vivo observations on the uptake rate of 17-monochloroacetylajmaline by human erythrocytes showed that about 30-35% of the drug is bound to red blood cells. These findings suggest a peculiar affinity of the drug for the erythrocytes, may be for the membrane or stromal phospholipids.

Cardiovascular effects and blood concentrations of ajmaline and its 17-monochloroacetate ester in cats.

The antiarrhythmic drugs ajmaline and its 17-monochloroacetate ester (MCAA; Rtimos-Elle) were studied in cats. MCAA was less than half as toxic as ajmaline. Non-lethal doses of MCAA decreased blood pressure before heart rate, whereas ajmaline initially decreased heart rate. Both drugs prolonged the PR, QRS and QT intervals of the EKG. Recovery of these effects was within one hr. MCAA (10 mg/kg) and ajmaline (4.05 mg/kg) were studied separately by a one and 10 min infusion in the same cat. The dose of MCAA was ten times the usual dose in man and that of ajmaline four times the usual clinical dose. More marked effects were observed with the one min infusion. Arrhythmias were usually observed with ajmaline, but not with MCAA, even though it was rapidly converted to ajmaline. Maximal cardiovascular effects of MCAA and ajmaline were observed within 3 min of the end of infusion, which was also the time of peak blood levels. The elimination of MCAA resembled the kinetics of a multi-compartment system after a one min infusion. Peak blood levels declined by one-half in 3 min. Ajmaline blood levels declined linearly, with a half-life of 100 min, after a one min infusion. The peak blood level of MCAA after an intraduodenal dose of 25 mg/kg occurred at 20 min, whereas the peak blood level of the ajmaline formed occurred at 4 hr. In conclusion, MCAA has some different pharmacological properties and different kinetics of elimination than ajmaline.

Determination of 17-monochloroacetylajmaline and its metabolite in plasma by TLC fluorescence detection.

A sensitive and specific method is described for the determination of 17-monochloroacetylajmaline (I) and its metabolite, ajmaline (II), in plasma. Method specificity is accomplished by combining an ion-pair extraction with chromatography followed by development and utilization of reaction product fluorescence of the isolated species on silica gel. Recovery of I and II added to plasma or water averaged 70%. The major loss in the assay resulted during a solvent evaporation step and was reproducible over the concentration interval studied. The limit of detectability for I is 0.06 mu-g/2 ml of plasma. The method was used to determine plasma levels of I and II in the dog following a dose of 10 mg/kg iv of I.