Resonance Rayleigh scattering technique as a detection method for the RP-HPLC determination of local anaesthetics in human urine.

A highly selective and sensitive method of reversed phase high-performance liquid chromatography (RP-HPLC) coupled with resonance Rayleigh scattering (RRS) was developed for the determination of procaine, bupivacaine and tetracaine. Separation of three local anaesthetics was achieved at 35 °C on a C18 column. The mobile phase was 30: 70 (v/v) acetonitrile/triethylamine-phosphoric acid buffer (pH 2.9) at flow rate of 0.3 mL/min. The RRS detection was conducted by taking advantage of the strong RRS enhancement of the local anaesthetics with erythrosine reaction in an acidic medium. Under optimum conditions, the limit of detection (S/N = 3) values were in the range of 2.4-11.2 ng/mL. Recoveries from spiked human urine samples were 95.8%-104.5%. The proposed method applied to the determination of local anaesthetics in human urine achieved satisfactory results. In addition, the mechanism of the reaction is fully discussed. Copyright © 2016 John Wiley & Sons, Ltd.

On-line preconcentration strategy for the simultaneous quantification of three local anesthetics in human urine using CZE.

The simultaneous determination of usually employed anesthetics (procaine, lidocaine, and bupivacaine) has been developed and validated using CE with ultraviolet detection at 212 nm. The separation of these three drugs has been achieved in less than 7 min, using a temperature of 25ºC and 25 kV, with a 150 mM citrate buffer (pH 2.5) as BGE. Field-amplified sample injection (FASI) has been used for on-line sample preconcentration. Ultrapure water and ACN 50/50 (v/v) mixture gave the greatest enhancement factor when it was employed as an injection solvent. Injection voltage and time were optimized, being 13 kV and 13 s, the optimum values, respectively. To avoid the possible irreproducibility associated with the electrokinetic injection, an internal standard such as tetracaine, was employed. The instrumental detection limits (LOD S/N = 3) for the compounds ranged between 2.6 and 7.0 µg L(-1) and the quantitation limits (LOQ S/N = 10) between 37.8 and 55.9 µg L(-1) . The detection limits obtained in real human urine samples ranged between 55.2 and 83.6 µg L(-1) and the quantitation limits between 196.0 and 276.0 µg L(-1) . The proposed method has demonstrated its applicability to the analysis of these local anesthetics in urine samples without any pretreatment, allowing the rapid determination of these target analytes.

Metabolism of benoxinate in humans.

The metabolism of benoxinate hydrochloride [2-(diethylamino)ethyl 4-amino-3-butoxybenzoate monohydrochloride; oxybuprocaine] was examined in humans after administration of a single oral dose. The drug was almost completely absorbed and was rapidly excreted in the urine (92.1% of dose in 9 h). Nine metabolites and unchanged drug were isolated from the urine and identified by comparison of TLC, GC, and GC-MS with authentic compounds. Any metabolites reflecting initial loss of the butyl side chain of benoxinate could not be detected. This suggests that the ester portion is metabolized more rapidly than the O-butyl side chain. 3-Butoxy-4-aminobenzoic acid, the hydrolyzed product of benoxinate, was primarily excreted (70-90% of dose) as the glucuronide together with a trace of the glycine conjugate (0.35% of dose). In addition, 3-butoxy-4-acetylaminobenzoic acid, 3-hydroxy-4-aminobenzoic acid, and 3-hydroxy-4-acetylaminobenzoic acid were identified, the latter two being detected partly as the glucuronides (1.20 and 1.43% of dose, respectively).

Differential pulse adsorption voltammetry for determination of procaine hydrochloride at a pumice modified carbon paste electrode in pharmaceutical preparations and urine.

Procaine hydrochloride was determined by the differential pulse voltammetry (DPV) using a 6% (m/m) pumice modified carbon paste electrode in 1.25 x 10(-3) mol x l(-1) KH(2)PO(4) and Na(2)HPO(4) buffer solution (pH 6.88, 25 degrees C). The anodic peak potential used was +0.980 V (vs. SCE). A good linear relationship was realized between the anodic peak current and procaine concentration in the range of 9.0 x 10(-7)-2.6 x 10(-5) mol x l(-1) with the detection limit of 5.0 x 10(-8) mol x l(-1). The recovery was 95.2-104.8% with the relative standard deviation of 3.2% (n=10). The pharmaceutical preparations, procaine hydrochloride injection and the urine samples were determined with the desirable results.

Determination of procaine in equine plasma and urine by high-performance liquid chromatography.

The variability in plasma and urine equine procaine measurement between three independent laboratories using current methods led to the development of a sensitive, reliable, and reproducible high-performance liquid chromatographic method. Standardbred mares were administered either a penicillin G procaine preparation intramuscularly or procaine hydrochloride subcutaneously, and blood and urine were collected at defined time intervals. By HPLC the detection limits for procaine in plasma and urine were 1 and 10 ng/mL, respectively. In contrast procaine in plasma could not be detected by GC-NPD, while the urinary detection limit was 50 ng/mL. The concentration of fluoride in the collection tubes and repetitive freeze-thawing modified plasma procaine measurement. Urinary pH was a factor in estimation of urine procaine levels with greater recovery and reproducibility of results at pH 5 as compared to pH 7. This HPLC method provides a simple, sensitive, and reliable quantitation of procaine in equine plasma and urine.

Veterinary aspects of doping.

Doping can improve or impair performance and can be done either deliberately or accidentally. Accidental doping to win is the offence which most concerns the veterinary surgeon. The distinction between legitimate therapy and assisting an unfit horse to win a race by giving it a drug is a fine one. General guidelines are presented for the veterinary surgeon in practice.

Plasma elimination and urinary excretion of procaine after administration of different products to standardbred mares.

Plasma and urinary concentrations of procaine were examined in Standardbred mares after subcutaneous administration of various doses (80 mg to 1600 mg) of procaine hydrochloride. Regardless of dose, peak plasma procaine values occurred within 1 h, but remained detectable in a dose-dependent manner, with procaine present at 1 h with the 80 mg dose and 6 h at the 1600 mg dose. Similarly, peak urinary procaine concentrations were attained within 1.5 to 3 h, irrespective of dose, while detection time was dose-dependent, being 23 h for 80-200 mg doses but as long as 30-54 h with the 1600 mg dose. When mares were given a single intramuscular injection of a penicillin G-procaine preparation (Ethacillin, Cillimycin, Penamycin, Derapen A, Azimycin or Diathal), peak plasma procaine concentrations varied and were reached from 10 min to 3 h in all cases, with detection from 3 to 20 h after drug administration. Although the peak urinary levels of procaine occurred between 30 mins and 6 h, detection in urine in most cases was as long as 78-120 h except for Diathal for which detection was limited to 54 h. Daily administration of a penicillin G-procaine preparation (Pen-Di-Strep) for 5 days produced a biphasic peak in plasma procaine at 3 and at 6-9 h with detection from 16 to 23 h after drug treatment. Although peak urinary procaine values were reached at similar times after single or multiple injections, the duration of detection was markedly longer (425 h) after the multiple-dose regimen.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulatory significance of procaine residues in plasma and urine samples: preliminary communication.

Plasma and urinary concentrations of procaine and the duration of response to procaine after its administration as a local anaesthetic to horses were studied. Following injection of a clinical dose of procaine HCl (80 mg), the concentration of procaine in plasma was less than the lower limit of quantitation and unsuitable for threshold determination. Therefore, the urinary concentration of procaine was determined after injection of a dose of 5 mg procaine HCl, the highest no-effect dose (HNED) of this agent. Free unconjugated procaine in equine urine reached a peak concentration of 23.7 ng/mL, while total (unconjugated plus conjugated) procaine peaked at 37.9 ng/mL (mean urine pH of 8.5). Because a basic drug may concentrate substantially in acidic urine, a threshold concentration of 25 ng/mL of unconjugated procaine is a reasonable and conservative threshold for procaine at this time. Horses were administered abaxial sesamoid blocks containing 2% procaine HCl (40, 80, 160 and 320 mg) and 2% procaine HCl (40 and 320 mg) with epinephrine (1:100,000) in local anaesthetic experiments. There was a significant local anaesthetic (LA) effect for all doses of procaine HCl with the duration of effect ranging from 30 min (40 mg) to 60 min (320 mg). The addition of epinephrine significantly increased the duration of local anaesthesia to 180 min for a 40 mg dose and 420 min for a 320 mg dose. Because epinephrine may extend the duration of local anaesthesia beyond a reasonable period of confinement for horses before the starting time of a race, the increased LA effect following the addition of epinephrine to procaine has regulatory significance.

Procaine in the urine of racing Greyhounds: possible sources.

Greyhounds (n = 25) were given procaine in the form of procaine HCl or procaine penicillin G or were fed meat prepared from a heifer given procaine penicillin G on 3 consecutive days before slaughter. Dogs given procaine HCl or procaine penicillin G were given daily doses equivalent to 9 mg of procaine/kg. Urine samples were collected from the dogs twice daily before dosing, during the dosing period, and for 4 days after final dose administration. All dogs excreted detectable concentrations of procaine in the urine, regardless of the dose form or route of administration. Blood plasma samples were prepared from 10 Greyhounds to determine procaine esterase activity. Hydrolysis of procaine by plasma esterases did not occur. Low plasma procaine esterase activity, coupled with rapid oral absorption of procaine, resulted in high urinary concentrations of the parent drug in dogs given procaine HCl or procaine penicillin G (9 mg/kg). Even in the dogs given relatively small doses (0.85 mg/kg) of procaine in the form of meat residues, urinary procaine concentrations were found. The results of these studies indicate that procaine is rapidly absorbed following oral administration and that meat from livestock given procaine penicillin before slaughter may serve as a source of urinary procaine in Greyhounds consuming the meat.

Oxybuprocaine and five metabolites simultaneously determined in urine by gas chromatography and gas chromatography-mass spectrometry after extraction with Extrelut.

We describe a gas-liquid chromatographic (GC) method for determination of oxybuprocaine, and a gas chromatographic-mass spectrometric (GC-MS) method for simultaneous determination of four of its nine metabolites in urine. We used an Extrelut column to simply and rapidly extract oxybuprocaine and its metabolites from urine. For the GC-MS analyses, we monitored the characteristic fragment ions at m/z 353, 395, 369, 411, and 235 for 3-butoxy-4-aminobenzoic acid (metabolite 2, M-2), 3-butoxy-4-acetylaminobenzoic acid (M-3), 3-hydroxy-4-aminobenzoic acid (M-4), 3-hydroxy-4-acetylaminobenzoic acid (M-5), and methaqualone (internal standard), respectively. We quantified the glucuronide of M-2 after enzymic treatment. The assay's selectivity and reproducibility (within-day and between-day CVs less than 8% for all metabolites) make it applicable to determine oxybuprocaine and its metabolites in human urine. Mean 9-h urinary excretion of oxybuprocaine and its five metabolites from four healthy volunteers was 89.2% after a 100-mg oral dose.

Recovery of procaine from biological fluids.

A published method for the recovery of procaine from human plasma using 5M NaOH gave very poor recoveries. Investigation showed that under the recommended extraction conditions procaine was rapidly hydrolysed. Extraction into benzene of samples buffered to pH 9.0 with borate buffer allowed essentially 100% recovery of procaine from equine plasma and urine.

Solid-phase cation exchange extraction of basic drugs from the urine of racing greyhounds.

The extraction of basic drugs from the urine of racing greyhounds has been carried out for many years using solvent extraction. This paper describes an extraction method that utilises a cation exchange column. Fourteen basic drugs were introduced onto the column in their ionised form, washed with organic and aqueous solvents and eluted with basic methanol. Three drugs, cyclizine, procaine and quinine were determined in authentic samples from racing greyhounds. A comparison of the solid-phase method with liquid-liquid extraction showed an increase in the efficiency of extraction and a reduction in both the time and cost incurred. The method requires a minimal volume of sample and solvent, little glassware and is easily automated.

Quantification of penicillin-G and procaine in equine urine and plasma using high-performance liquid chromatography.

A rapid and sensitive method for the extraction and quantification of penicillin-G and procaine in horse urine and plasma samples has been successfully developed. The method involves the use of solid-phase extraction (SPE) for penicillin-G, liquid-liquid extraction (LLE) for procaine, and high-performance liquid chromatography (HPLC) for the quantification of penicillin-G and procaine. The new method described here has been successfully applied in the pharmacokinetic studies of procaine, penicillin-G and procaine-penicillin-G administrations in the horse.