Simultaneous determination of procaine, lidocaine, ropivacaine, tetracaine and bupivacaine in human plasma by high-performance liquid chromatography.

A simple and sensitive high-performance liquid chromatography with ultraviolet detection (HPLC-UV) method has been developed and validated for simultaneous quantification of five local anesthetics in human plasma: procaine, lidocaine, ropivacaine, tetracaine and bupivacaine. In an ice-water bath, 500 microL plasma sample, containing 100 microg/mL neostigmine methylsulfate as anticholinesterase, was spiked with carbamazepine as internal standard and alkalized by sodium hydroxide. Liquid-liquid extraction with ethyl ether was used for plasma sample preparation. The chromatographic separation was achieved on a Kromosil ODS C18 column with a mobile phase consisting of 30 mM potassium dihydrogen phosphate buffer (0.16% triethylamine, pH adjusted to 4.9 with phosphoric acid) and acetonitrile (63/37, v/v). The detection was performed simultaneously at wavelengths of 210 and 290 nm. The chromatographic analysis time was 13 min per sample. The calibration curves of all five analytes were linear between 0.05 and 5.0 microg/mL (r(2) > or = 0.998). Precision ranged from 1.4% to 7.9% and accuracy was between 91.7% and 106.5%. The validated method is applicable for simultaneous determination of procaine, lidocaine, ropivacaine, tetracaine and bupivacaine for therapeutic drug monitoring and pharmacokinetic study.

The quantitation of procaine in equine plasma by liquid chromatography-linear ion trap mass spectrometry.

A method for the extraction and quantitation of procaine in equine plasma was developed for use with liquid chromatography-mass spectrometry (LC-MS). Procaine was isolated from equine plasma by liquid-liquid extraction at pH 11 with dichloromethane using procaine-d10 as an internal standard. Quantitation was achieved by LC-MS using a 3-microm C-18 column coupled to an electrospray ionization source on a linear ion-trap mass spectrometer. The limit of detection and limit of quantitation was determined to be 50 and 200 pg/mL, respectively. The lowest limit of detection determined by previous methods was 1 ng/mL. Administration samples were obtained as part of a larger study to determine a regulatory limit for procaine in racehorses and procaine concentrations were determined using this method.

Plasma concentration and local anesthetic activity of procaine hydrochloride following subcutaneous administration to horses.

OBJECTIVE: To determine the durations of the local anesthetic effect and plasma procaine concentrations associated with 5- and 10-mg doses of procaine hydrochloride (with or without 100 microg of epinephrine) administered SC over the lateral palmar digital nerves of horses. ANIMALS: 6 healthy adult horses. PROCEDURES: The hoof withdrawal reflex latency (HWRL) period was determined by use of a focused heat lamp before and after administration of procaine with and without epinephrine. Blood samples were collected immediately before determination of each HWRL period to assess plasma concentrations of procaine via liquid chromatography-mass spectrometry-mass spectrometry (LC-MS-MS). RESULTS: 10 but not 5 mg of procaine alone and 5 and 10 mg of procaine administered with epinephrine significantly prolonged the HWRL period (mean durations of effect, 5, 120 and 180 minutes, respectively), compared with baseline values. Plasma procaine concentrations did not correlate well with local anesthetic activity; for example, although the HWRL was prolonged to the maximum permitted duration of 20 seconds at 60 to 180 minutes following administration of the 5-mg dose of procaine with epinephrine in certain horses, plasma procaine concentrations were less than the limit of quantitation of the LC-MS-MS assay. CONCLUSIONS AND CLINICAL RELEVANCE: Small doses of procaine coadministered with epinephrine provided long-lasting local analgesia and resulted in plasma procaine concentrations that were not always detectable via LC-MS-MS. On the basis of these results, the use of regulatory limits or thresholds for procaine concentration in equine plasma samples obtained after racing should be seriously reconsidered.

[Analysis of 37 drugs in whole blood by HPLC after solid phase extraction].

OBJECTIVE: To develop a specific, sensitive, reproducible SPE-HPLC method for the determination of 37 drugs in whole blood. METHODS: With the doxapram as internal standard, Oasis column was used to extract drugs from whole blood. Two kinds of mobile phases were used in this study. Separations were achieved by a LiChrospher 100 RP-C18 (250 mm x 4.0 mm x 5 microm) column kept at 50 degrees C, the DAD detector was set at 230 nm and 250 nm. RESULTS: The limit of detection were 1-30 ng/mL. The method showed excellent linearity and the linear correlation coefficient was > or =0.997 98. The relative standard deviation for between-day and within-day assay were <10%. CONCLUSION: The method is effective, simple, reliable and has been used in real cases.

A sequential injection fluorometric procedure for the determination of procaine in human blood and pharmaceuticals.

An automated procedure for the assay of procaine hydrochloride in human blood and pharmaceuticals was developed using a sequential injection (SI) technique with fluorometric detection and fluorescamine as the fluorescence probe. A few microliters of fluorescamine and procaine hydrochloride solutions were used in the SI system leading to the formation of a derivative, which was then excited by a 400-nm LED and whose emitted fluorescence was monitored at a wavelength of 494 nm. A linear calibration graph was obtained with 10-200 ng mL(-1) (procaine) by loading 10.0 microL of sample solution and 5.0 microL of fluorescamine solution (both 0.125 % m/v). A detection limit of 2.6 ng mL(-1), defined as 3 times the blank standard deviation (3sigma), was achieved along with a sampling frequency of 25 h(-1) and a precision of 2.1 % RSD at the 50.0 ng mL(-1) level. Procaine contents in injection solutions from various pharmaceutical manufactures were analyzed and reasonable agreement was achieved between the values obtained by using the present procedure and the documented spectrophotometry, and both were coincident with the nominal concentrations. In addition, the degradation of procaine in human blood was investigated. A fast degradation of procaine in human blood was observed for the first 30 min, while afterwards the degradation was retarded.

Determination of trace procaine hydrochloride by differential pulse adsorptive stripping voltammetry with a Nafion modified glassy carbon electrode.

A sensitive and selective method for the determination of procaine hydrochloride with a Nafion-modified glassy carbon electrode has been developed. The voltammetric behavior of procaine hydrochloride on the Nafion-modified electrode indicated that the modified electrode not only increased the sensitivity of the determination of procaine hydrochloride, but also catalyzed the electrode process. Procaine hydrochloride was accumulated in Britton-Robinson buffer (pH 2.09) at a potential of -0.2 V (vs. SCE) for 180 s, and was then determined by differential pulse adsorptive stripping voltammetry. The effect of various parameters, such as the pH of the medium, the mass of drop-coated Nafion, the accumulation potential, the accumulation time and the scan rate, were investigated. Under the optimum conditions, a linear calibration graph was obtained in the concentration range of 6.0 x 10(-8) to 6.0 x 10(-6) mol l(-1) with a correlation coefficient of 0.9987. The relative standard deviation was 4.18% for eight successive determinations of 1.0 x 10(-7) mol l(-1) procaine hydrochloride, and the detection limit (three times signal to noise) was 7.0 x 10(-9) mol l(-1). A study of interfering substances was also performed, and the method was applied to the direct determinations of procaine hydrochloride in the injection solution of procaine hydrochloride and in rabbit serum.

Determination of the stability of drugs in plasma.

In order for a drug to be effective, enough of the active form must reach the target to elicit the desired effect. The first-pass effect for drugs taken orally is well known but the metabolism of drugs in plasma must also be considered during the drug discovery and development process. For instance, hydrolysis of a compound by plasma enzymes can significantly alter the bioavailability of the active compound. This unit describes methods for the determination of the stability of compounds in plasma.

High-performance liquid chromatographic analysis of the 2-chloroprocaine metabolite, diethylaminoethanol, in blood and serum.

BACKGROUND AND OBJECTIVES: 2-Chloroprocaine is rapidly metabolized in the blood to yield 2-chloro-para-aminobenzoic acid (an inactive metabolite) and diethylaminoethanol (DEAE). DEAE possesses local anesthetic activity. The only reported assay for DEAE is a colorimetric method. METHODS: Clinical samples of whole blood and serum were obtained from patients receiving stepped intravenous infusions of 3% 2-chloroprocaine. A high pH-dependent liquid-liquid extraction step with diethyl ether was used to eliminate interfering peaks in high-pressure liquid chromatography (HPLC) analysis. Separation and quantitation were performed using HPLC on a polymeric-reversed phase column with a mobile phase consisting of 10% or 20% acetonitrile (for whole blood or serum analysis, respectively) in 50 mm aqueous sodium phosphate buffer, pH = 11.50. The elution order of DEAE and its analogues was tested to interpret the HPLC separation mechanism. RESULTS: Extraction recovery of DEAE from whole blood was 67 +/- 13.5%, from serum, 71 +/- 12.2%, and from water, 75 +/- 2.9%. The high pH value of the mobile phase resulted in sharp, well-resolved peaks with run times of approximately 8 minutes using 20% acetonitrile. The lower limit of detection was 5 ng/mL of DEAE from a 1-mL sample. The elution order of DEAE and its analogues indicated that separation was based on the hydrophobicity of the analytes rather than polar group interactions occurring with silica-based stationary phase. CONCLUSIONS: A new, simple and rapid HPLC method for extraction and measurement of DEAE in whole blood or serum samples is reported here.

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.

Determination of ester-type local anesthetic drugs (procaine, tetracaine, and T-caine) in human serum by wide-bore capillary gas chromatography with nitrogen-phosphorus detection.

A sensitive method for simultaneous determination of ester-type local anesthetic drugs (procaine, tetracaine, and T-caine) has been developed using wide-bore capillary gas chromatography with nitrogen-phosphorus detection (GC-NPD). The extraction procedure, the experimental conditions for heptafluorobutyryl (HFB) derivative formation, and the percentage of the ester-type local anesthetic drugs from the human serum are described. The HFB derivatives of ester-type local anesthetic drugs showed sensitivity of approximately 2-3 fold higher than that without derivatization. The detection limits of HFB derivatives of the ester-type local anesthetic drugs were approximately 60-70 pg on column. Recoveries from the human serum were 85-94%. This method could be used to determine concentrations as low as 24-28 ng/mliters of the ester-type local anesthetic drugs.

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.

Rapid determination of chloroprocaine and its major metabolite, 2-chloroaminobenzoic acid, in plasma by high-performance liquid chromatography.

A sensitive and specific high-performance liquid chromatographic method for determination of the 2-chloroprocaine, local anesthetic of ester type, and its major metabolite 2-chloroaminobenzoic acid, has been developed and validated. A single-step extraction procedure is employed followed by high-performance liquid chromatographic separation using reversed-phase column and analysis using variable length UV detection. Lidocaine was used as internal standard for 2-chloroprocaine measurement and p-aminobenzoic acid was used as internal standard for 2-chloroaminobenzoic acid analysis. The analysis of spiked plasma demonstrated good accuracy and precision of the method with limit of detection 0.1 microgram/ml for 2-chloroprocaine and 0.5 microgram/ml for 2-chloroaminobenzoic acid. The method has been used for pharmacokinetic studies in laboratory animals.

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.

Procaine incorporation into human erythrocyte membrane--a spin label study.

The positioning of procaine among the components of human erythrocyte membranes was investigated by electron paramagnetic resonance (EPR) using several spin labelled molecules: fatty acids 5-, 12- and 16-doxyl-stearic acids (5DSA, 12DSA, 16DSA), procaine (SLP) and a quaternary ammonium salt (CAT4). It was proved that while procaine significantly modified EPR parameters of both CAT4 and 5DSA, it had no effect at the level of the twelfth or sixteenth carbon atom of the membrane lipid chains. Simultaneous incorporation of 5DSA and SLP and SLP in erythrocyte membranes revealed a shorter than 10 A distance between paramagnetic centers of 5DSA and SLP. It is inferred that procaine interacts with the membrane not only at lipid water interface, but also (at least) up to the fifth carbon atom of the hydrophobic tail of phopolipids. Procaine inclusion does not change the transition point (40 degrees C) at which a break appears in the temperature dependence of the erythrocyte volume, hence no strong interaction between the procaine and the cytoskeleton or the integral proteins responsible for this phase transition occurs. In the temperature domain of 25-42 degrees C, procaine seems to be freely diffusible within the membrane (Debye model can be applied).

Continuous caudal anesthesia for inguinal hernia repair in former preterm infants.

STUDY OBJECTIVE: To determine the feasibility of continuous caudal anesthesia with 2-chloroprocaine in conscious former preterm infants undergoing inguinal hernia repair. DESIGN: Prospective study. SETTING: University-affiliated children's hospital. PATIENTS: Ten former preterm infants, ASA physical status II and III, who were 35 to 49.5 weeks postconceptional age at the time of surgery. INTERVENTIONS: Caudal anesthesia was administered via an indwelling catheter using a loading dose of 1 ml/kg (30 mg/kg) of 3% 2-chloroprocaine, followed by incremental doses of 0.3 ml/kg (9 mg/kg) to achieve a level of T4 to T2. The block was maintained by a minimum infusion rate of 30 mg/kg/hr (1 ml/kg/hr) of the same local anesthetic solution. MEASUREMENTS AND MAIN RESULTS: The mean cumulative dose of 2-chloroprocaine was 2.8 +/- 1.0 ml/kg/hr (84 +/- 30 mg/kg/hr) infused over a mean duration of 95 +/- 35 minutes. Serum cholinesterase concentration and plasma 2-chloroprocaine concentration were measured in five infants. CONCLUSIONS: Three percent 2-chloroprocaine can be used effectively for continuous caudal anesthesia in conscious, former preterm infants for inguinal hernia and penoscrotal surgical procedures lasting 85 to 170 minutes.

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.

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)

Study on brain uptake of local anesthetics in rats.

Brain uptake of local anesthetics under steady-state plasma condition and/or following intravenous bolus administration was investigated in rats. All ester-type anesthetics examined such as ethyl (Et), propyl (Pr), butyl (Bu) esters of p-aminobenzoic acid (PABA), and procaine disappeared rapidly from plasma in a dose-dependent manner. Plasma profiles of these compounds were well explained by a 2-compartment model with a Michaelis-Menten type elimination process from a central compartment. On the other hand, lidocaine, amide-type anesthetic, showed a linear pharmacokinetic characteristic and its half life in the elimination phase was far longer than those of ester type agents. Extents of brain uptake (brain-to-plasma partition coefficient, Kp value) of these drugs were determined at 3 different steady-state plasma concentrations (1-15 microM). The Kp value of each drug was similar under the three different steady-state plasma concentrations. The Kp value increased in the following order; procaine (1.1) less than PABA-Et (1.9) less than lidocaine (2.2) less than PABA-Pr (2.7) less than PABA-Bu (3.6). A linear relationship was observed between the Kp value and the logarithmic value of the partition coefficient obtained in n-heptane/water or n-octanol/water partition system. The value of PABA-Et and PABA-Bu following intravenous bolus administration were varied with time elapsed but the mean values were almost same with those obtained under steady-state plasma conditions.

Effect of procaine hydrochloride on the electrophoretic mobility of human red blood cells.

The interaction of cationic anesthetics with biological membranes and the resulting alterations of membrane electrokinetic properties continue to be of current interest. The present study was designed to examine the effects of procaine hydrochloride (PRHCL) on the mobility of human red blood cells (RBC); electrophoretic measurements were made on RBC suspended in phosphate-buffered saline (PBS, pH = 5.0, 7.4, or 9.2), autologous plasma or 3 g% dextran T70/PBS (pH = 7.4), with PRHCL concentrations from 8 x 10(-6) to 8 x 10(-2) M. Low concentrations of PRHCL (8 x 10(-5)-8 x 10(-3) M) significantly (p less than 0.001) increased RBC mobility, with a maximal increase of 8.2% at 8 x 10(-4) M. Conversely, a higher PRHCL concentration (8 x 10(-2) M significantly (p less than 0.001) decreased RBC mobility. Both glutaraldehyde fixation and lipid extraction abolished any PRHCL-induced increase in RBC mobility; the observed increases in mobility for normal cells are, thus, consistent with a mechanism based on expansion of the RBC membrane glycocalyx. Microelectrophoretic methods were also used to study the effect of PRHCL (8 x 10(-4) and 8 x 10(-2) M) on RBC membrane calcium binding, with the results indicating that PRHCL competes with calcium for neuraminate binding sites. We conclude that the observed changes in RBC electrokinetic properties reflect incorporation of PRHCL into the RBC membrane; such changes may be of importance in modulating cell-cell interactions.