Br. J. clin. Pharmac.
LETTERS TO THE EDITORS
598
(1979), 8
THE MEASUREMENT OF LIGNOCAINE AT LOW CONCENTRATIONS IN PLASMA, A COMPARISON OF GAS LIQUID CHROMATOGRAPHY WITH ENZYME IMMUNOASSAY the comparison of a g.l.c. assay with EMIT using the modification. The g.l.c. assay was carried out as follows: 600 tl plasma and 50 VIl mepivacaine internal standard solution (10 gg ml- ) were pipetted into a screw cap tube. Sodium hydroxide (5M, 100 pl) and 5 ml diethyl ether were then added and the tube capped and shaken for 1O min. The tube was then centrifuged and the ether layer removed to a clean tapered glass tube and evaporated in a water bath at 600C. The residue was redissolved in 20 1l butyl acetate and 2 pl of this was injected in to the g.l.c. A Perkin Elmer F17 was the instrument used and it was fitted with a hot bead nitrogen detector. The column was lm x 4mm i.d. packed with 3% SP 2250 on Supelcoport 80-100 mesh (Chromatography Services, Wirral, U.K.), the temperature used was 235°C, with the detector and injector at 250°C. The nitrogen carrier gas flow was 40 ml min-' and the detector air and hydrogen supplies were set at 13 lb in-2. Under these
Lignocaine is extensively used in the treatment of cardiac arrhythmias and plasma concentrations of between 2 and 6 jg ml - ' are thought to be effective. Many techniques have been used for the analysis of lignocaine in plasma including thin layer chromatography (Lee, Nurok, Zlatis & Karmen, 1978), liquid chromatography (Lagerstrom & Persson, 1978), gas liquid chromatography (Walberg, 1978) and homogeneous enzyme immunoassay (EMIT) (Walberg, 1978). Of these the latter has the shortest sample preparation and analysis time (about 1 min) and is extremely simple to use. The EMIT assay is also accurate and precise but its intended range is lignocaine concentrations within the therapeutic window and this would be of little use in a clinical pharmacology study where low levels of the drug may be encountered. However by a minor modification in the procedure the useful range of the technique can be extended to cover plasma concentrations between 50 and 2000 ng ml- of lignocaine. This paper describes
2000
1800 0
1600
1400 0
S
- 1200 a) 0
0~~~~~~~~~~
,
.
*0
800
p
0
.'S
)
0
0
1000
I
./,*0
200 800
1000
1200
1400
1600
1800
2000
2200
EMIT lignocaine (ng/mI) Figure 1 Lignocaine: a comparison of g.l.c. with EMIT using an undiluted plasma sample. Intercept - 15.1, slope 0.996, r = 0.963, n = 98.
Br. J. clin. Pharmac. (1979), 8
conditions the retention time of lignocaine was 3.4 min and mepivacaine 6.6 min. The homogeneous immunoassay was a kit purchased from the Syva Corporation U.K., EMIT-cad. The assay was carried out according to the makers' instruction, except that samples of < 1000 ng ml-' were measured without initial dilution to increase the assay sensitivity six fold. The equipment used was a Gilford Stasar spectrophotometer coupled to a Syva computer printer 1000, a Syva pipetter-diluter was used for the sample and reagent measurements. The lignocaine standards used were those supplied with the EMIT-cad kit, the low level standards were made by dilution of these by fresh plasma to give concentrations of 0 to 1200 ng ml- . The calibration graphs obtained by both methods were linear over the concentration range and were reproducible from day to day. A total of 98 plasma samples were assayed on both systems. These were obtained from volunteers who had received lignocaine intramuscularly or subcutaneously during a clinical trial comparing routes of administration. The concentrations measured in these samples ranged between 50 and 2200 ng ml-I and a linear correlation of the results from the g.l.c. method with those from the EMIT procedure yielded the following equation. y = 0.996x - 15.1 where x is EMIT and y g.l.c.
The regression coefficient was r = 0.963, and the slope was not significantly different from unity (t = 0.14) nor the intercept from zero (t = 0.78). The results are illustrated in Figure 1 as the correlation graph. Although the reagent costs are high, EMIT has important advantages over other methods for the
LETTERS TO THE EDITORS
599
analysis of lignocaine; it requires only 50 gl of serum or plasma, it is a very simple technique to use and does not call for a great deal of user skill, and the analysis time is short. These factors combine to make EMIT a method of choice for the analysis of routine lignocaine plasma concentrations in patients. We have shown its use in research studies (Beckett, Burgess, Johnston & Warrington, 1978) and this paper confirms that it compares well with an established method. We would like to thank Syva U.K. for the loan of the EMIT equipment, Dr E. Cooke for the plasma samples and Professor P. Turner for his helpful discussion of the manuscript.
SUMAIN JAIN & A. JOHNSTON Department of Clinical Pharmacology, Bartholomew's Hospital, London, ECIA 7BE
St
Received July 16, 1979 References BECKETT, A.H., BURGESS, C.D., JOHNSTON, A. & WARRINGTON, S.J. (1978). The systemic availability of a
slow release rectal preparation of lignocaine. Br. J. clin. Pharmac., 6, 442P. LAGERSTROM, P.-O. & PERSSON, B.-A. (1978). Liquid chromatography in the monitoring of plasma levels of antiarrhythmic drugs. J. Chromatog., 149, 331-340. LEE, K.Y., NUROK, D., ZLATIS, A. & KARMEN, A. (1978). Simultaneous determination of antiarrhythmia drugs by high performance thin layer chromatography. J. Chromatog., 158, 403-410. WALBERG, C.B. (1978). Lidocaine by enzyme immunoassay. J. analyt. Tox., 2, 121-123.
KINETICS OF 5-FLUOROURACIL I read with interest the paper of Finch, Bending & Lant (1979), entitled 'Plasma levels of 5-fluorouracil after oral and intravenous administration in cancer patients'. In my view, the data presented are open to other interpretations than those chosen by the authors, and I regret that the summary was not a little more tentative. In Table 1, a summary of pharmacokinetic data obtained from 11 patients receiving 5-fluorouracil by the intravenous route shows more than one entry for four of these patients. Careful reading of the text reveals that some of the doses of drug were administered under very different circumstances,
namely in the middle of a 5 day course of quadruple chemotherapy. The results as tabulated show increased values of T* for second and subsequent doses, and this is surely suggestive of drug interaction. The text implies that the second study, a comparison of different doses given by the intravenous route, was carried out by giving the lower dose first in all cases. This is probably what happens in medical practice, and these results are important for that reason, but surely it is dangerous to speculate on causative mechanisms without reversing the dose order in a few cases to ensure that the effect reported
LETTERS TO THE EDITORS
598
(1979), 8
THE MEASUREMENT OF LIGNOCAINE AT LOW CONCENTRATIONS IN PLASMA, A COMPARISON OF GAS LIQUID CHROMATOGRAPHY WITH ENZYME IMMUNOASSAY the comparison of a g.l.c. assay with EMIT using the modification. The g.l.c. assay was carried out as follows: 600 tl plasma and 50 VIl mepivacaine internal standard solution (10 gg ml- ) were pipetted into a screw cap tube. Sodium hydroxide (5M, 100 pl) and 5 ml diethyl ether were then added and the tube capped and shaken for 1O min. The tube was then centrifuged and the ether layer removed to a clean tapered glass tube and evaporated in a water bath at 600C. The residue was redissolved in 20 1l butyl acetate and 2 pl of this was injected in to the g.l.c. A Perkin Elmer F17 was the instrument used and it was fitted with a hot bead nitrogen detector. The column was lm x 4mm i.d. packed with 3% SP 2250 on Supelcoport 80-100 mesh (Chromatography Services, Wirral, U.K.), the temperature used was 235°C, with the detector and injector at 250°C. The nitrogen carrier gas flow was 40 ml min-' and the detector air and hydrogen supplies were set at 13 lb in-2. Under these
Lignocaine is extensively used in the treatment of cardiac arrhythmias and plasma concentrations of between 2 and 6 jg ml - ' are thought to be effective. Many techniques have been used for the analysis of lignocaine in plasma including thin layer chromatography (Lee, Nurok, Zlatis & Karmen, 1978), liquid chromatography (Lagerstrom & Persson, 1978), gas liquid chromatography (Walberg, 1978) and homogeneous enzyme immunoassay (EMIT) (Walberg, 1978). Of these the latter has the shortest sample preparation and analysis time (about 1 min) and is extremely simple to use. The EMIT assay is also accurate and precise but its intended range is lignocaine concentrations within the therapeutic window and this would be of little use in a clinical pharmacology study where low levels of the drug may be encountered. However by a minor modification in the procedure the useful range of the technique can be extended to cover plasma concentrations between 50 and 2000 ng ml- of lignocaine. This paper describes
2000
1800 0
1600
1400 0
S
- 1200 a) 0
0~~~~~~~~~~
,
.
*0
800
p
0
.'S
)
0
0
1000
I
./,*0
200 800
1000
1200
1400
1600
1800
2000
2200
EMIT lignocaine (ng/mI) Figure 1 Lignocaine: a comparison of g.l.c. with EMIT using an undiluted plasma sample. Intercept - 15.1, slope 0.996, r = 0.963, n = 98.
Br. J. clin. Pharmac. (1979), 8
conditions the retention time of lignocaine was 3.4 min and mepivacaine 6.6 min. The homogeneous immunoassay was a kit purchased from the Syva Corporation U.K., EMIT-cad. The assay was carried out according to the makers' instruction, except that samples of < 1000 ng ml-' were measured without initial dilution to increase the assay sensitivity six fold. The equipment used was a Gilford Stasar spectrophotometer coupled to a Syva computer printer 1000, a Syva pipetter-diluter was used for the sample and reagent measurements. The lignocaine standards used were those supplied with the EMIT-cad kit, the low level standards were made by dilution of these by fresh plasma to give concentrations of 0 to 1200 ng ml- . The calibration graphs obtained by both methods were linear over the concentration range and were reproducible from day to day. A total of 98 plasma samples were assayed on both systems. These were obtained from volunteers who had received lignocaine intramuscularly or subcutaneously during a clinical trial comparing routes of administration. The concentrations measured in these samples ranged between 50 and 2200 ng ml-I and a linear correlation of the results from the g.l.c. method with those from the EMIT procedure yielded the following equation. y = 0.996x - 15.1 where x is EMIT and y g.l.c.
The regression coefficient was r = 0.963, and the slope was not significantly different from unity (t = 0.14) nor the intercept from zero (t = 0.78). The results are illustrated in Figure 1 as the correlation graph. Although the reagent costs are high, EMIT has important advantages over other methods for the
LETTERS TO THE EDITORS
599
analysis of lignocaine; it requires only 50 gl of serum or plasma, it is a very simple technique to use and does not call for a great deal of user skill, and the analysis time is short. These factors combine to make EMIT a method of choice for the analysis of routine lignocaine plasma concentrations in patients. We have shown its use in research studies (Beckett, Burgess, Johnston & Warrington, 1978) and this paper confirms that it compares well with an established method. We would like to thank Syva U.K. for the loan of the EMIT equipment, Dr E. Cooke for the plasma samples and Professor P. Turner for his helpful discussion of the manuscript.
SUMAIN JAIN & A. JOHNSTON Department of Clinical Pharmacology, Bartholomew's Hospital, London, ECIA 7BE
St
Received July 16, 1979 References BECKETT, A.H., BURGESS, C.D., JOHNSTON, A. & WARRINGTON, S.J. (1978). The systemic availability of a
slow release rectal preparation of lignocaine. Br. J. clin. Pharmac., 6, 442P. LAGERSTROM, P.-O. & PERSSON, B.-A. (1978). Liquid chromatography in the monitoring of plasma levels of antiarrhythmic drugs. J. Chromatog., 149, 331-340. LEE, K.Y., NUROK, D., ZLATIS, A. & KARMEN, A. (1978). Simultaneous determination of antiarrhythmia drugs by high performance thin layer chromatography. J. Chromatog., 158, 403-410. WALBERG, C.B. (1978). Lidocaine by enzyme immunoassay. J. analyt. Tox., 2, 121-123.
KINETICS OF 5-FLUOROURACIL I read with interest the paper of Finch, Bending & Lant (1979), entitled 'Plasma levels of 5-fluorouracil after oral and intravenous administration in cancer patients'. In my view, the data presented are open to other interpretations than those chosen by the authors, and I regret that the summary was not a little more tentative. In Table 1, a summary of pharmacokinetic data obtained from 11 patients receiving 5-fluorouracil by the intravenous route shows more than one entry for four of these patients. Careful reading of the text reveals that some of the doses of drug were administered under very different circumstances,
namely in the middle of a 5 day course of quadruple chemotherapy. The results as tabulated show increased values of T* for second and subsequent doses, and this is surely suggestive of drug interaction. The text implies that the second study, a comparison of different doses given by the intravenous route, was carried out by giving the lower dose first in all cases. This is probably what happens in medical practice, and these results are important for that reason, but surely it is dangerous to speculate on causative mechanisms without reversing the dose order in a few cases to ensure that the effect reported
