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Determination of succinylcholine in plasma by high-pressure liquid chromatography with electrochemical detection
British Journal of Anaesthesia 85 (4): 592±8 (2000)
Determination of succinylcholine in plasma by high-pressure liquid chromatography with electrochemical detection N. I. Pitts*, D. Deftereos and G. Mitchell Department of Physiology, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown 2193, South Africa *Corresponding author The plasma concentration of the neuromuscular blocking drug, succinylcholine, is dif®cult to measure. We have measured concentrations of the breakdown product of succinylcholine, choline, to assess whether choline concentration gives an accurate measure of succinylcholine concentration. Choline concentration was measured by HPLC and electrochemical detection in two blood or plasma samples, one in which succinylcholine hydrolysis was inhibited by 10±5 M physostigmine and another in which succinylcholine was completely hydrolysed in 20 min by 200 mU butyrylcholinesterase at 37°C. The difference in choline content between the two samples gives the succinylcholine concentration. Ninety-®ve per cent recovery of choline was achieved. Choline standard curves were linear from 156 pmol ml±1 to 200 nmol ml±1. Within-day and between-day mean coef®cients of variation for succinylcholine hydrolysis were small (mean (SD) 3.7% (1.2%) and 3.8% (1.6%), respectively). We conclude that this method of measuring succinylcholine concentration in blood is accurate, repeatable and relatively easy. Br J Anaesth 2000; 85: 592±8 Keywords: neuromuscular block; succinylcholine; measurement techniques Accepted for publication: June 5, 2000
Succinylcholine is a short-acting, depolarizing neuromuscular blocking agent1 that is used extensively in anaesthesia. Its duration of action is short because it is rapidly metabolised by butyrylcholinesterase (BuChE) (EC 3.1.1.8) in plasma.2 Effective blood concentrations of succinylcholine are dif®cult to determine because of its rapid hydrolysis. Succinylcholine is ®rst hydrolysed to yield succinylmonocholine and choline. Succinylmonocholine is then broken down to choline and the inactive succinic acid3 (Fig. 1, reaction 1). This second step shows little activity at physiological pH4 and is likely to yield only a small amount of choline after a bolus dose of succinylcholine.3 5 Estimates of succinylcholine in blood and tissues have involved bioassays,6 radiolabelled succinylcholine7 or expensive and sophisticated ion-pair extraction followed by demethylation, gas chromatography and mass spectrometry.8 9 These methods are costly and timeconsuming and are not practical for measuring succinylcholine concentrations in plasma in pharmacokinetic studies. Most studies of this compound have, therefore, dispensed with succinylcholine plasma concentrations and rely on following the time-course of neuromuscular blockade in response to controlled infusions and/or bolus intravenous injections.10±13
To examine succinylcholine overdose in large animals, such as elephants,14 we needed to measure the plasma succinylcholine concentrations as it is not practicable to assess neuromuscular blockade in these animals. We have, therefore, established a method for determining succinylcholine plasma concentrations by measuring the concentration of the succinylcholine cholinesterase reaction product, choline, instead of succinylcholine itself. Measurement of choline concentrations is easier and, as we show, gives a very accurate measure of succinylcholine concentration.
Materials and methods Acetylcholine and choline concentrations can be determined using high-pressure liquid chromatography with electrochemical detection.15 In the BAS acetylcholine/ choline analytical kit (Bioanalytical Systems, West Lafayette, IN, USA) these compounds are separated in a reversed-phase column and then passed through an immobilized enzyme reactor containing acetylcholinesterase (AChE) (EC 3.1.1.7) and choline oxidase. In the usual application of this system for the measurement of acetylcholine, the AchE yields choline, which is then oxidized by choline oxidase (Fig. 1, reaction 2). The subsequent reaction
Ó The Board of Management and Trustees of the British Journal of Anaesthesia 2000
Plasma succinylcholine determination
Fig 1 Reaction 1: Hydrolysis of succinylcholine (Succ) by butyrylcholinesterase (BuChE). The ®rst step yields succinylmonocholine (SmCh) and choline. SmCh is then hydrolysed to yield choline and succinate. Reaction 2: Hydrolysis of acetylcholine and choline by acetylcholinesterase (AchE) and choline oxidase, respectively, in the Bioanalytical Systems immobilized enzyme reactor (see text for details).
Fig 2 Post-column enzyme reactor and analytical column sequence. The trapping column removes physostigmine so that it does not poison the enzyme reactor.
Chromatographic conditions yields peroxide, which is detected at a platinum electrode. Although the molecular structure of succinylcholine is similar to that of acetylcholine, the highly speci®c enzyme AchE has no measurable activity against succinylcholine in this system,16 so only the oxidation of choline (reaction 2, step II) is relevant to the quanti®cation of succinylcholine. This method has been used to determine the ability of human plasma cholinesterase to hydrolyse succinylcholine.16 We propose that the succinylcholine concentration can be determined by hydrolysing succinylcholine in plasma and measuring the concentration of the reaction product, choline. The amount of choline already present in the plasma at that moment (endogenous choline together with any arising from prior succinylcholine hydrolysis) must be determined. To measure the pre-existing choline in a blood sample, plasma cholinesterase activity in the sample must be stopped immediately. In a duplicate, unblocked, aliquot, all the remaining succinylcholine is completely hydrolysed. The difference in choline content between these two measurements gives the succinylcholine concentration.
Chemicals Physostigmine salicylate and sodium perchlorate were purchased from Sigma Chemical Co. (Saint Louis, MO, USA). Succinylcholine was donated by Glaxo SA (Pty) Ltd (Johannesburg, South Africa). Succinylmonocholine was prepared by the reaction of choline iodide with succinic anhydride (Sigma).17 Choline, acetylcholine, AChE (EC 3.1.1.7) and choline oxidase (EC 1.1.3.17) were supplied in the BAS acetylcholine/choline analytical kit (MF-9053; Bioanalytical Systems). BuChE (EC 3.1.1.8; human origin; 100 U/2.1 mg) was purchased from Boehringer Mannheim Gmbh (Mannheim, Germany). Choline standards were prepared in the diluent recommended by BAS, namely 52 mM acetic acid (pH 5.5) with the antimicrobial agent Kathon (Bioanalytical Systems) at 0.01%.
The chromatographic ¯ow sequence is shown in Fig. 2. A Varian Star 9001 solvent delivery system (Varian Chromatography Systems, Sunnyvale, CA, USA) delivers mobile phase at a ¯ow rate of 0.7 ml min±1. The mobile phase was 0.05 M Tris/NaClO4 (pH 8.50) with 0.5% of the antimicrobial solution Kathon (Bioanalytical Systems). A 20 ml injection loop was used and a physostigmine trapping column (MF-6262, Bioanalytical Systems) was placed before the analytical column (acetylcholine analytical column; 96.0 mm35.0 mm i.d.; Bioanalytical Systems). Choline, which was retained in the analytical column for 11.1 min, was then passed through a 30 mm long immobilized enzyme column containing acetylcholine esterase (EC 3.1.1.7) and choline oxidase (60 U each). The reaction of the enzyme with its substrate yielded peroxide, which was measured at a platinum working electrode. The electrode potential was set at 0.5 mV relative to an Ag/AgCl reference electrode. The resulting current was monitored using an LC-4B amperometric detector (Bioanalytical Systems). Output was set at 5±20 nA full scale, depending on the concentrations measured and the electrode sensitivity. The detector output was ampli®ed and recorded with a STAR chromatography workstation interface and software (version 4.0; Varian).
Calibration curves Choline calibration curves were obtained by injecting serial dilutions of a 20 mM standard into the HPLC system. Similar concentrations of succinylcholine were injected into the HPLC apparatus. Calibration curves were generated by plotting the analyte peak-height against its concentration. The lower limit of quanti®cation was de®ned as the lowest concentration that could be measured with a coef®cient of variation (CV) of <15% for repeat determinations.
Succinylcholine hydrolysis To establish the optimal incubation time and enzyme activity to complete the in vitro hydrolysis of succinyl-
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choline, excess BuChE (20±600 mU) was incubated with 0.1 ml of succinylcholine standards (<200 mM) in 0.5 ml of 0.05 M phosphate buffer (pH 7.4) at 37°C. Excess BuChE was also incubated with both fresh human plasma and heatinactivated plasma (to destroy endogenous cholinesterases) under the same conditions. In these plasma standards, succinylcholine was added (<200 mM ®nal concentration) immediately before addition of the enzyme. After incubation for a speci®ed time (between 5 and 120 min), the reaction was stopped by addition of 1 ml ice-cold 0.1 M perchloric acid. Samples were then centrifuged at 50003g at 4°C for 5 min and stored at ±70°C until analysed for choline. Samples lacking enzyme or substrate or both were also assayed to determine spontaneous non-enzymatic hydrolysis of succinylcholine and endogenous choline concentration. A separate series of samples was used to establish the appropriate concentration of enzyme inhibitor. Physostigmine, an irreversible non-speci®c cholinesterase inhibitor, was added to these samples at concentrations of 10±6±10±3 M to block endogenous cholinesterase activity. Complete hydrolysis of succinylcholine to choline and succinic acid is a two-step reaction, yielding choline at each step.3 To con®rm the previous observations that succinylmonocholine would yield little choline in our technique,16 further plasma samples containing succinylcholine (129 mM) or succinylmonocholine (50 mM), or both, were incubated for <120 min with excess BuChE.
complete hydrolysis of the control sample in 20 min. The results of the assay were discarded if the averaged control differed from the expected value by >3%. (This occurred in only one batch when the waterbath thermostat malfunctioned.)
Precision and accuracy HPLC choline detection
Within-day precision was assessed by repeatedly injecting the same choline calibration standards into the HPLC system. Standards were prepared from serial dilutions of a 20 mM choline solution. Between-day precision was assessed by injecting the same choline calibration standards over a period of 3 weeks. Enzyme assay
Within-assay precision was assessed using succinylcholinespiked heat-inactivated plasma samples which were incubated with BuChE (200 mU) for 20 min at 37°C as before. HPLC assays were then completed in the same 24 h period. Between-assay precision was assessed using succinylcholine-spiked heat-inactivated plasma samples under the same assay conditions on ®ve different days. HPLC assays were again completed on the day of each enzyme assay.
Statistics
Estimation of plasma succinylcholine To con®rm that our technique can measure differences in choline content between two aliquots of a blood sample, the plasma concentration±time pro®le of succinylcholine was determined in vitro. Succinylcholine (10.5 mg) was added to 500 ml of human donor blood (haematocrit=0.46). This succinylcholine concentration would approximate a 1.5 mg kg±1 i.v. bolus dose for a 70 kg individual, immediately distributed in 5 litres of blood. Two 2 ml aliquots of blood were withdrawn from the pool immediately before the addition of succinylcholine and then after the addition of the succinylcholine at 30 s intervals for the ®rst 5 min and every 2 min thereafter. One aliquot was immediately treated with physostigmine (®nal concentration 0.01%) to stop further hydrolysis of succinylcholine. Both samples were placed on ice-water and centrifuged within 5 min at 4°C to harvest the plasma. The choline content of each aliquot was determined in duplicate by the previously standardized procedure (i.e. incubation with 200 mU BuChE, 37°C, 20 min). The aliquot blocked with physostigmine was incubated without BuChE, while the duplicate unblocked sample was incubated with 200 mU BuChE. The activity of this added enzyme was monitored by including a 124 mM succinylcholine control sample which was assayed in duplicate with each batch of eight samples. Based on the study of succinylcholine hydrolysis, a peak representing 150 pmol choline was expected for
Unpaired t-tests at P<0.05 were used to test for differences between groups.
Results Calibration data The peak height of the choline standard varied linearly with concentration (Table 1). Serial dilution of the 20 mM standard yielded a lower limit of quanti®cation of 156 pmol ml±1 (3.12 pmol injected; signal-to-noise ratio=4). By repeated injection of a 5 nmol ml±1 choline standard (n=6), the CV for the chromatographic procedure was determined to be 0.55%. No peaks were detected when succinylcholine was injected.
Precision of the HPLC method of choline detection Within-day reproducibility was determined from calibration curves prepared the same day in replicate (n=6). Betweenday reproducibility was determined from calibration curves prepared on different days (n=10). The same stock solutions were used throughout. Results are given in Table 1. For each calibration curve, the slope was signi®cantly different from zero (P<0.05).
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Plasma succinylcholine determination Table 1 HPLC detection of choline. A 20 mM choline standard was serially diluted to generate a standard curve. Standards in the concentration range 20 nmol ml±1 to 625 pmol ml±1 were injected into the HPLC apparatus. To determine within-day reproducibility, six independent calibration curves were established in 1 day. To determine between-day reproducibility, calibration curves established on 10 different days were compared. aLinear unweighted regression. y=bx+c.
Within-day reproducibility (n=6) Between-day reproducibility (n=10)
Variance of the linear regression analysisa (r2 (SD))
Slope (b) (Mean (SD))
Intercept (c) (Mean (SD))
0.9997 (1.03)310±4 (CV=0.01%) 0.9996 (3.62)310±4 (CV=0.04%)
2 (0) (CV=0%) 2 (0) (CV=0%)
7.2 (0.4) (CV=5.2%) 5.9 (0.9) (CV=15.2%)
Fig 3 Amount of choline formed from succinylcholine hydrolysis (where 100% represents the amount expected from complete hydrolysis of succinylcholine in the ®rst reaction step). A 0.1 ml sample in 0.5 ml phosphate buffer was incubated at 37°C under the following conditions: 100 mM succinylcholine standard with 20 or 200 mU BuChE, 124 nmol succinylcholine per ml of plasma with 200 mU BuChE in heatinactivated or fresh plasma or with 10±5 M physostigmine.
Succinylcholine hydrolysis Figure 3 shows representative data for the amount of choline produced from standards and spiked plasma samples incubated with excess BuChE. The ®gure shows that both standards and spiked plasma (0.1 ml) in 0.5 ml phosphate buffer require an incubation period of >20 min at 37°C in the presence of 200 mU BuChE to effect complete hydrolysis of succinylcholine. Increasing the amount of enzyme to 400 or 600 mU did not signi®cantly increase the extent of hydrolysis that occurred in <20 min (data not shown). Figure 3 also shows that the amount of choline produced by excess BuChE in spiked plasma (succinylcholine at 124.0 nmol ml±1) was not signi®cantly different between heat-inactivated and fresh plasma (122.1 (4.0) and 124.7 (2.9) nmol ml±1, respectively) after incubation for 20 min. This means that the 200 mU exogenous enzyme is suf®cient to hydrolyse all the succinylcholine present, so that the contribution made by any endogenous BuChE is not essential for the assay. Also shown in this ®gure is the per
Fig 4 Amount of choline formed from succinylmoncholine (SmCh) and succinylcholine (Succ) hydrolysis (where 100% represents the amount expected from complete hydrolysis of succinylcholine in the ®rst reaction step). A 0.1 ml fresh plasma sample in 0.5 ml 500 mM phosphate buffer was incubated with 200 mU BuChE at 37°C with succinylmonocholine at 50 mM or succinylcholine at 129 mM or both together.
cent hydrolysis of succinylcholine when 10±4 M physostigmine was present in the plasma. Physostigmine at 10±9, 10±6, 10±5 and 10±4 M inhibited the endogenous enzymatic hydrolysis of succinylcholine by 4%, 55%, 89% and >99% respectively in the ®rst 20 min. For the estimation of plasma succinylcholine, we therefore added physostigmine at 0.01% ®nal concentration (i.e. 1.54310±4 M) in the aliquots in which further succinylcholine hydrolysis was to be inhibited. Figure 4 shows the amount of choline formed from succinylmonocholine and succinylcholine. For the reaction conditions, complete hydrolysis of the succinylcholine standard in only the ®rst step of the reaction was expected to yield a chromatogram peak representing 150.0 pmol of choline. Complete hydrolysis of the succinylmonocholine standard was expected to yield a peak representing 58.4 pmol. Within the ®rst 20 min, the choline produced from the combined succinylcholine+succinylmonocholine standard does not exceed that produced by the succinylcholine standard alone. Further evidence that the choline measured
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Pitts et al. Table 2 Within- and between-assay reproducibility of succinylcholine analysis. The recovery of succinylcholine from plasma was estimated from the amount of choline liberated from the reaction with butyrylcholinesterase. The mean recovery of succinylcholine from quintuplicate human plasma samples was used to establish the within-day assay reproducibility. The mean recovery of succinylcholine from triplicate plasma samples was also completed on ®ve different days to establish the between-assay reproducibility of the results Theoretical concentration (nmol ml±1)
124.0 62.0 31.0 15.5 7.8
Within-assay reproducibility (n=5)
Between-assay reproducibility (n=5)
Mean (SD) experimental concentration (nmol ml±1)
Recovery (%)
CV (%)
Mean (SD) experimental concentration (nmol ml±1)
Recovery (%)
CV (%)
121.2 (3.5) 61.2 (1.2) 29.4 (1.3) 14.1 (0.7) 7.1 (0.3)
97.8 98.7 94.8 91.0 91.0
2.9 2.0 4.4 5.0 4.2
125.3 (2.3) 58.3 (2.7) 30.3 (1.8) 14.1 (0.4) 7.4 (0.3)
101.0 94.0 97.7 91.0 94.9
1.8 4.6 5.9 2.8 4.1
comes almost exclusively from the ®rst hydrolysis reaction is contained in Table 2 (see below).
Precision of the succinylcholine assay The within-assay reproducibility of the hydrolysis reaction for succinylcholine was determined in replicate samples (n=5) at succinylcholine concentrations ranging from 7.8 to 124.0 nmol ml±1 with 200 mU BuChE for 20 min. To test between-assay reproducibility, duplicate samples in the same concentration range were incubated with BuChE for 20 min on ®ve different days over a period of 2 weeks. Results are given in Table 2. Even at the lower concentrations of succinylcholine, no more choline can be detected than is expected from only the ®rst step of the hydrolysis reaction.
Plasma succinylcholine Human donor blood rapidly but incompletely hydrolysed succinylcholine. Figure 5 shows the difference in choline concentration between the aliquots, expressed as the amount of succinylcholine present at that time. The volume of the donor blood was 500 ml and, at a haematocrit of 0.46, it can be assumed that 10.5 mg succinylcholine was distributed in 270 ml plasma. The expected succinylcholine plasma concentration at time zero was therefore about 98 nmol ml±1. Hydrolysis appeared to be delayed over the ®rst minute, but this was probably because of incomplete mixing of the succinylcholine in the sample. After 1 min, the mean (SD) measured succinylcholine concentration was 91.1 (0.8) nmol ml±1. A linear ®t from 1 to 5 min (y=±21.6x+114.8; n=18; r2=0.9737) indicates that succinylcholine was hydrolysed at a rate of about 21.6 nmol ml±1 min±1. Beyond 5 min it appears that there is diminished plasma cholinesterase activity, leaving about 12% of the initial dose of succinylcholine unhydrolysed after 10 min.
Fig 5 Concentration of succinylcholine in plasma. Succinylcholine (10.5 mg) was added to a 500 ml pool of donor blood. Duplicate aliquots were withdrawn at intervals and the plasma was assayed for choline (see text for details).
Discussion This method for estimating plasma succinylcholine concentrations requires the complete hydrolysis of any succinylcholine remaining in the sample and the accurate measurement of choline concentration. We needed to hydrolyse succinylcholine to produce choline because the AChE (EC 3.1.1.7) in the enzyme reactor column has little activity against succinylcholine. Under our chromatographic conditions, no succinylcholine peaks could be detected. A similar result has been found by others using this Bioanalytical Systems kit.16 This lack of succinylcholine hydrolytic activity in the enzyme column raises serious doubts concerning reports in which it has been claimed that succinylcholine concentrations in plasma have been determined directly using this Bioanalytical Systems acetylcholine/choline detection kit.18
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In our method, it is assumed that the choline generated during the hydrolysis of succinylcholine comes only from the ®rst step of that reaction. The kit we have used enabled us to produce consistent standard curves for choline and to measure reliably the choline content of any sample. To estimate plasma succinylcholine concentrations accurately, we needed to stop the further hydrolysis of succinylcholine in one aliquot of a blood sample, but allow hydrolysis to continue to completion in a second aliquot of the same sample. The difference in choline concentrations is a measure of succinylcholine concentration. We found that 10±4 M physostigmine completely inhibits endogenous BuChE activity. Two review articles19 20 state that 10±5 M physostigmine will completely inhibit BuChE activity, but our data suggest that higher concentrations are needed. For the aliquot in which complete succinylcholine hydrolysis was desired, preliminary observations indicated that relying only on the endogenous BuChE activity to accomplish this hydrolysis gave variable results. However, when 200 mU of human BuChE was incubated with the samples for 20 min, the mean accuracy of succinylcholine estimation was improved, to 95% (Table 2). The large amount of BuChE needed may appear to represent a low activity of the exogenous BuChE, but we used an incubation pH of 7.4. The optimum pH for the enzymatic hydrolysis of succinylcholine by BuChE is between 8 and 9,4 but nonenzymatic degradation of succinylcholine at this pH is considerable.4 16 At high pH, excess choline would thus be produced in the physostigmine-blocked aliquot, thereby reducing the accuracy of the technique. The assumption that virtually all choline is produced from succinylcholine and that negligible hydrolysis of SmCh is occurring, is supported by our ®nding that recovery of choline never exceeds that expected from hydrolysis of succinylcholine (Table 2). When samples containing a 2.5:1 mixture of succinylcholine and succinylmonocholine are incubated together with BuChE, the recovery still does not differ from that expected from succinylcholine alone. Two previous studies also found that hydrolysis of succinylmonocholine is slower than that of succinylcholine.3 5 Even more importantly, these studies concluded that the af®nity of the enzyme for the succinylcholine was such that succinylmonocholine was catabolized only after almost complete hydrolysis of succinylcholine. The optimal pH for the enzymatic hydrolysis of succinylmonocholine is between 5 and 6,4 and it is possible that only after a signi®cant amount of choline has been generated in the in vitro sample (reaction 1, step I) does the pH begin to favour succinylmonocholine hydrolysis. Our assumption that the measured choline comes only from the ®rst-step hydrolysis of succinylcholine is thus likely to be true, especially at physiological pH. Our estimate of plasma succinylcholine concentration relies on the accurate measurement of the difference in choline content between a hydrolysed aliquot and an inhibited aliquot, so it is important to note the limitations
of this estimate. One possible source of error is in the HPLC determination, which we have shown is <1%. The other source of error arises from the hydrolysis of succinylcholine in the uninhibited aliquot. The mean CV in our examination of the within-assay reproducibility is <4%, and of course includes the 1% variation in the HPLC assessment. This means that only when the choline concentration of the inhibited aliquot lies within 4% of the choline concentration of the hydrolysed sample will the accuracy of this method be compromised. Preliminary in vivo data have shown that, following succinylcholine administration, a difference of <5% between the hydrolysed and inhibited aliquots typically takes >8 min to develop. We also used our technique to assess the disappearance of succinylcholine from whole blood. We know of one in vitro study where the amount of succinylcholine present in human blood was estimated at intervals.6 That study showed that approximately 85% of the succinylcholine was hydrolysed within the ®rst 30 s after mixing with blood. After 2 min, only 5% of the succinylcholine remained. The succinylcholine in that study was, however, estimated by bioassay, so lack of an effect cannot be related to absolute disappearance of the compound. A recent study used a different HPLC method to measure plasma succinylcholine concentration directly after an i.v. bolus dose in humans.21 In that study, the clearance of succinylcholine was about 23 nmol ml±1 min±1 over the ®rst 3 min. The net rate of succinylcholine loss we measured (21.6 nmol ml±1 min±1) is similar. The issue of accuracy of the assessment is relevant to the results we obtained for the in vitro hydrolysis of succinylcholine in donor blood. Since our data show that the minimum difference in choline concentration between the two aliquots (i.e. the succinylcholine estimate) was about 12%, this must re¯ect an inability of the blood to hydrolyse completely all the succinylcholine present. This phenomenon has not been evident in in vivo studies where the intact circulation still facilitates clearance of both succinylcholine and choline from the blood.21 22 However, in a study of succinylcholine overdose in dogs, cessation of regular heartbeats and a loss of arterial pressure after 4 min resulted in no further clearance of succinylcholine from the plasma over the following 6 min.8 This suggests that plasma cholinesterase activity was also diminished in that study. We conclude from our results that this assay accurately detects almost all choline produced from succinylcholine and is therefore an accurate measure of succinylcholine concentration. To produce these accurate results, excess BuChE must be added to the sample to ensure conversion of succinylcholine to choline. Under these assay conditions, none of the measured choline is expected to be derived from succinylmonocholine. This technique can, therefore, be used to obtain reliable plasma succinylcholine data in humans and other mammals in a relatively easy and simple manner.
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References 1 Larijani GE, Gratz I, Silverberg M, Jacobi AG. Clinical pharmacology of the neuromuscular blocking agents. DICP Ann Pharmacother 1991; 25: 54±64 2 Taylor P. The cholinesterases. J Biol Chem 1991; 266: 4025±8 3 Whittaker VP, Wijesundera S. The hydrolysis of suxamethonium by cholinesterase. Biochem J 1952; 52: 475±9 4 goedde HW, Altland K. Suxamethomium sensitivity. Ann N Y Acad Sci 1971; 179: 695±703 5 Foldes FF. Succinylmonocholine iodide: its enzymatic hydrolysis and neuromuscular activity. Proc Soc Exp Biol Med 1953; 83: 187±9 6 Kvisselgaard N, Moya F. Estimation of succinylcholine blood levels. Acta Anaesthiol Scand 1961; 5: 1±11 7 DelSanto G. Kinetics of distribution of radioactive labelled muscle relaxants. Anesthesiology 1968; 29: 435±43 8 Nordgren I, Baldwin K, Forney R Jr. SuccinylcholineÐtissue distribution and elimination from plasma in the dog. Biochem Pharmacol 1984; 33: 2519±21 9 Baldwin KA, Forney R Jr. Correlation of plasma concentration and effects of succinylcholine in dogs. J Forensic Sci 1988; 33: 470±9 10 Walts LF, Dillon JB. Clinical studies on succinylcholine chloride. Anesthesiology 1967; 28: 372±6 11 Cook DR, Windgard LB Jr, Taylor FH. Pharmacokinetics of succinylcholine in infants, children and adults. Clin Pharmacol Ther 1976; 20: 493±8 12 Smith CE, Donati F, Beavon DR. Dose±response curves for
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succinylcholine: single versus cumulative techniques. Anesthesiology 1988; 69: 338±42 Curtis MB, Eickner SE. Pharmacodynamic properties of succinylcholine in greyhounds. Am J Vet Res 1991; 52: 898±902 Hattingh J, Wright PG, De Vos V et al. Cessation of electroencephalographic activity in animals exposed to a lethal dose of suxamethonium. S Afr J Zool 1985; 20: 123±8 Maruyama Y, Ikarashi Y, Blank CL. Recent developments in the determination of acetylcholine and choline. Trends Pharmacol Sci 1987; 8: 84±6 Ikarashi Y, Hada T, Leong Way E, MaruyamaY. Determination of succinylcholine hydrolytic enzyme activity in human plasma. J Chromatog 1990; 533: 23±33 Phillips AP. Preparation of the monocholine ester of succinic acid and some related derivatives. J Am Chem Soc 1953; 75: 4725±7 Hoshi K, Hashimoto Y, Matsukawa S. Pharmacokinetics of succinylcholine in man. Tohoku J Exp Med 1993; 170: 245±50 Silk E, King J, Whittaker M. Assay of cholinesterase in clinical chemistry. Ann Clin Biochem 1979; 16: 57±75 Chatonnet A, Lockridge O. Comparison of butyrylcholinesterase and acetylcholinesterase. Biochem J 1989; 260: 625±34 Gao H, Roy S, Donati F, Varin F. Determination of succinylcholine in human plasma by high performance liquid chromatography with electrochemical detection. J Chromatog B 1998; 718: 129±34 Lagerwerf AJ, van Linthout LEH, Vree TB. Rapid determination of succinylcholine in human plasma by high performance liquid chromatography with ¯uorescence detection. J Chromatog 1991; 570: 390±5
Determination of succinylcholine in plasma by high-pressure liquid chromatography with electrochemical detection N. I. Pitts*, D. Deftereos and G. Mitchell Department of Physiology, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown 2193, South Africa *Corresponding author The plasma concentration of the neuromuscular blocking drug, succinylcholine, is dif®cult to measure. We have measured concentrations of the breakdown product of succinylcholine, choline, to assess whether choline concentration gives an accurate measure of succinylcholine concentration. Choline concentration was measured by HPLC and electrochemical detection in two blood or plasma samples, one in which succinylcholine hydrolysis was inhibited by 10±5 M physostigmine and another in which succinylcholine was completely hydrolysed in 20 min by 200 mU butyrylcholinesterase at 37°C. The difference in choline content between the two samples gives the succinylcholine concentration. Ninety-®ve per cent recovery of choline was achieved. Choline standard curves were linear from 156 pmol ml±1 to 200 nmol ml±1. Within-day and between-day mean coef®cients of variation for succinylcholine hydrolysis were small (mean (SD) 3.7% (1.2%) and 3.8% (1.6%), respectively). We conclude that this method of measuring succinylcholine concentration in blood is accurate, repeatable and relatively easy. Br J Anaesth 2000; 85: 592±8 Keywords: neuromuscular block; succinylcholine; measurement techniques Accepted for publication: June 5, 2000
Succinylcholine is a short-acting, depolarizing neuromuscular blocking agent1 that is used extensively in anaesthesia. Its duration of action is short because it is rapidly metabolised by butyrylcholinesterase (BuChE) (EC 3.1.1.8) in plasma.2 Effective blood concentrations of succinylcholine are dif®cult to determine because of its rapid hydrolysis. Succinylcholine is ®rst hydrolysed to yield succinylmonocholine and choline. Succinylmonocholine is then broken down to choline and the inactive succinic acid3 (Fig. 1, reaction 1). This second step shows little activity at physiological pH4 and is likely to yield only a small amount of choline after a bolus dose of succinylcholine.3 5 Estimates of succinylcholine in blood and tissues have involved bioassays,6 radiolabelled succinylcholine7 or expensive and sophisticated ion-pair extraction followed by demethylation, gas chromatography and mass spectrometry.8 9 These methods are costly and timeconsuming and are not practical for measuring succinylcholine concentrations in plasma in pharmacokinetic studies. Most studies of this compound have, therefore, dispensed with succinylcholine plasma concentrations and rely on following the time-course of neuromuscular blockade in response to controlled infusions and/or bolus intravenous injections.10±13
To examine succinylcholine overdose in large animals, such as elephants,14 we needed to measure the plasma succinylcholine concentrations as it is not practicable to assess neuromuscular blockade in these animals. We have, therefore, established a method for determining succinylcholine plasma concentrations by measuring the concentration of the succinylcholine cholinesterase reaction product, choline, instead of succinylcholine itself. Measurement of choline concentrations is easier and, as we show, gives a very accurate measure of succinylcholine concentration.
Materials and methods Acetylcholine and choline concentrations can be determined using high-pressure liquid chromatography with electrochemical detection.15 In the BAS acetylcholine/ choline analytical kit (Bioanalytical Systems, West Lafayette, IN, USA) these compounds are separated in a reversed-phase column and then passed through an immobilized enzyme reactor containing acetylcholinesterase (AChE) (EC 3.1.1.7) and choline oxidase. In the usual application of this system for the measurement of acetylcholine, the AchE yields choline, which is then oxidized by choline oxidase (Fig. 1, reaction 2). The subsequent reaction
Ó The Board of Management and Trustees of the British Journal of Anaesthesia 2000
Plasma succinylcholine determination
Fig 1 Reaction 1: Hydrolysis of succinylcholine (Succ) by butyrylcholinesterase (BuChE). The ®rst step yields succinylmonocholine (SmCh) and choline. SmCh is then hydrolysed to yield choline and succinate. Reaction 2: Hydrolysis of acetylcholine and choline by acetylcholinesterase (AchE) and choline oxidase, respectively, in the Bioanalytical Systems immobilized enzyme reactor (see text for details).
Fig 2 Post-column enzyme reactor and analytical column sequence. The trapping column removes physostigmine so that it does not poison the enzyme reactor.
Chromatographic conditions yields peroxide, which is detected at a platinum electrode. Although the molecular structure of succinylcholine is similar to that of acetylcholine, the highly speci®c enzyme AchE has no measurable activity against succinylcholine in this system,16 so only the oxidation of choline (reaction 2, step II) is relevant to the quanti®cation of succinylcholine. This method has been used to determine the ability of human plasma cholinesterase to hydrolyse succinylcholine.16 We propose that the succinylcholine concentration can be determined by hydrolysing succinylcholine in plasma and measuring the concentration of the reaction product, choline. The amount of choline already present in the plasma at that moment (endogenous choline together with any arising from prior succinylcholine hydrolysis) must be determined. To measure the pre-existing choline in a blood sample, plasma cholinesterase activity in the sample must be stopped immediately. In a duplicate, unblocked, aliquot, all the remaining succinylcholine is completely hydrolysed. The difference in choline content between these two measurements gives the succinylcholine concentration.
Chemicals Physostigmine salicylate and sodium perchlorate were purchased from Sigma Chemical Co. (Saint Louis, MO, USA). Succinylcholine was donated by Glaxo SA (Pty) Ltd (Johannesburg, South Africa). Succinylmonocholine was prepared by the reaction of choline iodide with succinic anhydride (Sigma).17 Choline, acetylcholine, AChE (EC 3.1.1.7) and choline oxidase (EC 1.1.3.17) were supplied in the BAS acetylcholine/choline analytical kit (MF-9053; Bioanalytical Systems). BuChE (EC 3.1.1.8; human origin; 100 U/2.1 mg) was purchased from Boehringer Mannheim Gmbh (Mannheim, Germany). Choline standards were prepared in the diluent recommended by BAS, namely 52 mM acetic acid (pH 5.5) with the antimicrobial agent Kathon (Bioanalytical Systems) at 0.01%.
The chromatographic ¯ow sequence is shown in Fig. 2. A Varian Star 9001 solvent delivery system (Varian Chromatography Systems, Sunnyvale, CA, USA) delivers mobile phase at a ¯ow rate of 0.7 ml min±1. The mobile phase was 0.05 M Tris/NaClO4 (pH 8.50) with 0.5% of the antimicrobial solution Kathon (Bioanalytical Systems). A 20 ml injection loop was used and a physostigmine trapping column (MF-6262, Bioanalytical Systems) was placed before the analytical column (acetylcholine analytical column; 96.0 mm35.0 mm i.d.; Bioanalytical Systems). Choline, which was retained in the analytical column for 11.1 min, was then passed through a 30 mm long immobilized enzyme column containing acetylcholine esterase (EC 3.1.1.7) and choline oxidase (60 U each). The reaction of the enzyme with its substrate yielded peroxide, which was measured at a platinum working electrode. The electrode potential was set at 0.5 mV relative to an Ag/AgCl reference electrode. The resulting current was monitored using an LC-4B amperometric detector (Bioanalytical Systems). Output was set at 5±20 nA full scale, depending on the concentrations measured and the electrode sensitivity. The detector output was ampli®ed and recorded with a STAR chromatography workstation interface and software (version 4.0; Varian).
Calibration curves Choline calibration curves were obtained by injecting serial dilutions of a 20 mM standard into the HPLC system. Similar concentrations of succinylcholine were injected into the HPLC apparatus. Calibration curves were generated by plotting the analyte peak-height against its concentration. The lower limit of quanti®cation was de®ned as the lowest concentration that could be measured with a coef®cient of variation (CV) of <15% for repeat determinations.
Succinylcholine hydrolysis To establish the optimal incubation time and enzyme activity to complete the in vitro hydrolysis of succinyl-
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choline, excess BuChE (20±600 mU) was incubated with 0.1 ml of succinylcholine standards (<200 mM) in 0.5 ml of 0.05 M phosphate buffer (pH 7.4) at 37°C. Excess BuChE was also incubated with both fresh human plasma and heatinactivated plasma (to destroy endogenous cholinesterases) under the same conditions. In these plasma standards, succinylcholine was added (<200 mM ®nal concentration) immediately before addition of the enzyme. After incubation for a speci®ed time (between 5 and 120 min), the reaction was stopped by addition of 1 ml ice-cold 0.1 M perchloric acid. Samples were then centrifuged at 50003g at 4°C for 5 min and stored at ±70°C until analysed for choline. Samples lacking enzyme or substrate or both were also assayed to determine spontaneous non-enzymatic hydrolysis of succinylcholine and endogenous choline concentration. A separate series of samples was used to establish the appropriate concentration of enzyme inhibitor. Physostigmine, an irreversible non-speci®c cholinesterase inhibitor, was added to these samples at concentrations of 10±6±10±3 M to block endogenous cholinesterase activity. Complete hydrolysis of succinylcholine to choline and succinic acid is a two-step reaction, yielding choline at each step.3 To con®rm the previous observations that succinylmonocholine would yield little choline in our technique,16 further plasma samples containing succinylcholine (129 mM) or succinylmonocholine (50 mM), or both, were incubated for <120 min with excess BuChE.
complete hydrolysis of the control sample in 20 min. The results of the assay were discarded if the averaged control differed from the expected value by >3%. (This occurred in only one batch when the waterbath thermostat malfunctioned.)
Precision and accuracy HPLC choline detection
Within-day precision was assessed by repeatedly injecting the same choline calibration standards into the HPLC system. Standards were prepared from serial dilutions of a 20 mM choline solution. Between-day precision was assessed by injecting the same choline calibration standards over a period of 3 weeks. Enzyme assay
Within-assay precision was assessed using succinylcholinespiked heat-inactivated plasma samples which were incubated with BuChE (200 mU) for 20 min at 37°C as before. HPLC assays were then completed in the same 24 h period. Between-assay precision was assessed using succinylcholine-spiked heat-inactivated plasma samples under the same assay conditions on ®ve different days. HPLC assays were again completed on the day of each enzyme assay.
Statistics
Estimation of plasma succinylcholine To con®rm that our technique can measure differences in choline content between two aliquots of a blood sample, the plasma concentration±time pro®le of succinylcholine was determined in vitro. Succinylcholine (10.5 mg) was added to 500 ml of human donor blood (haematocrit=0.46). This succinylcholine concentration would approximate a 1.5 mg kg±1 i.v. bolus dose for a 70 kg individual, immediately distributed in 5 litres of blood. Two 2 ml aliquots of blood were withdrawn from the pool immediately before the addition of succinylcholine and then after the addition of the succinylcholine at 30 s intervals for the ®rst 5 min and every 2 min thereafter. One aliquot was immediately treated with physostigmine (®nal concentration 0.01%) to stop further hydrolysis of succinylcholine. Both samples were placed on ice-water and centrifuged within 5 min at 4°C to harvest the plasma. The choline content of each aliquot was determined in duplicate by the previously standardized procedure (i.e. incubation with 200 mU BuChE, 37°C, 20 min). The aliquot blocked with physostigmine was incubated without BuChE, while the duplicate unblocked sample was incubated with 200 mU BuChE. The activity of this added enzyme was monitored by including a 124 mM succinylcholine control sample which was assayed in duplicate with each batch of eight samples. Based on the study of succinylcholine hydrolysis, a peak representing 150 pmol choline was expected for
Unpaired t-tests at P<0.05 were used to test for differences between groups.
Results Calibration data The peak height of the choline standard varied linearly with concentration (Table 1). Serial dilution of the 20 mM standard yielded a lower limit of quanti®cation of 156 pmol ml±1 (3.12 pmol injected; signal-to-noise ratio=4). By repeated injection of a 5 nmol ml±1 choline standard (n=6), the CV for the chromatographic procedure was determined to be 0.55%. No peaks were detected when succinylcholine was injected.
Precision of the HPLC method of choline detection Within-day reproducibility was determined from calibration curves prepared the same day in replicate (n=6). Betweenday reproducibility was determined from calibration curves prepared on different days (n=10). The same stock solutions were used throughout. Results are given in Table 1. For each calibration curve, the slope was signi®cantly different from zero (P<0.05).
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Plasma succinylcholine determination Table 1 HPLC detection of choline. A 20 mM choline standard was serially diluted to generate a standard curve. Standards in the concentration range 20 nmol ml±1 to 625 pmol ml±1 were injected into the HPLC apparatus. To determine within-day reproducibility, six independent calibration curves were established in 1 day. To determine between-day reproducibility, calibration curves established on 10 different days were compared. aLinear unweighted regression. y=bx+c.
Within-day reproducibility (n=6) Between-day reproducibility (n=10)
Variance of the linear regression analysisa (r2 (SD))
Slope (b) (Mean (SD))
Intercept (c) (Mean (SD))
0.9997 (1.03)310±4 (CV=0.01%) 0.9996 (3.62)310±4 (CV=0.04%)
2 (0) (CV=0%) 2 (0) (CV=0%)
7.2 (0.4) (CV=5.2%) 5.9 (0.9) (CV=15.2%)
Fig 3 Amount of choline formed from succinylcholine hydrolysis (where 100% represents the amount expected from complete hydrolysis of succinylcholine in the ®rst reaction step). A 0.1 ml sample in 0.5 ml phosphate buffer was incubated at 37°C under the following conditions: 100 mM succinylcholine standard with 20 or 200 mU BuChE, 124 nmol succinylcholine per ml of plasma with 200 mU BuChE in heatinactivated or fresh plasma or with 10±5 M physostigmine.
Succinylcholine hydrolysis Figure 3 shows representative data for the amount of choline produced from standards and spiked plasma samples incubated with excess BuChE. The ®gure shows that both standards and spiked plasma (0.1 ml) in 0.5 ml phosphate buffer require an incubation period of >20 min at 37°C in the presence of 200 mU BuChE to effect complete hydrolysis of succinylcholine. Increasing the amount of enzyme to 400 or 600 mU did not signi®cantly increase the extent of hydrolysis that occurred in <20 min (data not shown). Figure 3 also shows that the amount of choline produced by excess BuChE in spiked plasma (succinylcholine at 124.0 nmol ml±1) was not signi®cantly different between heat-inactivated and fresh plasma (122.1 (4.0) and 124.7 (2.9) nmol ml±1, respectively) after incubation for 20 min. This means that the 200 mU exogenous enzyme is suf®cient to hydrolyse all the succinylcholine present, so that the contribution made by any endogenous BuChE is not essential for the assay. Also shown in this ®gure is the per
Fig 4 Amount of choline formed from succinylmoncholine (SmCh) and succinylcholine (Succ) hydrolysis (where 100% represents the amount expected from complete hydrolysis of succinylcholine in the ®rst reaction step). A 0.1 ml fresh plasma sample in 0.5 ml 500 mM phosphate buffer was incubated with 200 mU BuChE at 37°C with succinylmonocholine at 50 mM or succinylcholine at 129 mM or both together.
cent hydrolysis of succinylcholine when 10±4 M physostigmine was present in the plasma. Physostigmine at 10±9, 10±6, 10±5 and 10±4 M inhibited the endogenous enzymatic hydrolysis of succinylcholine by 4%, 55%, 89% and >99% respectively in the ®rst 20 min. For the estimation of plasma succinylcholine, we therefore added physostigmine at 0.01% ®nal concentration (i.e. 1.54310±4 M) in the aliquots in which further succinylcholine hydrolysis was to be inhibited. Figure 4 shows the amount of choline formed from succinylmonocholine and succinylcholine. For the reaction conditions, complete hydrolysis of the succinylcholine standard in only the ®rst step of the reaction was expected to yield a chromatogram peak representing 150.0 pmol of choline. Complete hydrolysis of the succinylmonocholine standard was expected to yield a peak representing 58.4 pmol. Within the ®rst 20 min, the choline produced from the combined succinylcholine+succinylmonocholine standard does not exceed that produced by the succinylcholine standard alone. Further evidence that the choline measured
595
Pitts et al. Table 2 Within- and between-assay reproducibility of succinylcholine analysis. The recovery of succinylcholine from plasma was estimated from the amount of choline liberated from the reaction with butyrylcholinesterase. The mean recovery of succinylcholine from quintuplicate human plasma samples was used to establish the within-day assay reproducibility. The mean recovery of succinylcholine from triplicate plasma samples was also completed on ®ve different days to establish the between-assay reproducibility of the results Theoretical concentration (nmol ml±1)
124.0 62.0 31.0 15.5 7.8
Within-assay reproducibility (n=5)
Between-assay reproducibility (n=5)
Mean (SD) experimental concentration (nmol ml±1)
Recovery (%)
CV (%)
Mean (SD) experimental concentration (nmol ml±1)
Recovery (%)
CV (%)
121.2 (3.5) 61.2 (1.2) 29.4 (1.3) 14.1 (0.7) 7.1 (0.3)
97.8 98.7 94.8 91.0 91.0
2.9 2.0 4.4 5.0 4.2
125.3 (2.3) 58.3 (2.7) 30.3 (1.8) 14.1 (0.4) 7.4 (0.3)
101.0 94.0 97.7 91.0 94.9
1.8 4.6 5.9 2.8 4.1
comes almost exclusively from the ®rst hydrolysis reaction is contained in Table 2 (see below).
Precision of the succinylcholine assay The within-assay reproducibility of the hydrolysis reaction for succinylcholine was determined in replicate samples (n=5) at succinylcholine concentrations ranging from 7.8 to 124.0 nmol ml±1 with 200 mU BuChE for 20 min. To test between-assay reproducibility, duplicate samples in the same concentration range were incubated with BuChE for 20 min on ®ve different days over a period of 2 weeks. Results are given in Table 2. Even at the lower concentrations of succinylcholine, no more choline can be detected than is expected from only the ®rst step of the hydrolysis reaction.
Plasma succinylcholine Human donor blood rapidly but incompletely hydrolysed succinylcholine. Figure 5 shows the difference in choline concentration between the aliquots, expressed as the amount of succinylcholine present at that time. The volume of the donor blood was 500 ml and, at a haematocrit of 0.46, it can be assumed that 10.5 mg succinylcholine was distributed in 270 ml plasma. The expected succinylcholine plasma concentration at time zero was therefore about 98 nmol ml±1. Hydrolysis appeared to be delayed over the ®rst minute, but this was probably because of incomplete mixing of the succinylcholine in the sample. After 1 min, the mean (SD) measured succinylcholine concentration was 91.1 (0.8) nmol ml±1. A linear ®t from 1 to 5 min (y=±21.6x+114.8; n=18; r2=0.9737) indicates that succinylcholine was hydrolysed at a rate of about 21.6 nmol ml±1 min±1. Beyond 5 min it appears that there is diminished plasma cholinesterase activity, leaving about 12% of the initial dose of succinylcholine unhydrolysed after 10 min.
Fig 5 Concentration of succinylcholine in plasma. Succinylcholine (10.5 mg) was added to a 500 ml pool of donor blood. Duplicate aliquots were withdrawn at intervals and the plasma was assayed for choline (see text for details).
Discussion This method for estimating plasma succinylcholine concentrations requires the complete hydrolysis of any succinylcholine remaining in the sample and the accurate measurement of choline concentration. We needed to hydrolyse succinylcholine to produce choline because the AChE (EC 3.1.1.7) in the enzyme reactor column has little activity against succinylcholine. Under our chromatographic conditions, no succinylcholine peaks could be detected. A similar result has been found by others using this Bioanalytical Systems kit.16 This lack of succinylcholine hydrolytic activity in the enzyme column raises serious doubts concerning reports in which it has been claimed that succinylcholine concentrations in plasma have been determined directly using this Bioanalytical Systems acetylcholine/choline detection kit.18
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In our method, it is assumed that the choline generated during the hydrolysis of succinylcholine comes only from the ®rst step of that reaction. The kit we have used enabled us to produce consistent standard curves for choline and to measure reliably the choline content of any sample. To estimate plasma succinylcholine concentrations accurately, we needed to stop the further hydrolysis of succinylcholine in one aliquot of a blood sample, but allow hydrolysis to continue to completion in a second aliquot of the same sample. The difference in choline concentrations is a measure of succinylcholine concentration. We found that 10±4 M physostigmine completely inhibits endogenous BuChE activity. Two review articles19 20 state that 10±5 M physostigmine will completely inhibit BuChE activity, but our data suggest that higher concentrations are needed. For the aliquot in which complete succinylcholine hydrolysis was desired, preliminary observations indicated that relying only on the endogenous BuChE activity to accomplish this hydrolysis gave variable results. However, when 200 mU of human BuChE was incubated with the samples for 20 min, the mean accuracy of succinylcholine estimation was improved, to 95% (Table 2). The large amount of BuChE needed may appear to represent a low activity of the exogenous BuChE, but we used an incubation pH of 7.4. The optimum pH for the enzymatic hydrolysis of succinylcholine by BuChE is between 8 and 9,4 but nonenzymatic degradation of succinylcholine at this pH is considerable.4 16 At high pH, excess choline would thus be produced in the physostigmine-blocked aliquot, thereby reducing the accuracy of the technique. The assumption that virtually all choline is produced from succinylcholine and that negligible hydrolysis of SmCh is occurring, is supported by our ®nding that recovery of choline never exceeds that expected from hydrolysis of succinylcholine (Table 2). When samples containing a 2.5:1 mixture of succinylcholine and succinylmonocholine are incubated together with BuChE, the recovery still does not differ from that expected from succinylcholine alone. Two previous studies also found that hydrolysis of succinylmonocholine is slower than that of succinylcholine.3 5 Even more importantly, these studies concluded that the af®nity of the enzyme for the succinylcholine was such that succinylmonocholine was catabolized only after almost complete hydrolysis of succinylcholine. The optimal pH for the enzymatic hydrolysis of succinylmonocholine is between 5 and 6,4 and it is possible that only after a signi®cant amount of choline has been generated in the in vitro sample (reaction 1, step I) does the pH begin to favour succinylmonocholine hydrolysis. Our assumption that the measured choline comes only from the ®rst-step hydrolysis of succinylcholine is thus likely to be true, especially at physiological pH. Our estimate of plasma succinylcholine concentration relies on the accurate measurement of the difference in choline content between a hydrolysed aliquot and an inhibited aliquot, so it is important to note the limitations
of this estimate. One possible source of error is in the HPLC determination, which we have shown is <1%. The other source of error arises from the hydrolysis of succinylcholine in the uninhibited aliquot. The mean CV in our examination of the within-assay reproducibility is <4%, and of course includes the 1% variation in the HPLC assessment. This means that only when the choline concentration of the inhibited aliquot lies within 4% of the choline concentration of the hydrolysed sample will the accuracy of this method be compromised. Preliminary in vivo data have shown that, following succinylcholine administration, a difference of <5% between the hydrolysed and inhibited aliquots typically takes >8 min to develop. We also used our technique to assess the disappearance of succinylcholine from whole blood. We know of one in vitro study where the amount of succinylcholine present in human blood was estimated at intervals.6 That study showed that approximately 85% of the succinylcholine was hydrolysed within the ®rst 30 s after mixing with blood. After 2 min, only 5% of the succinylcholine remained. The succinylcholine in that study was, however, estimated by bioassay, so lack of an effect cannot be related to absolute disappearance of the compound. A recent study used a different HPLC method to measure plasma succinylcholine concentration directly after an i.v. bolus dose in humans.21 In that study, the clearance of succinylcholine was about 23 nmol ml±1 min±1 over the ®rst 3 min. The net rate of succinylcholine loss we measured (21.6 nmol ml±1 min±1) is similar. The issue of accuracy of the assessment is relevant to the results we obtained for the in vitro hydrolysis of succinylcholine in donor blood. Since our data show that the minimum difference in choline concentration between the two aliquots (i.e. the succinylcholine estimate) was about 12%, this must re¯ect an inability of the blood to hydrolyse completely all the succinylcholine present. This phenomenon has not been evident in in vivo studies where the intact circulation still facilitates clearance of both succinylcholine and choline from the blood.21 22 However, in a study of succinylcholine overdose in dogs, cessation of regular heartbeats and a loss of arterial pressure after 4 min resulted in no further clearance of succinylcholine from the plasma over the following 6 min.8 This suggests that plasma cholinesterase activity was also diminished in that study. We conclude from our results that this assay accurately detects almost all choline produced from succinylcholine and is therefore an accurate measure of succinylcholine concentration. To produce these accurate results, excess BuChE must be added to the sample to ensure conversion of succinylcholine to choline. Under these assay conditions, none of the measured choline is expected to be derived from succinylmonocholine. This technique can, therefore, be used to obtain reliable plasma succinylcholine data in humans and other mammals in a relatively easy and simple manner.
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