In vitro distribution of ketoprofen enantiomers in articular tissues of osteoarthritic patients

The distribution of ketoprofen enantiomers in joint tissues was studied as a function of their relative tissular affinities using the multi-chamber distribution dialysis system described by Bickel et al. Selected off-cuts of synovial membrane, joint capsule, cartilage and ligament were obtained from ten patients suffering from osteoarthritis of the knee (n=3) or hip (n=7). Sörensen solution (4 ml) spiked with racemic ketoprofen (2 μg ml⁻¹) was dialysed against 1 ml of the four solutions of tissue homogenates (0.4 g ml⁻¹). Ketoprofen enantiomers were quantified in buffer and tissue solutions by high-performance liquid chromatography. The distribution of ketoprofen enantiomers in the Bickel's multi-compartment model indicated that there was a non-stereoselective affinity of ketoprofen enantiomers for their potential target tissues. Despite the interindividual variability in articular tissues, the concentrations (±S.D.) of R- and S-ketoprofen were significantly higher in synovial membrane (8.69 (4.76) μg g⁻¹ for S, 9.14 (5.57) μg g⁻¹ for R), joint capsule (5.71 (2.49) μg g⁻¹ for S, 5.49 (2.62) μg g⁻¹ for R) and ligament (6.28 (3.61) μg g⁻¹ for S, 6.40 (3.64) μg g⁻¹ for R) than in articular cartilage (3.67 (1.75) μg g⁻¹ for S, 3.70 (1.67) μg g⁻¹ for R). There were no significant differences in the distribution of R- and S-ketoprofen between the solutions of joint capsule, synovium and ligament tissues. These data may be related to differences in ketoprofen affinity for the different constituents of joints. This in vitro distribution profile is similar to that reported in vivo for other non-steroidal anti-inflammatory drugs.     

Interaction between accumulation of cadmium selenium in the tissues of turbot Scophthalmus maximus

The interaction between accumulation of waterborne cadmium and selenite in juvenile turbot, Scophthalmus maximus, was investigated in the laboratory. Intestine, kidney and liver of turbot exposed to 150 μg Cd 1⁻¹ accumulated cadmium linearly with time over 5 wk at rates of 0.50, 0.014 and 0.11 μg Cd g⁻¹ dry wt. d⁻¹, respectively. Gills, skin and muscle reached steady-state cadmium levels of ca. 15, 0.8 and 0.25 μg Cd g⁻¹ after 1–3 wk of exposure. Plasma and erythrocytes reached steady-state concentrations of ca. 0.1 and 1–2 μg Cd ml ⁻¹ after 1–2 wk of exposure. Exposure to 105 μg Se-SeO₃²⁻1⁻¹ did not consistently alter selenium concentrations in gills, skin, liver, muscle and erythrocytes of juvenile turbot. Kidneys accumulated selenium linearly (0.029 μg Se g⁻¹ dry wt. d⁻¹) with time over 5 wk, while intestine reached a steady-state level after 2 wk. Selenium concentrations in the plasma were maintained close to the ambient level throughout the exposure time. Concurrent exposure to selenite augmented cadmium accumulation rates in gills, kidney and liver and reduced cadmium accumulation in intestine and erythrocytes; cadmium accumulation in spleen, skin, muscle and plasma was not affected. Concurrent exposure to cadmium depleted erythrocytes and partly skin of selenium and reduced accumulation of selenium in kidney and plasma, whereas selenium accumulation patterns in gills, intestine, liver, muscle and spleen were not affected by exposure to cadmium.     

Development of an assay for the extraction and quantification of nine 5-n-alkyl-5-ethyl barbituric acids in various rat tissues

Methods were developed to quantify a series of nine homologous 5-n-alkyl-5-ethyl barbituric acids in 15 rat tissues. Tissue homogenates were spiked with one of four multicomponent mixtures (methyl to n-propyl, n-propyl to n-pentyl, n-pentyl to n-heptyl and n-pentyl to n-nonyl). Liquid–liquid extraction was used to extract the homologues from the rat tissues. Reverse phase HPLC with UV detection at 214 nm was used to separate and quantify the individual barbiturates. The limit of detection for each respective homologue was 1 μg g⁻¹ except skin and bone (2 μg g⁻¹). The methodology developed reduced a potential 135 individual assays to a more manageable 16.     

Residue determination of two co-administered antibacterial agents — cephalexin and colistin — in calf tissues using high-performance liquid chromatography and microbiological methods

Residues of two antibacterial agents, cephalexin and colistin, co-administered by intramuscular injection to calves, were quantified in four different tissues (muscle, fat, liver and kidney) by column switching HPLC and by a microbiological method. For cephalexin assay, tissue samples with cephradin as internal standard were homogenized in a 5% trichloroacetic acid solution and filtrates were injected onto a concentration pre-column filled with LiChroprep RP-18 (25–40 μm). A clean-up step was incorporated by flowing a mobile phase (methanol—0.01 M phosphate buffer (pH 3.0); 15:85, v/v) through the enrichment column before elution on a LiChrospher RP-18e (5 μm) column with a methanol—phosphate buffer (30:70, v/v) at a flow rate of 1 ml min⁻¹. Spectrometric detection was at 260 nm. An additional “off-line” washing step of extracts with methylene chloride was operated to achieve higher selectivity in the case of liver and kidney samples. The limit for quantitative assay was 0.045 μg g⁻¹ with relative standard deviations in the range 5–8% and recoveries within 70%.

For microbiological assay of colistin, samples were homogenized in 0.1 M hydrochloric acid–acetonitrile mixtures (3:1, v/v, for kidney and liver; 3:2, v/v, for fat and muscle). The supernatants were assayed by the cylinder plate method after evaporation to dryness under vacuum. Bordetella bronchiseptica ATCC 4617 was chosen as test organism. After a 3-h diffusion step at room temperature, the medium was incubated at 37°C for 18 h and then the diameter of the growth inhibition zones was measured. Sensitivity reached 0.10–0.15 μg g⁻¹. Results from the analysed samples over a 7–28 day period after drug administration show that no cephalexin was found at concentrations higher than the quantitation limit in the four test tissues and that colistin was found in muscle (injection site only) for 15 days and in kidney for 21 days.     

Levels of nickel and other potentially allergenic metals in Ni-tested commercial body creams

It is extensively well-known that Ni and other metals occurring as impurities in cosmetic products might give rise to contact dermatitis in subjects with pre-existing allergy. The present study on the content of 13 metals (Cd, Co, Cr, Cu, Hg, Ir, Mn, Ni, Pb, Pd, Pt, Rh, and V) in moisturizing creams, labelled as “Ni-tested” (i.e., Ni content <100 ng g⁻¹) and available on the Italian market, provides a basis for assessing their safety for consumers. Quantification of metals was performed by sector field inductively coupled plasma mass spectrometry after microwave-assisted acid digestion of products. The developed method had limits of quantification less than 0.8 ng g⁻¹ for all the elements; recovery was in the interval 88% (Cd, Co) to 110% (Hg), and precision was always under 7%. Nickel was present in all the products with levels between 17.5 and 153 ng g⁻¹; three skin creams were slightly above the concentration reported on the label. The other elements were at levels below 1 μg g⁻¹. The highest concentrations, in ng g⁻¹, of Co, Cr, Cu, and Mn were 222, 303, 51.2, and 59.9, respectively. Mean Cd, Pb, and V were below 5 ng g⁻¹, while Hg was absent in all the samples. Among the new emergent allergens, Ir and Rh were in traces or even undetectable, while Pt had levels of 2.65 and 6.28 ng g⁻¹ in two creams and Pd was equal to 1.07 ng g⁻¹ in one product. The overall results are below the sensitizing limit proposed for consumer products and, thus, probably have no significant toxicological effects. Nevertheless, some creams presented amounts of Co and Cr comparable to those of Ni and therefore they have to be monitored in consideration of their cross-reactivity as well.     

Simultaneous separation and determination of active components in Cordyceps sinensis and Cordyceps militarris by LC/ESI-MS

A simple and rapid isocratic LC/MS coupled with electrospray ionization (ESI) method for simultaneous separation and determination of adenine, hypoxanthine, adenosine and cordycepin in Cordyceps sinensis (Cs) and its substitutes was developed. 2-Chloroadenosine was used as internal standard for this assay. The optimum separation for these analytes was achieved using the mixture of water, methanol and formic acid (85:14:1, v/v/v) as a mobile phase and a 2.0×150 mm Shimadzu VP-ODS column. Selective ion monitoring (SIM) mode ([M+H]⁺ at m/z 136, 137, 268, 252 and 302) was used for quantitative analysis of above four active components. The regression equations were liner in the range of 1.4–140.0 μg ml⁻¹ for adenine, 0.6–117.5 μg ml⁻¹ for hypoxanthine, 0.5–128.5 μg ml⁻¹ for adenosine and 0.5–131.5 μg ml⁻¹ for cordycepin. The limits of quantitation (LOQ) and detection (LOD) were, respectively 1.4 and 0.5 μg ml⁻¹ for adenine, 0.6 and 0.2 μg ml⁻¹ for hypoxanthine, 0.5 and 0.1 μg ml⁻¹ for adenosine and cordycepin. The recoveries of four constituents were from 93.5 to 107.0%. The nucleoside contents of various types of natural Cs and its substitutes were determined and compared with this developed method.     

PAHs and metals in the soils of inner-city and suburban New Orleans, Louisiana, USA

Representative soil samples of an inner-city and suburban community (n = 19 each) are evaluated for 16 polycyclic aromatic hydrocarbons—PAHs (naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benz(a)anthracene, chrysene, benzo[k]fluoranthene, benzo[j]fluoranthene, benzo(a)pyrene, indeno[1,2,3-cd]pyrene, dibenz[a,h]anthracene and benzo[g,h,i]perylene) and nine metals (Pb, Zn, Cd, Mn, Ni, Cu, Cr, Co and V). Surface (2.5 cm deep) samples were air-dried and sieved (2 mm USGS #10). Accelerated solvent extraction was used for PAH preparation prior to analysis with gas chromatography–mass spectrometry. Metals were extracted at a 5:1 ratio of 1 mol nitric acid to soil, shaken at room temperature, centrifuged, filtered and analyzed by inductively coupled plasma-atomic emission spectrometry (ICP-AES). Total PAHs (median 2927 ng g⁻¹ versus 731 ng g⁻¹) and the total metals (median 1323 μg g⁻¹ versus 183 μg g⁻¹) summarize differences (P < 0.0001) between the inner-city and suburb, respectively. A strong association exists between PAHs and metals for all 38 soil samples (correlation coefficient = 0.831, P < 0.00001). In terms of the specific sites of accumulation, both PAHs and metals show the same pattern: busy streets > foundations > residential streets > open areas. This study provides real-world data about various chemical mixtures which may be a factor of possible health disparities in sensitive populations, especially children, in different communities of New Orleans.     

Lethal and sublethal effects of malathion on three life stages of the grass shrimp, Palaemonetes pugio

The effects of malathion exposure on three life stages of the grass shrimp (Palaemonetes pugio) were evaluated. After 96-h exposures, malathion was most toxic to newly hatched larvae with an LC₅₀ of 9.06 μg l⁻¹ followed by an LC₅₀ of 13.24 μg l⁻¹ for 18-day-old larvae and an LC₅₀ of 38.19 μg l⁻¹ for adult shrimp. In a separate bioassay, to simulate field conditions, newly hatched larvae were exposed to malathion at 6 h day⁻¹ every 5 days at a salinity of 10‰ until metamorphosis to postlarvae. After four pulse dose exposures, mortality was highest in the two highest concentrations, of 15.0 and 30.0 μg l⁻¹. The number of instars to postlarvae was significantly lower in the highest concentration compared to control. The findings indicate that malathion may not directly affect growth in a measurable way but may alter natural metamorphic rhythms at the highest concentrations. Whole body acetylcholinesterase (AChE) activity was measured on Day 0 and Day 15 from the pulse exposure test. AChE activities were not significantly different from controls. Other factors than just AChE inhibition may have contributed to malathion toxicity.     

Flow injection spectrophotometric determination of Al in hemodialysis solutions

A flow analysis (FA) system with spectrophotometric detection for Al determination in hemodialysis solutions was developed. The method was based on the reaction of Al with eriochrome cyanine R (ECR). The complex formed associated with cetyltrimethylammonium bromide (CTAB) — a cationic surfactant, which showed enough sensitivity to execute the direct analyte determination. All interferences were eliminated with the matrix matching calibration. The system presented the following analytic parameters: sensitivity (m) of 8.10 × 10⁻⁴ L μg⁻¹, limit of detection (LOD) of 3.24 μg L⁻¹ (3σ), linear correlation coefficient of 0.9966 and linear range response from 10.8 to 650 μg L⁻¹. The accuracy of the proposed method was checked by comparison with electrothermal atomic absorption spectrometry (ET-AAS) method. There were no differences among the results obtained from both methods, at a confidence level of 95% (paired t-test). Recovery tests were also made, values obtained were from 90.4 to 109 of recovery for Al-spiked samples.     

Activity of the enzymes of the antioxidative system in cadmium-treated Oxya chinensis (Orthoptera Acridoidae)

One purpose in this research was to determine the toxic effects of Cd on antioxidant enzymes of Oxya chinensis (Orthoptera: Acridoidae). Changes in the activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and guaiacol peroxidase (GPx) were measured in O. chinensis insects injected with Cd²⁺. Fifth-nymphs of O. chinensis insects were injected with Cd²⁺ at different concentrations (0, 0.55 × 10⁻⁴, 1.10 × 10⁻⁴, 1.65 × 10⁻⁴, 2.20 × 10⁻⁴, and 2.75 × 10⁻⁴ g g⁻¹). An increase in SOD activity in O. chinensis was observed at 1.10 × 10⁻⁴ to 2.75 × 10⁻⁴ g g⁻¹ Cd²⁺. The SOD activity was lower at 2.20 × 10⁻⁴and 2.75 × 10⁻⁴ g g⁻¹ than that at 1.10 × 10⁻⁴ and 1.65 × 10⁻⁴ g g⁻¹. It appears that SOD had a positive protective effect at low Cd²⁺ concentrations, and that this effect disappeared at high Cd²⁺ concentrations. CAT activity was accelerated to varying degrees at 1.10 × 10⁻⁴ to 2.75 × 10⁻⁴ g g⁻¹ for males and at 1.10 × 10⁻⁴, 2.20 × 10⁻⁴, and 2.75 × 10⁻⁴g g⁻¹ for females. CAT showed a strong detoxification effect with all treatments. GPx activity decreased with increasing Cd²⁺ concentration with all treatments for males and at 2.20 × 10⁻⁴ and 2.65 × 10⁻⁴g g⁻¹ for females. We showed that GPx activity had a weak detoxification function with all treatments for males and at high Cd²⁺ for females. Thus, CAT had a strong detoxification effect, whereas SOD had a medium and GPx had a weak detoxification effect. Among the three enzymes, CAT played an important role in the damaging mechanisms of reactive oxygen species in O. chinensis insects. Alterations of the antioxidant enzyme level under environmental stresses are suggested as indicators of biotic and abiotic stress.     

Determination of trace elements in a large series of spent peritoneal dialysis fluids by atomic absorption spectrometry

An analytical procedure is reported for the determination of six elements in a large series of spent dialysis fluid samples. Determinations of aluminium, chromium, copper, manganese and iron were made by electrothermal atomic absorption spectrometry (ETAAS) with Zeeman background correction, while zinc was analysed by flame atomic absorption spectrometry (FAAS). Because of the complex matrix with high salt content and a high content of proteins, the measurement parameters were optimised for each particular element determined by ETAAS. The samples were collected in polyethylene eppendorf cups and stored in a freezer at −20°C. When the elements were determined by ETAAS the standard addition method was applied in the calibration procedure. The sample (10 μl) was injected into a cuvette and careful drying and long ashing of samples at temperatures between 850 and 1000°C performed. Triton X-100 was added before each determination to reduce the matrix effects of the proteins. Zinc was determined by FAAS in an air–acetylene flame under the usual recommended procedure, calibrating with aqueous standards. The limits of detection (3σ basis) were 1.0 μg l⁻¹ for aluminium, 0.20 μg l⁻¹ for chromium, 0.40 μg l⁻¹ for copper, 0.20 μg l⁻¹ for manganese, 0.50 μg l⁻¹ for iron and 5.0 μg l⁻¹ for zinc. The reproducibility of the measurements for aluminium, copper, iron and zinc was better than ±3.0%. It was worse for manganese and chromium (±6.0 and ±12.0%, respectively), since these two elements were present in very low concentrations in all the samples analysed.     

Selective tolerance of group III and IV somatosympathetic reflexes to the effects of alfentanil

The effect of alfentanil on responses in renal sympathetic nerves evoked by supramaximal electrical stimulation of the radial nerve, has been observed in 6 dogs anaesthesised with -chloralose, paralysed with suxamethonium and ventilated artificially. During an initial infusion of alfentanil the responses of the late group IV (C fibre) and early group III (Aδ) were abolished by mean doses of 68 μg kg⁻¹ (SEM 3.2 μg kg⁻¹) and 797 μg kg⁻¹ (SEM 120 μg kg⁻¹), respectively. Recovery was allowed to occur to approximately 50% of control values (mean time 76 ± 14.3 min). The preparations were then conditioned with 7 incremental doses from 7.5 to 120 μg kg⁻¹ (i.v.) (total dose 308.5 μg kg⁻¹), administered at intervals of 10 min, and subsequently tested with large bolus doses (up to 2000 μg kg⁻¹) of alfentanil. In two preparations, the responses of both group IV and group III became completely tolerant to the effects of alfentanil while in the other four the response of the group IV was still eliminated by the drug and the response of group III showed selective tolerance. The heart rate and arterial pressure were reduced by 45 and 29%, respectively during the initial infusion of alfentanil. Thereafter there were no further significant changes in the circulation until the administration of naloxone (2 mg i.v.), which restored the sympathetic responses, heart rate and arterial pressure to control values.     

Spectrophotometric determination of pefloxacin in pharmaceutical preparations

It has been established that the antibiotic pefloxacin (Abaktal) methane-sulphonate reacts with Fe(III) at pH 1.00–8.00 to form a water-soluble complex with maximum absorbance at 360 nm. The composition of the complex, determined spectrophotometrically by the application of Job's, molar-ratio and Bent—French's methods, was pefloxacin: Fe(III) = 1:1 (pH = 2.50; λ = 360 nm; μ = 0.1 M). The relative stability constant, obtained by the methods of Sommer and Asmus was 10^5.02 (pH = 2.50; λ = 360 nm; μ = 0.1 M). The molar absorptivity of the complex at 360 nm was found to be 4.8 × 10³ l mol⁻¹ cm⁻¹, Beer's law was followed for pefloxacin concentrations of 2.15–85.88 μg ml⁻¹. The lower sensitivity limit of the method was 2.15 μg ml⁻¹. The relative standard deviation (n = 10) was 0.57–1.07%. The method can be applied to the rapid and simple determination of pefloxacin in aqueous solutions and tablets.     

Determination of catecholamines by flow-injection analysis and high-performance liquid chromatography with chemiluminescence detection

A chemiluminescence (CL) detection of catecholamines [norepinephrine (NE), epinephrine (E), dopamine (DA) and l-dopa (LD)] is described for the flow-injection (FI) and high-performance liquid chromatographic (HPLC) determination of these compounds. The detection method is based on the inhibition effect of catecholamines (CAs) on the CL reaction of luminol with iodine in the alkaline medium. The proposed FI method allows the determination of CAs in pharmaceutical preparations for the purpose of drug quality control. The calibration curves show good linearity in the concentration range of: 1.1–20.0 μg l⁻¹ (NE), 0.5–5.0 μg l⁻¹ (E), 0.6–9.0 μg l⁻¹ (DA) and 0.6–10.0 μg l⁻¹ (LD). The limits of detection (defined as a signal-to-noise ratio of 3) are: 0.34 μg l⁻¹ (NE), 0.15 μg l⁻¹ (E) and 0.18 μg l⁻¹ (DA, LD). The HPLC procedure was successfully applied for the determination of catecholamines (NE, E, DA) in human urine after solid-phase extraction (SPE). In a simple run time CAs can be determined in 20 min. The chromatographic linear ranges are: 5.0–72.0 μg l⁻¹ (NE), 5.0–48.0 μg l⁻¹ (E) and 5.0–96.0 μg l⁻¹ (DA). The limits of detection for three urinary CAs are: 0.71 μg l⁻¹ (NE), 0.26 μg l⁻¹ (E) and 0.73 μg l⁻¹ (DA).     

Spectrophotometric determination of oxytetracycline in pharmaceutical preparations using sodium molybdate as analytical reagent

A spectrophotometric method is proposed for the determination of oxytetracycline in pharmaceutical preparations. The method is based on the measurement of the absorbance of the molybdate—oxytetracycline complex at 404 nm (pH 5.50; μ = 0.1 M; 20°C). The composition of the complex (1:1) was determined by the application of the spectrophotometric methods of Job and Bent—French (pH 5.50; λ = 390 nm; μ = 0.1 M). The relative stability constant (K′ = 10^4.6) of the complex was obtained by the methods of Sommer and Nash (pH 5.50; λ = 390 nm; μ = 0.1 M; 20°C). The molar absorptivity of the complex was 9.5 × 10³ l mol⁻¹ cm⁻¹. Beer's law was obeyed over the concentration range 2.48–34.78 μg ml⁻¹. The relative standard deviation RSD (n = 10) was 0.27–0.39%. The method proposed can be applied to the assay of oxytetracycline in capsules. The detection limit of oxytetracycline is 2.5 μg ml⁻¹.     

In vitro assay for human toxicity of cereulide, the emetic mitochondrial toxin produced by food poisoning Bacillus cereus

The in vitro boar spermatozoon test was compared with the LC ion trap MS analysis for measuring the cereulide content of a pasta dish, implemented in serious emetic food poisoning caused by Bacillus cereus. Both assays showed that the poisonous food contained approximately 1.6 μg of cereulide g⁻¹ implying the toxic dose in human as 8 μg kg⁻¹ body weight. The threshold concentration of cereulide provoking visible mitochondrial damage in boar sperm exposed in vitro was 2 ng of cereulide ml⁻¹ of extended boar sperm. The same threshold value was found for cereulide extracted from the food and from the cultured bacteria. This shows that other constituents of the food did not enhance or mask the effects of cereulide. Exposure of four human cell lines (HeLa, Caco-2, Calu-3 and Paju) to cereulide showed that the threshold concentration for the loss of mitochondrial membrane potential in human cells was similar to that observed in boar sperm. Human cells and boar sperm were equally sensitive to cereulide. The results show that boar spermatozoan assay is useful for detecting cereulide concentrations toxic to humans. Spermatozoa in commercially available extended fresh boar and cryopreserved bull semen were compared, boar sperms were 100 times more sensitive to cereulide than bull sperms.     

A capillary GC-MS method for analysis of phenytoin and [C3]-phenytoin from plasma obtained from pulse dose pharmacokinetic studies

Stable isotope analogues of phenytoin are useful for pulse dose pharmacokinetic studies in epilepsy patients. A simultaneous assay was developed to quantitate phenytoin (5,5-diphenylhydantoin) and its stable isotope analogue [¹³C₃]-phenytoin (5,5-diphenyl-2,4,5-¹³C₃-hydantoin) from plasma. Quantitation was achieved by GC-MS analysis of liquid/liquid extracted plasma samples, with [²H₁₀]-phenytoin (5,5-di(pentadeuterophenyl)-hydantoin) as an internal standard. The total coefficients of variance (C.V._t) were <7% for phenytoin (2.5–40 μg ml⁻¹) and <10.3% for [¹³C₃]-phenytoin (0.1–6.0 μg ml⁻¹). The accuracy of the assay varied from 87.8–100.1% (phenytoin, 2.5–40 μg ml⁻¹) and 89.6–116.3% ([¹³C₃]-phenytoin, 0.02–6.0 μg ml⁻¹). The assay was tested under in vivo conditions by administration of a pulse dose of the stable isotope analogue to a single rat dosed to steady-state with fosphenytoin, a phenytoin prodrug. The results of the in vivo experiment demonstrate the usefulness of this assay for future pharmacokinetic studies in special population epilepsy patients.     

Analysis of various nucleosides in plasma using solid phase extraction and high-performance liquid chromatography with UV detection

The National Cancer Institute (NCI) has screened many nucleosides for antiviral activity to the HIV-1 virus. Drugs demonstrating antiviral activity are tested in animal models to evaluate their toxicity and pharmacokinetic characteristics. These drugs are subsequently evaluated for efficacy in human clinical trials. Sensitive analytical methodology is needed to quantify nucleosides in plasma and other biological matrices in support of these studies. Battelle has modified and validated a reversed phase high-performance liquid chromatography (HPLC) method for several of these nucleosides that could be easily adapted for similar compounds. Methods have been validated for 6-chloro-2′,3′-dideoxyguanosine (6ClddG), 6-chloro-2′,3′-dideoxyinosine (6ClddI) and their primary metabolites 2′,3′-dideoxyguanosine (ddG) and 2′,3′-dideoxyinosine (ddI) in both rat and dog plasma containing EDTA. The method has also been validated for 2′-fluoro-2′,3′-dideoxyara-adenosine (βFlddA) and its primary metabolite 2′-β-fluorodideoxyinosine (βFddI) in rat plasma containing heparin. Calibration plasma standards were prepared over ranges of 0.1–10 μg ml⁻¹ for βFlddA and βFddI, 0.1–50 μg ml⁻¹ for 6ClddG and ddG, and 0.25–50 μg ml⁻¹ for 6ClddI and ddI in plasma containing 4 μg ml⁻¹ pentostatin. The addition of pentostatin to the plasma samples inhibits in-vitro deamination of the drug after collection. Quality control (QC) standards were prepared containing the appropriate anticoagulant and 4 μg ml⁻¹ pentostatin at concentrations within each of the bracketed calibration ranges in plasma. These methods have been successfully applied to plasma samples generated during various animal studies.     

In vitro dermal and transdermal delivery of doxycycline from ethanol/migliol 840 vehicles

This work investigated the feasibility of dermal and transdermal delivery of doxycycline from vehicles containing Migliol 840 (M840) and ethanol. Delivery of the drug via the skin would provide a useful alternative to oral delivery, which has many undesirable side-effects, such as oesophageal ulceration and disturbance of the normal gut flora. Potential applications include malaria prophylaxis, and the treatment of acne vulgaris, Lyme disease and Reiter syndrome. Experiments were performed to determine the permeation of doxycycline across excised full-thickness human skin and heat-separated epidermal membranes from saturated solutions in ethanol, 1:1 and 2:1 ethanol/M840. Unusual burst behaviour was observed using an ethanol vehicle, possibly as a result of the formation of dimers at saturation. Doxycycline permeated to a higher degree from ethanolic vehicles when M840 is present, suggesting that M840 is capable of enhancing the permeation of doxycycline. The flux across full-thickness skin was highest from a 2:1 ethanol:M840 vehicle (2.41 μg cm⁻² h⁻¹), sufficient to deliver 282 μg l⁻¹ using an area of application of 30 cm². The data also produced unexpected results in that permeability across heat separated skin was an order of magnitude greater than across full-thickness skin (28.75 μg cm⁻² h⁻¹ for the 2:1 ethanol:M840 vehicle). Depth profiling indicated that the drug distributed quite evenly throughout the epidermis. The mean amount of doxycycline recovered from the epidermis at the end of a permeation experiment was 458.4 μg ml⁻¹. This was far higher than the volume of extractable lipid present in the same unit area, approximately 52.3 μg ml⁻¹ and indicated that a large proportion of the drug must have been located within the proteinaceous domain. The data therefore suggest (1) significant amounts of doxycycline can be administered into and across the skin; (2) M840 is a potentially useful enhancing vehicle; and (3) the transcellular route was of significance.     

Simultaneous LC determination of paracetamol and related compounds in pharmaceutical formulations using a carbon-based column

A simple, rapid and convenient high performance liquid chromatographic method, which permits the simultaneous determination of paracetamol, 4-aminophenol and 4-chloracetanilide in pharmaceutical preparation has been developed. The chromatographic separation was achieved on porous graphitized carbon (PGC) column using an isocratic mixture of 80/20 (v/v) acetonitrile/0.05 M potassium phosphate buffer (pH 5.5) and ultraviolet detection at 244 nm. Correlation coefficient for calibration curves in the ranges 1–50 μg ml⁻¹ for paracetamol and 5–40 μg ml⁻¹ for 4-aminophenol and 4-chloroacetanilide were >0.99. The sensitivity of detection is 0.1 μg ml⁻¹ for paracetamol and 0.5 μg ml⁻¹ for 4-aminophenol and 4-chloroacetanilide. The proposed liquid chromatographic method was successfully applied to the analysis of commercially available paracetamol dosage forms with recoveries of 98–103%. It is suggested that the proposed method should be used for routine quality control and dosage form assay of paracetamol in pharmaceutical preparations. The chromatographic behaviour of the three compounds was examined under variable mobile phase compositions and pH, the results revealed that selectivity was dependent on the organic solvent and pH used. The retention selectivity of these compounds on PGC was compared with those of octadecylsilica (ODS) packing materials in reversed phase liquid chromatography. The ODS column gave little separation for the degradation product (4-aminophenol) from paracetamol, whereas PGC column provides better separation in much shorter time.