Journal of Analytical Toxicology,Vol. 26, January/February2002
The Detection and Identification of Quaternary Nitrogen
Muscle Relaxantsin Biological Fluidsand Tissuesby
Ion-Trap LC-ESI-MS
C.H.M. Kerskes1,2, K.J. Lusthof1, RG.M. Zweipfenning 1, and J.R Franke2,*
7The Netherlands Forensic Institute, Department Toxicology, P.O. Box 3110, 2280 GC Rijswijk, The Netherlands and 2University
Centre for Pharmacy, Department of Bioanalysis and Toxicology,Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands
[ Abstract ]
Quaternary nitrogen muscle relaxants pancuronium, rocuronium,
vecuronium, gallamine, suxamethonium, mivacurium, and
atracurium and its metabolites were extracted from whole blood
and other biological fluids and tissues by using a solid-phase
extraction procedure. The extracts were examined by using highperformance liquid chromatography-electrospray ionization mass
spectrometry (LC-ESI-MS). The drugs were separated on a ODS
column in a gradient of ammonium acetate buffer (pH 5.0) and
acetonitrile. Full-scan mass spectra of the compounds showed
molecular ions, and MS-MS spectra showed fragments typical of
the particular compounds. LC-ESI-MS allowed an unequivocal
differentiation of all muscle relaxants involved. The method was
applied in a case of rocuronium and suxamethonium administration
in a Caesarian section and in a case of intoxication by pancuronium
injection. In both cases, the administered drugs could be detected
and identified in the supplied samples.
performed by using high-performance liquid chromatography
(HPLC). Detection, however, poses a problem because many
compounds have no UVabsorption. Although some compounds
may be detected by UVabsorption or fluorescence (e.g., mivacurium, atracurium, and laudanosine, a metabolite of atracurium), screening for and identification of an array of muscle
relaxants in one run is not possible using HPLC-UV or
HPLC-fluorescencedetection.
HPLC coupled with mass spectrometry (MS) appears to be
potentially well suited for the screening and identification of
quaternary nitrogen muscle relaxants. Electrospray ionization
(ESI) generally is the interface of choice in the case of charged
species such as quaternary amines.
Therefore, we studied the applicability of LC-ESI-MS (ion
trap) to the screening of whole blood for the presence of the
muscle relaxants rocuronium, pancuronium, vecuronium,
mivacurium, atracurium, gallamine, and suxamethonium. In
addition, other fluids and tissues, specificallyurine, serum, liver,
brain, muscle, and bile were examined in two forensic cases.
Introduction
In clinical and forensic toxicology,the detection and identification of quaternary nitrogen muscle relaxants is of importance.
The extraction of quaternary nitrogen muscle relaxants is considered to be difficult; methods have been described in the literature using liquid-liquid and solid-phase extractions (1-10).
Liquid-liquid extractions require ion pair formation between the
muscle relaxants and, for example, potassium iodide. Solidphase extractions may be achieved by using silica-based C18 or
C8 sorbent. Most of the methods described were optimized to
extract one or two compounds. However,a general extraction
method applicable to a large number of quaternary nitrogen
muscle relaxants has not yet been described.
Separation of quaternary nitrogen muscle relaxants could be
* Author to whom correspondenceshould be addressed. E-mail:[email protected].
Experimental
Reagents
Ammonium carbonate (extra pure) and ammonium acetate
(pro Analysi) were supplied by Merck (E.Merck, Darmstadt,
Germany). Methanol, acetonitrile, and hexane were HPLC grade
and supplied by Rathburn Chemicals Ltd. (Walkerburn,
Scotland).
Drugstandards
The standards used were injection fluids.
Pancuronium dibromide (Pavulon| rocuronium bromide
(Esmeron| vecuronium bromide (Norcuron| were suppliedin
ampoules of 2, 10, and 4 mg/mL, respectively(Organon Teknika
BV,Boxtel,The Netherlands).Atracurium dibesilate (Tracrium|
and mivacurium chloride Mivacron| were supplied in
Reproduction(photocopying)of editorialcontentof thisjournalis prohibitedwithoutpublisher'spermission.
29
Journalof AnalyticalToxicology,Vol.26, January/February2002
ampoules of 10 and 2 mg/mL, respectively (Glaxo Wellcome,
Zeist, The Netherlands). Suxamethonium chloride was supplied
in a 50-mg/mL ampoule (Hospital "OLVG" Leiden, The
Netherlands) and a 20-mg/mL ampoule (Hospital "Reinier de
Graaf', Delft, The Netherlands).
Gallamine triethiodide (Flaxedil| was supplied in a 20-mg/mL
ampoule (Specia/Rhone-Poulenc, France).
These injection fluids were diluted to the required concentration with methanol in 5-mL silanized bottles. The final solutions
were kept at -18~
Buffers
Ammonium acetate buffer (pH 5.0, 50mM) was prepared by
dissolving 3.85 g of ammonium acetate in about 900 mL of water
(Millipore), adjusting the pH to 5.0 with acetic acid (1M), and
making up to 1000 mL with water.
Ammonium carbonate buffer (pH 9.3, 0.01M) was prepared by
dissolving 0.47 g of ammonium carbonate in about 475 mL of
water (Millipore), adjusting the pH to 9.3 with 0.1M potassium
hydroxide,and making up to a final volume of 500 mL with water.
Sample pretreatment
Urine, bile, and liver samples were first incubated with 5 IJL [3glucuronidase/aryl sulfatase (from Helix pomatia, Merck) at
36~ for 3 h. A 1.0-mL portion of sample (whole blood, urine,
bile, or serum) was transferred to a 10-mL polypropylene tube.
Two milliliters of ammonium carbonate buffer was added to the
sample. The mixture was vortex mixed for I rain and centrifuged
for 5 min at 4000 rpm (Rotina 48 centrifuge, Hettich). The
supernatant was used.
Table I. Gradient Used in the HPLC Procedure
Time (min)
Flow (mL/min)
%buffer
% AcN
Tissues (liver,brain, or muscle) were minced by using a Turrax
mixer, after addition of a double volume of ammonium carbonate buffer the mixture was centrifuged for 5 min at 4000 rpm.
The supernatant was used.
Extraction
For the solid-phase extraction experiments, the vacuum manifold system was purchased from Supelco (Bellefonte,PA).Solidphase extraction was performed on BondElut C18 HF cartridges
(10 and 3 mL, bed volume 200 rag) purchased from Varian
Analytichem (Harbor City, CA). At first, the cartridges were
rinsed with 2 mL of methanol and 2 mL of ammonium carbonate
buffer. The pretreated sample was introduced on the cartridge,
followed by rinsing twice with 1.5 mL ammonium carbonate
buffer. The cartridges were vacuum dried for 5 min. Then 50 ~L
hexane was brought onto the cartridge, followed by vacuum
drying for 5 min. The retained drugs were eluted under gravity
force with 1 mL of methanol containing 0.1M acetic acid. The
elutes were dried in a vacuum (evaporator)/centrifuge (Savant
SpeedVacAES2000) for 25 rain (temperature high, radiant cover
off, cryopumping off). The dried residues were reconstituted in
100 IJL of a solution of ammonium acetate buffer and acetonitrile (7:3) and centrifuged for 5 rain at 4000 rpm.
LC procedure
HPLC was performed on an Inertsil ODS2 column
(Chrompack) using a mobile phase of ammonium acetate buffer
(pH 5.0, 50raM) and acetonitrile in the gradient shown in TableI.
The LC-MS system consisted of a SpectraSystem AS3000
autosampler and a P4000 quaternary narrowbore gradient pump
that was connected to a classical LCQ ion-trap MS (Thermoquest, San Jose, CA).
MS tuning
Tuning of the MS was performed in two steps. First, an auto0.00
0.40
95
5
tuning procedure was done for each compound. The following
5.00
0.40
70
30
parameters were optimized automatically in this procedure: cap15.00
0.40
70
30
illary voltage, tube lens offset voltage, first octapole direct cur25.00
0.40
65
35
rent
offset voltage, interoctapole lens voltage, and second
30.00
0.40
30
70
octapole
direct current offset voltage. Table II shows the results
33.00
0.40
30
70
of
this
autotuning
procedure.
33.50
0.40
95
5
After this, a manual tuning of the capillary temperature and
35.00
0.40
95
5
the sheath gas flowwas performed. The results of this procedure
are shown in Table II, For MS-MS measurements,
the collision energy was optimized. For all comTable II. Tuning: Parameters Optimized by Autotuning and Manual Tuning
pounds, the optimal collision energy lay between
Autotuning
Manual tuning
32 and 37%.
In this way, a tune file was obtained for each
tube
first
second inter
sheath capillary
lens octapole octapole octapole capillary gas tempcompound. In cases where screening for one of
Compound
m/z
offset offset offset lens voltage flow erature these muscle relaxants had to be performed, a
method that used the suxamethonium tune file
Suxamethonium 145.13 55 -4.75
-10.5
-16
46
45
125
from 0 to 6 min and the atracurium tune file from
Gallamine
284.51
55 -4.25
-7.5
-16
5
75
15o
6 to 35 min was applied. This method turned out
Rocuronium
529.33
55
-3.50
-6.5
-16
3
75
2oo
to give the best results overall.
Pancuronium
286.20
10 -1.75
-6.5
-16
42
65
225
If the identity of the muscle relaxant is known,
Vecuronium
557.40
40
-3.75
-6.5
-16
21
55
25O
200
the
tune file of the specific drug can be used.
Atracurium
464.22
5
-3.25
-6.5
-20
39
85
2oo
Depending
on the drug, this can be in MS,
Mivacurium
514.20
35
-3.00
-6.5
-28
3
85
MS-MS, or MS-MS-MS mode.
30
Journal of Analytical Toxicology, Vol. 26, January/February 2002
Results and Discussion
Qualitative MS data
All muscle relaxants examined are quaternary nitrogen compounds. Massspectra of these compoundsshow a basepeakat the
molecular mass divided by the charge of the molecule. A protonated molecular ion (m/z = M+I) is not observed for these compounds.
In Table III the most intense ions (relative abundance more
than 10%) of the mass spectra are listed for each compound. For
pancuronium, vecuronium, and rocuronium, only the molecular ion is observed in MS mode. When MS-MS is applied, the
fragmentation pattern of the parent ion leads to an unequivocal
identification of these muscle relaxants. Rocuronium only shows
one peak in MS-MS. When MS-MS-MS is applied, a specific
fragmentation pattern is observed.
I
~-3
:['
i
u~
lr
For mivacurium, a small peak with a mass-to-charge ratio of
342.3 is observed in MS mode, together with the basepeak of the
molecular ion. When MS-MS is applied to the molecular ion
peak, a few fragments appear, rn/z 671.3 being the most abundant.
The MS spectrum of atracurium shows three peaks, which correspond to the molecular ions of atracurium and two of its degradation products, laudanosine and a quaternary monoacrylate.
Laudanosine is protonated at the pH of the eluent. Therefore, the
peak of this compound is visible at m/z M+I.
Suxamethonium also shows a molecular ion peak in its MSspectrum. When MS-MS is applied a peak with a mass-to-charge
ratio of 115.6 is visible. MS-MS-MS and MS-MS-MS-MS show
mass-to-charge ratios of 204 and 145, respectively.
Gallamine is the only compound that only shows a signal in
MS mode and not when MS-MS is applied.
it
lu
lvt 8
,i
,o -3
Tlrml (mln)
Figure 1. Chromatogram of a cocktail of all examined muscle relaxants. Peak identification: 1, suxamethonium; 2, gallamine; 3, laudanosine; 4, rocuronium; 5, pancuronium; 6, vecuronium;
7, atracurium; 8, mivacurium; and 9, quaternary monoacrylate.
Table III. Mass Spectra Data*
Compound
MS
MS-MS
Pancuronium
286.4
Vecuronium
557.5
Rocuronium
529.3
236.6; 472.3; 430.2;
206.7; 100.3
497.4; 398.2; 338.2;
416.2; 458.2; 356.3
487.4
Suxamethonium
Mivacurium
145.2
514.4; 342.3
Atracurium
464.3; 358.2;
570.2
Laudanosine
Quaternary
monoacrylate
Gallamine
358.2
570.2
115.6
671.3; 357.1; 342.2;
600.3; 428.2; 325.2
MS-MS-MS
427.4; 3763; 358.2; 418.2;
487.3; 400.3; 340.3; 445.4
_t
327.0; 307.2; 370.2;
358.1 ;296.1
206.2; 327.0
370.2; 327.0; 412.1;
256.1
284.7
* Most intense ions (relative abundance more than 10%) of MS, MS-MS, and MS-MS-MS spectra are listed in
descending order of intensity.
* MS-MS--MS and MS-MS-MS-MS data for suxamethonium are mentioned in the text.
tC development
Table IV shows the retention times of the
muscle relaxants and their possible degradation
products, when using the gradient described.
Figure I shows a chromatogram of a mixture of
all muscle relaxants examined.
The gradient was optimized towards separation
of the compounds. However, given the limited
choice of volatile eluents, full baseline separation
of all compounds and their degradation products
was not possible. However, compounds with
overlapping peaks could be identified based on
their different mass spectra.
Rocuronium, vecuronium, and pancuronium
show good separation. Only one of the degradation products of atracurium (laudanosine) interferes with the rocuronium peak. Mivacurium and
atracurium show no baseline separation
(between 16 and 22 rain) because these compounds consist of several isomers.
Solid-phaseextraction
BondElut C18 HF cartridges contain C18
bonded silica. Quaternary nitrogen muscle relaxants have on one hand a very polar group or
groups and on the other hand a large apo]ar site.
Residual silanol groups on the sorbent will
become negatively charged under alkaline conditions. These negatively charged silanol groups
interact with the positively charged quaternary
nitrogens. The apolar site of the molecule interacts with the C18 chains of the sorbent.
Therefore, the compounds were eluted under
acidic conditions, when the silanol groups are
protonated. This has the advantage that the
column could be washed with a non acidic solvent, without risking a loss in recovery. Elution
was accomplished with 0.1M acetic acid in
methanol
The recovery of the extraction procedure has
been determined for pancuronium, rocuronium,
31
Journal of Analytical Toxicology,VoL 26, January/February2002
vecuronium and gallamine. The recoverieswere 89, 97, 92, and
95%, respectively.
Because atracurium and mivacurium degrade under alkaline
conditions, degradation will take place during the extraction
process. In biological samples, however, these substances are
alreadyconvertedto their degradationproducts to a large extent.
These products are easilydetected by using our method because
they have a specificmass spectrum, they e]ute within 35 rain and
they are extracted with the solid-phase extraction procedure
described.The presence of the degradationproducts givesstrong
evidencefor the (past) presence of atracurium or mivacurium in
the body.
Propofol/h, 7:59 a.m.; 100 mg Suxamethonium, 8:00 a.m.; and
40 mg Esmeron| (rocuronium bromide), 8:10 a.m. Serum and
urine sampleswere taken at 8:35 a.m.
Methods
The method describedin the previoussection was used to analyze the biological samples. The screening method for muscle
relaxantswas used. Detectionwas in MS and in MS-MS mode.
Results
In the chromatogram of extracted serum, a peak with a massto-charge ratio of 529 was seen. This corresponds with the
molecular mass of rocuronium. Confirmationwas obtained by
measuring the same sample in MS-MS mode. A peak with m/z
487 was observed,correspondingwith the MS-MS spectrum of a
rocuronium standard. Suxamethonium was not detected in the
serum sample.
In the chromatogram of extracted urine, two peakswith massto-charge ratios of 529 and 145, respectively,were seen. When
detection in MS-MS mode was applied, both peaks could be
identified (Figure 2). The first peak with m/z 115 was attributed
to suxamethonium,and the second peakwith m/z 487 to rocuronium.
Suxamethonium was detectable in urine 35 min after administration, but wasn't detectable in serum at the same time.
Rocuroniumwas detectablein both samples25 rain after administration.
A Case of Rocuronium and
Suxamethonium Administration
Casehistory
The following drugs were administered to a woman undergoing a Caesarian section: 2 g Cefazoline, 7:45 a.m.; 20 IJg
Sufentanil, 7:59 a.m.; 160 mg Propofol followed by 700 mg
Table IV. RetentionTimesMuscle Relaxants
Compound
Retentiontime(min)
Suxamethonium
Gallamine
Laudanosine
Rocuronium
Pancuronium
Vecuronium
Atracurium
Mivacurium
Quaternary monoacrylate
1.6
4.5
7.9
7.9
8.6
9.5
17.1, 17.8, 18.4
18.5, 20.5, 22.2
23.9, 25.6
A Case of Intoxication by Pancuronium Injection
Case history
A 30-year-oldman was found dead in his home. The police discovered 14 ampoules of Pavulon| In the trash bin. They also discoveredan empty 10-mLsyringeand a needle in the kitchen. The
cause of death appeared to be administration of the contents of
Rz: 0.00. ~,00
e.01
Tm: 141
~,t~Imm
1.~
~.~
41~
~.~
~
(D~.~ 10.44
12.64
14.a
15.04
1012
IJ.N
~.~
2~.zt4 z4.~t
?,~.44 x ~
30.12
31.12
3321
~z+ss,~.
4e75 MS
~2ml
11.741
*'1"
1000
[LG4 .IO.4D
12.~1
14.14
I~+~
17.45
ILIO
1.~1
"l
i I.w
~+84
~].21
24.17
~.4S
~ 1 7 7 . ~.21_
31.$2
3.1.31
t~.21SE$
1 ~$.0.
116.0 MS
2,41
4.~
4~
Time (rain)
Figure 2. MS-MS spectrumof urine sample.The urine samplewas taken 35 min afteradministrationof suxamethoniumand 25 min afteradministrationof rocuronium.
The upper chromatogram is full scan MS-MS with parent 145 from 0 to 6 min and parent 529 from 6 to 35 min. The middle picture is the samechromatogram with
only mass487 displayed,and the picture on the bottom is the samechromatogram with only mass115 displayed.
32
Journal of Analytical Toxicology, Vol. 26, January/February2002
Because the compound concerned was known, the pancuronium tune file was used. Detection was in MS-MS mode.
Results
The chromatogram of the heart blood extract and the mass
spectrum in MS-MS mode of the pancuronium peak at 8.59 min
are displayed in Figure 3.
Pancuronium was detected and identified in all biological
specimens, namely femoral and heart blood, bile, urine, liver,
muscle, and brain tissue. Both blood samples (heart and femoral
blood) were full of white sugarlike crystals, which appeared to
consist of pancuronium to a large extent. This was determined by
washing these crystals with water, dissolving a small amount in
ammonium acetate buffer/acetonitrile (7:3) and injecting this
solution directly into the MS, after filtration. This resulted in the
MS and MS-MS spectra of pancuronium.
The approximate concentrations of pancuronium in the biological samples are given in TableV. Calibration lines were made
in blood. Because extraction yields may differ from biological
specimen to specimen, the data given in Table V are only indicative. For accurate quantitative results per specimen and per substance, validation procedures have to be carried out. The
concentration in brain tissue is very low, as expected for a quaternary nitrogen compound.
In the various samples, two peaks were consistently observed.
They presumably correspond with the metabolites 3- and 17hydroxypancuronium. The mass-to-charge ratio of the base peak
in the mass spectra of these metabolites corresponds with the
molecular mass of hydroxypancuronium divided by 2.
the ampoutes. However, Pavulon ampoules contain 2 mL of a
pancuronium dibromide solution with a concentration of 2
mg/mL. For this reason, it was impossiblethat the contents of all
14 ampoules pancuronium dibromide were administered in one
injection.
The place of injection in the arm was covered by a bandage.
Autopsy findings were unremarkable, and no signs of violence
were observed.Biologicalspecimens including blood, urine, bile,
brain, muscle, and liver were collected for toxicologicalanalysis.
Methods
The previously described method was used to analyze the biological samples. Blood samples spiked with pancuronium were
analyzed for comparison.
Table V. Approximate Concentrations Found in Biological
Fluids and Tissues in a Case of Intoxication by
Pancuronium Injection
Biological sample
Concentration
Femoral blood
Heart blood
Urine
Bile
Liver
Brain
Muscle
0.7 mg/L
0.7 mgtL
1.8 mg/L
0.4 mg/L
2.4 mg/kg
0.1 mg/kg
0.2 mg/kg
01 I11/2000 12:01:37
F:~QUATS_ESIk...~rBst m o n s t e r l l ~ l l r ~ l o e d
RT; 0+0O- 35~00
1OO-
hllrtbloed 991028025
!
Nk:
%44E4
r
g0-
~
MS
Ra~blQad
8o
i 'o
60
50
~
4O
30
30
10
'~,32 1 ~0
2
3,83 4,61
4
7.~1. 8"3~
e
IS
~8
.!1:04
10
13.76 14.63
12
14
17.12 17.07
16
19.67 20.98 22+g?
10
20
32
25.43
24
~6
27.80 26.91 30,30 32.59 33.42
28
30
32
34
Time (rain)
hsrlbloed#651-718 RT:8.37-9.13 A~ 68 NL: 7,9$E4
T: * C ESI Full ms2 [email protected] [ 100.00-500.00)
230,6
90
50
1001
205+7
430,3
70
60
50
40
30
20
100,3
10~1--'
1229 143.7 165.7 185.6
0-~ ~,.~'.,...., ,',.,i..'..lpL.,l~......;+r ..'l .. ~'-,-:.-1-:.L:
100
150
200
[i
2~5a
~sp., 20,.2
250
~004 33p~ 34..4 3,0.3
300
350
.4,~2
400
,~,0. ,,,6
450
,500,
500
nVz
Figure 3. Chromatogram in MS-MS mode and MS-MS spectrumof the peak at 8.59 min an extractedsampleof heart blood. In the upper picture, the chromatogram
in MS-MS mode with parent 286 is displayed. The correspondingMS-MS spectrum of the peak is also displayed.
33
Journal of Analytical Toxicology, Vol. 26, January/February 2002
Conclusions
The system described is suited to identifyquaternary nitrogen
muscle relaxants in whole blood. Other specimens (such as
urine, serum, bile, liver, muscle, and brain tissue) may also be
used. However,for accurate quantitative results per specimen
and per substance, validation procedures have to be carried out.
In a possible case of assisted suicide, pancuronium was
detected and identifiedin a number of biologicalspecimens. The
results showed that the cause of death was the result of the
administration of pancuronium (Pavulon injection). In another
case, suxamethonium was detected in urine 35 min after administration, but not in serum. This is a result of the short half-life
of quaternary nitrogen compounds in general. Because of the
short half-life,these compounds may be present in higher concentrations in urine than in blood after a time interval as small
as 10 min. Wheneverpossible, both samples should be analyzed.
In cases of atracurium and mivacurium, identification of the
metabolites will generally be the only way to prove the administration of these compounds.
References
1. C. Schopfer and A. Benakis. Simplified method for the determination of atracurium and laudanosine in pig plasma by high-performance liquid chromatography and fluorimetric detection.
J. Chromatogr. 526:223-227 (1990).
2. E. Neill and C. Jones. Determination of atracurium besylate in
34
human plasma. J. Chromatogr. 274:409-412 (1983).
3. A. Brown, C. James, R. Welch, and J. Harrelson. Stereoselective
high-performance liquid chromatographic assay with fluorometric
detection for the isomers of mivacurium in human plasma.
J. Chromatogr. S78:302-308 (1992).
4. M. Lacroix, T. Tu, F. Donati, and F. Varin. High-performance liquid
chromatographic assayswith fluorometric detection for mivacurium
isomers and their metabolites in human plasma. J. Chromatogr. B
663:297-307 (1995).
5. L. Wingard, E. Abouleish, D. West, and T. Goehl. Modified fluorometric quantitation of pancuronium bromide and metabolites in
human maternal an umbilical serums. J. Pharm. Sci. 68:914-916
(1979).
6. E. Briglia, P. Davis, M. Katz, and L. Dal Cortivo. Attempted murder
with pancuronium. J. Forensic Sci. 35:1468-1476 (1990).
7. J. Greying, J. Jonkman, F. Fiks, and R. de Zeeuw. Determination of
oxyphenonium bromide in plasma and urine by means of ion-pair
extraction, derivatization and gas chromatography-electron capture
detection. J. Chromatogr. 554:39-46 (1991).
8. M. Nisikawa, M. Tatsuno, S. Suzuki, and H. Tsuchihashi.The analysis
of quaternary ammonium compounds in human urine by direct
inlet electron impact ionization massspectrometry. Forensic ScL Int.
51:131-138 (1991).
9. M. Weindlmayr-Goettel, G. Weberhofer, H. Girly, and H. Kress.
Determination of mivacurium in plasma by high-performance
liquid chromatography. J. Chromatogr. B 685:123-127 (1996).
10. T. Lukaszewski. The extraction and analysis of quaternary ammonium compounds in biological material by GC and GC-MS. J. Anal
Toxicol. 9:101-108 (1985).
11. F. Varin, J. Ducharme, and J. Besner. Determination of atracurium
and laudanosine in human plasma by high-performance liquid
chromatography. J. Chromatogr. 529:319-327 (1990).
Manuscript received February 27, 2001 ;
revision received July 3, 2001.