Bupivacaine, ropivacaine and levobupivacaine: Analytical techniques and applied clinical studies.

摘要

Bupivacaine ((R, S)-1-butyl-2-piperidylformo-2', 6'-xylidide), an anilide type local anaesthetic is manufactured in the standard racemic form and is widely used in the practice of regional anaesthesia. Despite its popularity as a local anesthetic, it has the potential to produce severe cardiotoxicity. Enantiomers, which are a pair of chiral isomers that are direct, nonsuperimposable mirror images of each other, vary in their chemical, pharmacological and toxicological profiles due to different stereospecific recognition in the body. Single enantiomeric drugs, when compared to racemic drugs, exert similar clinical effects but produce decreased risks of cardiac and neurotoxicity. This has led to the development of the single enantiomeric drugs ropivacaine ((S)-1-propyl-2-piperidylformo-2', 6'-xylidide) and levobupivacaine ((S)-1-butyl-2-piperidylformo-2', 6'-xylidide). Since local anaesthetics are extensively bound (>90%) to plasma protein in blood such as album and alpha1-acid-glycoprotein, it is only the free form of the flowing drug that can exert its pharmacological effects and are believed to be closely related to systemic toxicity. Although the safety and efficacy of these newer local anaesthetics have been ascertained in the literatures, but there are limited data on their pharmacokinetic profiles; thus it is envisioned that further pharmacokinetic trials would be required to elucidate their pharmacological and clinical effects. The aim of this thesis was to develop sensitive, reproducible and reliable methods of local anaesthetic assays to support such clinical trials.;The first method developed is the simultaneous determination of ropivacaine and bupivacaine in human plasma using high performance chromatography (HPLC). Most published methods of determining ropivacaine in human plasma use gas chromatography and a review of literature to date shows no data describing the use of HPLC to simultaneously determine both drugs. This is the first report describing a simple, isocratic, reversed-phase, liquid-liquid extraction procedure of high-performance liquid chromatographic method that allows the simultaneous detection of both local anaesthetics in one single injection. The chromatography was achieved using a reversed-phase chromatographic system with a Waters Novapak C18 column. 0.5 ml plasma was used for the sample preparation procedures. Bupivacaine and ropivacaine concentrations ranging from 10ng/ml to 3000 ng/ml and fixed amounts of pentycaine (internal standard) were spiked into the plasma samples for calculating the calibration graphs. Calibration graphs were linear over the range 10-3000 ng/ml (r=0.9978 for bupivacaine and r=0.9986 for ropivacaine). The within-day (intra-assay) coefficient of variation of the assay varied between 13.84% at 100 ng/ml, 1.84% at 500 ng/ml and 3.34% at 2000 ng/ml for bupivacaine; and 5.29% at 100 ng/ml, 1.38% at 500 ng/ml and 3.93% at 2000 ng/ml for ropivacaine. The between-day (inter-assay) coefficient of variation was 8.43% at 100 ng/ml, 4.06% at 500 ng/ml and 9.15% at 2000 ng/ml for bupivacaine, and 5.66% at 100 ng/ml, 4.40% at 500 ng/ml and 8.14% at 2000 ng/ml for ropivacaine. The limit of detection for both drugs was 10 ng/ml.;The second analytical methodology describes the assay of levobupivacaine in human plasma using HPLC. Calibration graphs relating peak height ratios and concentrations were linear over the range 10-3000 ng/ml (r=0.9995). The chromatography was achieved with an XTerra MS C18 column with the ultraviolet monitor set at 210 nm. The sample preparation steps were similar to the first analytical method, but with a different internal standard used. Precision and accuracy were assessed by performing analysis on replicate control plasma samples. The within-day (intra-assay) coefficient of variation of the assay varied between 4.25% at 50 ng/ml, 3.38% at 500 ng/ml, 3.76% at 1000 ng/ml and 3.14% at 2000 ng/ml. The between-day (inter-assay) coefficient of variation of the assay varied between 4.68% at 50 ng/ml, 4.94% at 500 ng/ml, 4.25% at 1000 ng/ml and 2.94% at 2000 ng/ml. The limit of detection was 10 ng/ml.;The third analytical methodology details the development and validation of a chiral analytical technique. This is the first report describing the development of a simple, isocratic, reversed-phase, liquid-liquid extraction procedure of a direct chiral method that allows the simultaneous detection of either free or total concentrations of bupivacaine enantiomers and ropivacaine in one single injection. It is also a novel technique to assay bupivacaine enantiomers with the use of vancomycin CSP column and liquid chromatography-mass spectrometry (LC-MS/MS) analysis, which achieved the lowest published detection limit with the lowest volume of plasma used. Calibration graphs were linear over the range 0.1-2000 ng/ml. Precision and accuracy were assessed by performing analysis on replicate control plasma samples. The within-day (intrassay) coefficient of variations of the assay for the drugs ropivacaine, levobupivacaine, dextrobupivacaine varied from 2.20% to 5.78%, 1.96% to 9.64%, 1.78% to 6.34%, respectively, for concentrations between 0.5 ng/ml to 2000 ng/ml. The between-day (interassay) coefficient of variations of the assay for the drugs ropivaciane, levobupivacaine, dextrobupivacaine varied from 3.66% to 9.61%, 3.18% to 8.34%, 2.22% to 10.59%, respectively, for concentrations between 0.5 ng/ml to 2000 ng/ml. The limit of detection was 0.05 ng/ml.;The fourth analytical technique describes the successful development of an ultrafiltration protein binding procedure to detect the free levels of the local anaesthetics in human plasma. Sample plasma was deposited in the ultrafiltration apparatus and ultrafiltrate containing the free local anaesthetics was forced thru a membrane under a fixed-angle rotor centrifugal force. Experiments were done to establish the optimum parameters for the ultrafiltration apparatus' binding capacities. The validated procedures use 0.5 ml plasma as the starting volume and it was deposited into the ultrafiltration apparatus. It was then subjected to 1750g centrifugal force for 20 minutes at centrifugal temperature of 37°C. The resultant ultrafiltrate was processed according to the described LC-MS/MS method to detect the free local anaesthetic levels.;The assays described in the thesis have been applied to numerous clinical research projects. Out of the various studies, the following will be discussed: Ropivacaine undergoes slower systemic absorption from the caudal epidural space in children than bupivacaine; Arterial and venous pharmacokinetics of ropivacaine with and without epinephrine after thoracic paravertebral block; Pharmacokinetics of levobupivacaine after thoracic paravertebral block.