J. Moncrieff

Determination of pharmacological levels of harmane, harmine and harmaline in mammalian brain tissue, cerebrospinal fluid and plasma by high-performance liquid chromatography with fluorimetric detection.

Increased blood aldehyde levels, as occur in alcohol intoxication, could lead to the formation of beta-carbolines such as harmane by condensation with indoleamines. Endogenous beta-carbolines, therefore, should occur in specific brain areas where indoleamine concentrations are high, whilst exogenous beta-carbolines should exhibit an even distribution. The author presents direct and sensitive methods for assaying the beta-carbolines harmane, harmine and harmaline in brain tissue, cerebrospinal fluid and plasma at picogram sample concentrations using reversed-phase high-performance liquid chromatography with fluorimetric detection and minimal sample preparation. Using these assay methods, it was found that the distribution of beta-carbolines from a source exogenous to the brain results in a relatively even distribution within the brain tissue.

Determination of cetirizine in serum using reversed-phase high-performance liquid chromatography with ultraviolet spectrophotometric detection.

A method using reversed-phase high-performance liquid chromatography with ultraviolet detection for the determination of ceterizine in serum is described. The method is sensitive down to 50 ng/ml (250-microliter loop). Sample preparation involves only serum deproteination with perchloric acid and injection of the centrifuged supernatant. Elution is at pH 2.5 with acetonitrile-methanol-0.05 M phosphate buffer (33:9:58, v/v) on a 25 cm x 4.6 mm I.D. Spherisorb S5 ODS2 column. Detection is at 211 nm, its lambda max. For levels above 300 ng/ml the serum sample size is 100 microliter and a 200-microliter sample is necessary for concentrations less than 300 ng/ml. At the 2 micrograms/ml concentration the intra-assay relative standard deviation is better than 2.2%, whilst the inter-assay deviation is 2.6% over eight samples. At 200 ng/ml the intra-assay relative standard deviation is 6% over seven samples. Detector response is linear from 100 ng/ml to 10 micrograms/ml (100-microliter loop).

Polymorphism of the 4-Hydroxylation of Debrisoquine in the San Bushmen of Southern Africa:

1 The metabolic oxidation of debrisoquine has been studied in a group of 96 San Bushmen. 2 The amounts of debrisoquine and 4-hydroxy-debrisoquine excreted in 0-8 h urine were measured and the metabolic ratio (% dose as debrisoquine/% dose as 4-hydroxy-debrisoquine) calculated. 3 On the basis of Caucasian criteria, that metabolic ratios > 12.6 represent poor metabolizers, 19% of the Bushmen were poor metabolizers in contrast to the 8-10% found in Caucasian studies. 4 Probit plots showed four modes may be present in the data, which may represent at least three isozymes of the relevant enzyme which may also differ from the Caucasian isozymes.

Non-correlation between Debrisoquine and Metoprolol Polymorphisms in the Venda

1 The metabolic 4-hydroxylation of debrisoquine has been studied in a group of 98 black African villagers in Vendaland. 2 The metabolic α-hydroxylation of metoprolol has been studied in 94 of the same black African villagers. 3 A 4% prevalence of poor oxidative metabolism of debrisoquine and a 7.4% incidence of poor oxidation of metoprolol were found. The 4% result for debrisoquine differs considerably from the 19% found in San Bushmen, 30% in Hong Kong Chinese, 9% in Britains and 0% in Nigerians and Japanese, whilst the 7.4% result for metoprolol compares with 8.4% in Britains but differs from 0% in Nigerians and 4.1% in San Bushmen. 4 None of the poor oxidative metabolizers of debrisoquine were also poor oxidative metabolizers of metoprolol. This is contrary to results in British and Nigerian subjects where defective oxidation of metoprolol co-segrates with that of debrisoquine. 5 No similarities were found between the Venda metabolic ratio (MR) distributions and either extensive or poor MR distributions in Britains or Nigerians.

Extractionless determination of diclofenac sodium in serum using reversed-phase high-performance liquid chromatography with fluorimetric detection

The author describes a method of using reversed-phase high-performance liquid chromatography with fluorimetric detection for the assay of diclofenac sodium in serum. The method is sensitive down to 20 ng/ml (250-microliters loop). Elution is at pH 6.2 with methanol in 0.05 M phosphate buffer (43:57, v/v) on a 25-cm Spherisorb S5 ODS2 column. Detection is at an excitation wavelength of 282 nm and an emission wavelength of 365 nm. Serum sample size is 100 microliters. Sample protein, to which diclofenac is highly bound, is first denatured by heat and then with methanol to release the diclofenac prior to centrifugation and injection of 100 microliters (or 250 microliters) of the clear supernatant. Harmol, with similar fluorescence and polarity characteristics to diclofenac, is a satisfactory internal standard. At the 1 micrograms/ml level intra-sample reproducibility is better than 2%, whilst inter-sample reproducibility is 4.6%. Detector response is linear from 40 ng/ml to 20 micrograms/ml (100-microliters loop).

Determination of theophylline in serum and saliva in the presence of caffeine and its metabolites

Because of marked variability in its metabolic clearance and its narrow therapeutic range (10-20 micrograms/ml) investigation of each patients clearance of theophylline is desirable. The author reports here a rapid reversed-phase high-performance liquid chromatographic (HPLC) method to determine, within 3 min, the theophylline in serum and saliva in the 0.1-50 micrograms/ml range. A fast HPLC column, 10 x 4.6 mm, packed with 3-microns spherical ODS packing is used with acetonitrile-methanol-buffer pH 4.7 (4:7:89) to achieve separation of theophylline from paraxanthine and matrix components. Since theophylline is a major pediatric bronchodilator, the feasibility of assay in saliva was investigated as an alternative route for determining the clearance is stressed asthmatic children. Using this method it was found that the ratio of theophylline in simultaneous serum and saliva samples is very consistent over time in the same person (+/- 3.99%), but inter-individually this consistency is reduced ten-fold. Simultaneous serum and saliva samples need be taken only once to obtain the ratio and the kinetics followed further with salivary samples only.

Metoprolol α-Hydroxylation Polymorphism in the San Bushmen of Southern Africa

1 The metabolic oxidation of metoprolol has been studied in a group of 98 San Bushmen. 2 The amounts of metoprolol and α-hydroxy metoprolol excreted in 0-8 h urine collection, after dosing with 100 mg metoprolol, were measured and the metabolic ratio (% dose excreted as metoprolol/% dose excreted as α-hydroxy metoprolol) calculated. 3 Frequency distribution and probit plots of the metabolic rate data showed a bimodal distribution with 4.1% of the population exhibiting slow metabolism with an MR > 10. 4 These results are much less than found in Caucasians (8.4%) but very different from the unimodal distribution found for Nigerians. 5 A previous study in the same group of Bushmen had revealed that 18 of 96 subjects were poor or non-metabolizers of debrisoquine to 4-hydroxy debrisoquine, but only one of the poor metoprolol metabolizers was a poor metabolizer of debrisoquine. 6 On the basis of these results, the claim of debrisoquine type of polymorphism for β-adrenoceptor antagonists found in Caucasians cannot be extrapolated to the San Bushmen, and one must query the use of debrisoquine as measure of oxidative status in any group other than Caucasians.

Determination of dapsone in serum and saliva using reversed-phase high-performance liquid chromatography with ultraviolet or electrochemical detection

A simple, extractionless method for the determination of dapsone in serum and saliva is described. Reversed-phase high-performance liquid chromatography is used with UV detection at 295 nm or electrochemical detection at 0.7 V. Diazoxide in buffer is the internal standard for UV detection and practolol for electrochemical detection. Sample preparation is minimal with protein precipitation of serum samples whilst saliva samples are simply diluted with addition of an internal standard. Low-level serum and saliva samples are front-cut on-line with a 3 cm laboratory-made precolumn in the loop position on a standard Valco injection valve. Isocratic separation is achieved on a 250 mm x 4.6 mm I.D. stainless-steel Spherisorb S5 ODS-1 column. The mobile phase for high levels of dapsone is acetonitrile-elution buffer (12:88, v/v) at 2 ml/min and a column temperature of 40 degrees C for both serum and saliva separations. For the low-level assays using electrochemical detection and solid-phase clean-up, the mobile phase is acetonitrile-methanol-elution buffer (9:4:87, v/v/v). The UV and electrochemical detection limits are 25 ng/ml and 200 pg/ml, respectively, in both serum and saliva. This simple method is applicable to the routine monitoring of dapsone levels in serum from leprotic patients and electrochemical detection gives a simple, reliable method for the monitoring of trough values in subjects on anti-malarial prophylaxis.

Absence of Polymorphism of Sparteine Oxidation in the South African Venda

1 This study has found no occurrence of poor metabolism of sparteine within a South African Venda populaton of 97 subjects. 2 On the basis of MR (metabolic ratio) the mean and distribution of the results are very similar to those found in Ghanaians.5,14 3 The distribution is also similar to that for fast metabolizers in Caucasians.14 4 It is concluded that different P450 cytochromes are responsible for immediate oxidation of debrisoquine and sparteine, but that both may be activated by the same P450 reductase.

Fraction of theophylline in sustained-release formulation which is absorbed from the large bowel

SummaryIn a cross-over study of six healthy male volunteers, 500 mg theophylline was administered either as plain tablets or in a sustained release preparation. On each occasion 2 g of non-enteric coated sulphasalazine was administered simultaneously as the time of appearance of sulphapyridine, the product of hydrolysis, in the blood provides an approximation of the oral — caecal transit time. The mean fraction absorbed — time profile was calculated from serial serum concentration measurements of theophylline by a modification of the Wagner-Nelson equation. The mean cumulative fraction of the dose absorbed following administration of the plain tablets was maximal at 3 h i.e. approximately 3 h ahead of the mean oral-caecal transit time, which was 5.9 h. Thus complete absorption occurred in the small intestine.With the sustained — release formulation, approximately only half of the dose was absorbed at the time the medication reached the large bowel i.e. at about 5.4 h. Absorption continued and at least 38% of the administered dose was additionally absorbed over the next 25 h.A reliable lengthened dosage interval is therefore possible with this particular sustained — release formulation.