Students

Name: April Wong Sum Yee

Post:

Part-time Ph.D
(While working as a laboratory technician in our department)

Enrolled: 2000 - 2006

Origin: Hong Kong

First Degree:

BSc (Cell Biology and Genetics) from University of British Columbia, Vancouver, B.C. Canada

Supervisor: Prof. Manoj Karmakar, Prof. Tony Gin

Title of Thesis:

Bupivacaine, Ropivacaine and Levobupivacaine: Analytical Techniques and Applied Clinical Studies

Outline of the Thesis:

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 α1-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 research 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 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 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.

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 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.

Current Employment:

Chemist, Racing Laboratory, Hong Kong Jockey Club, Shatin

Publications arising from her thesis:

(A) Scientific Papers:

Karmakar MK, Aun CST, Wong ELY, Wong ASY, Chan SKC, Yeung CK. Ropivacaine undergoes slower systemic absorption from the caudal epidural space in children than bupivacaine. Anesthesia & Analgesia 2002; 94: 259-265.
Lee BB, Ngan Kee WD, Lau WM, Wong ASY. Epidural infusions for labor analgesia: A comparison of 0.2% ropivacaine, 0.1% ropivacaine, and 0.1% ropivacaine with fentanyl. Regional Anesthesia and Pain Medicine 2002; 27: 31-36
Lee BB, Ngan Kee WD, Plummer JL, Wong ASY. The effect of the addition of epinephrine on early systemic absorption of epidural ropivacaine in humans. Anesthesiology 2002; 95: 1402-1407.
Morley AP, Chung DC, Wong ASY, Short TG. The sedative and electroencephalographic effects of regional anaesthesia. Anaesthesia 2000; 55(9): 864-869.
Wong ASY, Ngan Kee WD, Karmakar MK, Tam TP, Gin T. Enantioselective liquid chromatographic-mass spectrometric determination of bupivacaine and ropivacaine in human plasma by vancomycin chiral stationary phase. (Pending acceptance)

(B) Conference Abstracts:

Karmakar MK, Ho AMH, Cheung YK, Gin T, Chung CK, Wong ASY, Sammy. Pharmacokinetics of Levobupivacaine after Thoracic Paravertebral Block. 13th World Congress of Anesthesiologists p.P0510. France, 2004.4.
Karmakar MK, Ho AMH, Law KB, Gin T, Chung CK, Wong ASY, Ng F. Pharmacokinetics of Ropivacaine after Thoracic Paravertebral Block. IMRAPT 2002; 14 (N.2): 40
Karmakar MK, Ho AH, Law KB, Gin T, Chung CDK, Wong ASY, Ng FF. Pharmacokinetics of ropivacaine after thoracic paravertebral block. The World Congress of the Regional Anaesthesia & Pain Therapy, Barcelona, Spain, May 2002.
Wong ASY, Karmakar MK, Gin T. Simultaneous Determination of Bupivacaine and Ropivacaine in Plasma Using High Performance Liquid Chromatography. International symposium on regional anesthesia & pain medicine 2000:247.
Morley AP, Chung DC, Wong ASY, Short TG. The sedative and electroencephalographic effects of regional anaesthesia. 6th Annual Meeting of the European Society of Anesthesiologist, Barcelona, April 1998.