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A modified method for the fluorimetric determination of pancuronium bromide in plasma
511
Clinica Chimica Acta, 79 (1977) 511-512 0 Elsevier/North-Holland Biomedical Press
BRIEF TECHNICAL NOTE CCA 8696
A MODIFIED METHOD FOR THE FLUORIMETRIC OF PANCURONIUM BROMIDE IN PLASMA
DETERMINATION
M .J. WATSON * and K. McLEOD Departments of Clinical Biochemistry and of Anaesthesia, Royal Victoria Infirmary, Newcastle upon Tyne NE1 4LP (U.K.) (Received
February 25th, 1977)
The fluorimetric analysis of bisquartemary steroids using rose bengal depends upon the complex which they form with the dye [1,2]. This complex is readily extracted from plasma using a solution of phenol in chloroform. Addition of acetone is advised at this stage in view of the unstable nature of the complex in chloroform/phenol. The fluorescence is then determined at the appropriate excitation and emission wave lengths. In our initial measurements in plasma of the non-depolarising muscle relaxant, pancuronium bromide, we closely followed the method described by Kersten et al. [2]. We took aliquots (0.5 ml) of plasma and of plasma standards and scaled the reagents proportionally. Owing to the large number of assays we performed during the study of the pharmacokinetics of the relaxant [3] and in subsequent investigations on the behaviour of the drug [4,5] it was found expedient to consider a more rapid adaptation of the method. Contrary to the precautions described by Kersten et al., the purified rose bengal in stock solution was stable at 4°C for up to 4 days in the dark. It was also found unnecessary to keep the working solution of phenol in chloroform under nitrogen. Consistent results were obtained by a short extraction time of 15 min followed by a brief centrifugation. The shorter extraction time is important in view of the instability of the fluorescence in chloroform/phenol which diminishes by 20-2576 within 30 min. The necessity for modification was further heightened by the fact that analytical reagent acetone was unreliable as a stabilising solvent for the drugrose bengal complex. The intensity of the fluorescence, in some instances, decreased to very low levels following addition of the chloroform layer.(0.5 ml) to acetone (3.5 ml). This was contrary to the earlier observations [l]. Several batches of acetone were investigated and not all could be guaranteed to stabilise or enhance the fluorescence. The fluorescence of the plasma blank was * To
whom corresuondence should be addressed.
512
usually negligible, one batch of acetone, however, gave an exceptionally high value. The acetone in this case was examined by nuclear magnetic resonance spectroscopy and found to be pure. In view of the nature of the drug-dye complex the solvent ethyl alcohol (analytical reagent) of similar dielectric constant to acetone, was chosen as a substitute for acetone. This proved to be wholly satisfactory. The fluorescence was stabilised and moreover enhanced many fold as described for acetone [l], despite dilution factors. The problem of evaporation in the use of acetone was thus eliminated and furthermore the presence of acetone is highly undesirable in laboratories performing analysis dependent upon the presence of an 0x0 grouping. The maximum excitation wave length in ethyl alcohol was found to be at 544 nm with maximum emission at 566 nm. This is quite comparable to the fluorescence measured in acetone. The Hg line of 546 nm may be used for excitation thus enabling a relatively simple filter fluorimeter to be used for the analysis. The method sensitivity is also comparable and is to within 0.02 mg pancuronium bromide per litre of plasma. References 1 2 3 4 5
Cohen, E.N. (1963) J. Lab. Clin. Med. 61.338-345 Kersten, U.W.. Metier. D.K.F. and Agoston. S. (1973) Clin. Chin Acta 44. 59-66 McLeod, K., Watson. M.J. and Rawlins. M.D. (1976) Br. J. Anaesth. 48, 341-345 Booth. P.N.. Watson, M.J. and McLeod, K. (1977) Anaesthesia 32. 320-323 McLeod. K., Ryan. D.W.. Watson, M.J. and Hull, C.J. (1977) Br. J. Am&h. 49, 185-186
Clinica Chimica Acta, 79 (1977) 511-512 0 Elsevier/North-Holland Biomedical Press
BRIEF TECHNICAL NOTE CCA 8696
A MODIFIED METHOD FOR THE FLUORIMETRIC OF PANCURONIUM BROMIDE IN PLASMA
DETERMINATION
M .J. WATSON * and K. McLEOD Departments of Clinical Biochemistry and of Anaesthesia, Royal Victoria Infirmary, Newcastle upon Tyne NE1 4LP (U.K.) (Received
February 25th, 1977)
The fluorimetric analysis of bisquartemary steroids using rose bengal depends upon the complex which they form with the dye [1,2]. This complex is readily extracted from plasma using a solution of phenol in chloroform. Addition of acetone is advised at this stage in view of the unstable nature of the complex in chloroform/phenol. The fluorescence is then determined at the appropriate excitation and emission wave lengths. In our initial measurements in plasma of the non-depolarising muscle relaxant, pancuronium bromide, we closely followed the method described by Kersten et al. [2]. We took aliquots (0.5 ml) of plasma and of plasma standards and scaled the reagents proportionally. Owing to the large number of assays we performed during the study of the pharmacokinetics of the relaxant [3] and in subsequent investigations on the behaviour of the drug [4,5] it was found expedient to consider a more rapid adaptation of the method. Contrary to the precautions described by Kersten et al., the purified rose bengal in stock solution was stable at 4°C for up to 4 days in the dark. It was also found unnecessary to keep the working solution of phenol in chloroform under nitrogen. Consistent results were obtained by a short extraction time of 15 min followed by a brief centrifugation. The shorter extraction time is important in view of the instability of the fluorescence in chloroform/phenol which diminishes by 20-2576 within 30 min. The necessity for modification was further heightened by the fact that analytical reagent acetone was unreliable as a stabilising solvent for the drugrose bengal complex. The intensity of the fluorescence, in some instances, decreased to very low levels following addition of the chloroform layer.(0.5 ml) to acetone (3.5 ml). This was contrary to the earlier observations [l]. Several batches of acetone were investigated and not all could be guaranteed to stabilise or enhance the fluorescence. The fluorescence of the plasma blank was * To
whom corresuondence should be addressed.
512
usually negligible, one batch of acetone, however, gave an exceptionally high value. The acetone in this case was examined by nuclear magnetic resonance spectroscopy and found to be pure. In view of the nature of the drug-dye complex the solvent ethyl alcohol (analytical reagent) of similar dielectric constant to acetone, was chosen as a substitute for acetone. This proved to be wholly satisfactory. The fluorescence was stabilised and moreover enhanced many fold as described for acetone [l], despite dilution factors. The problem of evaporation in the use of acetone was thus eliminated and furthermore the presence of acetone is highly undesirable in laboratories performing analysis dependent upon the presence of an 0x0 grouping. The maximum excitation wave length in ethyl alcohol was found to be at 544 nm with maximum emission at 566 nm. This is quite comparable to the fluorescence measured in acetone. The Hg line of 546 nm may be used for excitation thus enabling a relatively simple filter fluorimeter to be used for the analysis. The method sensitivity is also comparable and is to within 0.02 mg pancuronium bromide per litre of plasma. References 1 2 3 4 5
Cohen, E.N. (1963) J. Lab. Clin. Med. 61.338-345 Kersten, U.W.. Metier. D.K.F. and Agoston. S. (1973) Clin. Chin Acta 44. 59-66 McLeod, K., Watson. M.J. and Rawlins. M.D. (1976) Br. J. Anaesth. 48, 341-345 Booth. P.N.. Watson, M.J. and McLeod, K. (1977) Anaesthesia 32. 320-323 McLeod. K., Ryan. D.W.. Watson, M.J. and Hull, C.J. (1977) Br. J. Am&h. 49, 185-186