Molecularly imprinted microspheres synthesized by a simple, fast, and universal suspension polymerization for selective extraction of the topical anesthetic benzocaine in human serum and fish tissues.

A simple, fast, and universal suspension polymerization method was used to synthesize the molecularly imprinted microspheres (MIMs) for the topical anesthetic benzocaine (BZC). The desired diameter (10-20 µm) and uniform morphology of the MIMs were obtained easily by changing one or more of the synthesis conditions, including type and amount of surfactant, stirring rate, and ratio of organic to water phase. The MIMs obtained were used as a molecular-imprinting solid-phase-extraction (MISPE) material for extraction of BZC in human serum and fish tissues. The MISPE results revealed that the BZC in these biosamples could be enriched effectively after the MISPE operation. The recoveries of BZC on MIMs cartridges were higher than 90% (n = 3). Finally, an MISPE-HPLC method with UV detection was developed for highly selective extraction and fast detection of trace BZC in human serum and fish tissues. The developed method could also be used for the enrichment and detection of BZC in other complex biosamples.

Benzocaine-induced methemoglobinemia in an acute-exposure rat model.

Tricaine methanesulfonate, a sedative for temporarily immobilizing fish, has a 21-day withdrawal time. Benzocaine has been proposed as an alternative sedative because a withdrawal period may not be required. Since benzocaine is known to induce methemoglobinemia, the potential for orally administered benzocaine to induce methemoglobin was assessed in rats. Sprague-Dawley rats were given a single gavage administration of 64mg benzocaine hydrochloride per kg bw and then euthanized at intervals up to 120min. Plasma levels of benzocaine were relatively low at all times, whereas methemoglobin peaked at 24min. Additional rats were orally gavaged with 0-1024mg benzocaine hydrochloride per kg bw and euthanized after 24min. Plasma levels of benzocaine increased from 0.01µM at 2mg per kg bw to 2.9µM at 1024mg per kg bw. Methemoglobin levels did not differ from controls at doses up to 32mg per kg bw in females and 64mg per kg bw in males, whereupon the value increased to ∼80% at 1024mg per kg bw. These data were used to estimate the potential impact of benzocaine residues in fish and suggest that the consumption of fish treated with benzocaine hydrochloride will not cause methemoglobinemia in humans.

Death of an infant involving benzocaine.

This report describes the death of a four-month-old Hispanic male which may be related to benzocaine toxicity. A toxicological evaluation revealed benzocaine at a concentration of 3.48 mg/L, and postmortem methemoglobin of 36% (normal 0.4-1.5). Methemoglobinemia is a complication of benzocaine toxicity. In light of the toxicology findings, the coroner investigated the source of the benzocaine and discovered that the child was treated with Zenith Goldline Allergen Ear Drops containing 0.25% w/v benzocaine and 5.4% w/v antipyrine. There was an admission by a caregiver that on the day prior to the child's death, he had been treated with three times the prescribed dose. Blood benzocaine concentrations in nine other unrelated cases were determined and concentrations ranged from <0.05-5.3 mg/L (mean 1.48 mg/L). Seven of the nine cases were positive for drugs of abuse, and one additional case was described as a known drug user. Methemoglobin in these benzocaine positive cases ranged from 6-69%; however, methemoglobin concentrations in postmortem cases are frequently elevated and should be interpreted with caution. The unknown significance of the benzocaine, and the circumstances of the case raise questions about the ultimate attribution of this death to SIDS.

Effect of temperature on the pharmacokinetics of benzocaine in rainbow trout (Oncorhynchus mykiss) after bath exposures.

The pharmacokinetics of benzocaine during bath exposures at 1 mg/L were determined in rainbow trout acclimated at 6 degrees C, 12 degrees C or 18 degrees C for at least 1 month. Individual fish were exposed to benzocaine in a recirculating system for 4 h and pharmacokinetic parameters were estimated in a unique manner from the concentration of benzocaine in the bath water vs. time curve. Elimination from plasma was also determined after the 4 h exposure. The uptake clearance and metabolic clearance increased with increased acclimatization temperatures (uptake clearance 581 +/- 179 mL/min/kg at 6 degrees C and 1154 +/- 447 mL/min/kg at 18 degrees C; metabolic clearance 15.2 +/- 4.1 mL/min/kg at 6 degrees C and 22.3 +/- 4.2 mL/min/kg at 18 degrees C). The apparent volume of distribution had a trend for increasing with temperature that was not significant at the 5% level (2369 +/- 678 mL/kg at 6 degrees C to 3260 +/- 1182 mL/kg at 18 degrees C). The elimination half-life of benzocaine in plasma was variable and did not differ significantly with temperature (60.8 +/- 30.3 min at 6 degrees C to 35.9 +/- 13.0 min at 12 degrees C). Elimination of benzocaine from rainbow trout is relatively rapid and even more rapid at higher acclimatization temperatures based on calculated metabolic clearances and measured plasma concentrations, but was not evident by measurement of terminal plasma half-lifes.

Determination of benzocaine in rainbow trout plasma.

A liquid chromatographic method is described for analysis of benzocaine (BZ), a proposed fish anesthetic, in rainbow trout plasma. Mean recoveries of BZ from plasma samples fortified at 44-10 100 ng/mL were 96-100%. The method detection limit is 10 ng/mL, and the limit of quantitation is 37 ng/mL. Acetylation of BZ occurs in whole blood after storage at room temperature (i.e., 21 degrees C) for 10 min. However, no acetylation of BZ was detected in plasma samples held at room temperature for 4 h. Mean method precision for plasma samples with incurred BZ residue is similar to that for fortified samples in the same concentration range (relative standard deviations of 0.9 and 1.2%, respectively).

Determination of highest no effect dose (HNED) for local anaesthetic responses to procaine, cocaine, bupivacaine and benzocaine.

The highest no effect doses (HNEDs) for the local anaesthetic (LA) effects of procaine, cocaine, bupivacaine and benzocaine were determined using the heat lamp/hoof withdrawal model of Kamerling et al. (1985b) and the abaxial sesamoid block model of local anaesthesia. The heat lamp rapidly (4 or 5 s) increased the temperature of the superficial skin layers of the pastern to about 90 degrees C, at which point the animal sharply withdrew its hoof. Effective LA blockade precluded this response and superficial skin temperatures exceeded 120 degrees C. Thermal stimulus experiments were routinely terminated after 10 s of exposure to prevent undue tissue damage. Following abaxial sesamoid block with bupivacaine, the HNED for that drug was about 0.25 mg/site. Increasing the dose to 2 mg/site apparently produced complete and prolonged LA blockade. Analogous work showed that the HNED for procaine was about 2.5 mg/site. Similarly, the dose response curve for procaine was parallel with that of bupivacaine but was shifted 10-fold to the right. The duration of the LA response following procaine injection was less than for bupivacaine with the statistically significant response following 40 mg/site injection lasting less than 45 min. Cocaine was less potent than procaine, showing a shallower dose response curve. The HNED for cocaine was less than 5 mg/site, although at this dose the duration of action was extremely short (< 7.5 min). Benzocaine had no significant LA action when a dose of 800 mg was applied topically as a 5% preparation. These results show that the HNEDs for bupivacaine and procaine are remarkably low, that cocaine is somewhat less potent as a LA than might be expected, and that 5% topical benzocaine has no significant pharmacology. The small doses of bupivacaine and procaine producing effective local anaesthesia suggests that developing plasma thresholds for these agents is likely to be very challenging.

Capillary gas chromatographic method for the determination of n-butyl-p-aminobenzoate and lidocaine in plasma samples.

A fast capillary gas chromatographic method with nitrogen-selective detection is described that allows selective and reproducible quantification of n-butyl-p-aminobenzoate (BAB) and lidocaine in plasma. The sampling and sample storage conditions are critical for the quantification of BAB. Diisopropyl fluorophosphate, an organo-phosphorus pesticide, has to be added during sampling to prevent the rapid decomposition of BAB by cholinesterases.

Preliminary study to assay plasma amethocaine concentrations after topical application of a new local anaesthetic cream containing amethocaine.

Plasma concentrations of amethocaine were measured after topical application of amethocaine cream 2 g (5% w/w) to the dorsum of the right hand of 10 adult volunteers. The cream was applied for 240 min and plasma was assayed for amethocaine and its metabolite p-n-butylaminobenzoic acid at 0, 30, 60, 90, 120 and 240 min in all 10 volunteers, and at 360 min in seven volunteers, by high pressure liquid chromatography. No amethocaine was detected in the plasma of seven volunteers. Plasma concentrations of amethocaine up to 0.20 mg litre-1 were observed in three volunteers. No significant side effects were seen and pain scores on insertion of a 16-gauge cannula were 0 in all subjects. We conclude that the absence of clinical toxicity in the 10 healthy volunteers was a reflection of slow absorption and tissue hydrolysis of amethocaine after topical dermal application.

Determination of benzyl alcohol and its metabolite in plasma by reversed-phase high-performance liquid chromatography.

A study undertaken following recent reports of deaths in neonatal children associated with the use of benzyl alcohol resulted in the development of a stability-indicating high-performance liquid chromatographic assay of benzyl alcohol in plasma using benzocaine as internal standard. Thawed plasma samples were diluted and subjected to solid-phase extraction using Extrelut and eluted with ethyl acetate. The evaporated eluate was reconstituted with mobile phase and chromatographed on a C18 column with water-acetonitrile-glacial acetic acid as mobile phase and detection at 254 nm. Baseline separation was achieved within 12 min for benzyl alcohol, benzaldehyde, benzoic acid, hippuric acid and benzocaine. Peak-height ratios were linear over 80-640 ng of benzyl alcohol injected (r = 0.998) and over 10-80 ng of benzoic acid injected (r = 0.999). Benzaldehyde and hippuric acid were not quantitated because these compounds were not detectable in actual dog plasma. Validation studies by spiking dog plasma with benzyl alcohol and benzoic acid gave overall percent recoveries (+/- relative standard deviation, n = 4) of 98.3 +/- 3.0 and 101.4 +/- 7.6%, respectively. The method was applied to the assay of actual plasma samples. Since benzyl alcohol is very susceptible to oxidation to benzaldehyde and benzoic acid, its purity in bulk liquid samples can be determined by this method.