Simultaneous Determination of Bupivacaine, Mepivacain, Prilocaine and Ropivacain in Human Serum by Liquid Chromatography-Tandem Mass Spectrometry
Simultaneous Determination of Bupivacaine, Mepivacain, Prilocaine and Ropivacain in Human Serum by Liquid Chromatography-Tandem Mass Spectrometry
Simultaneous Determination of Bupivacaine, Mepivacain, Prilocaine and Ropivacain in Human Serum by Liquid Chromatography-Tandem Mass Spectrometry
Abstract
A liquid chromatography–tandem mass spectrometric (LC–MS-MS) method with a rapid and simple sample preparation was developed
and validated for the simultaneous determination of the local anesthetics bupivacaine, mepivacaine, prilocaine and ropivacaine in human
serum. An external calibration was used. The mass spectrometer was operated in the multiple reaction monitoring mode. A good quadratic
response over the range of 1.0–200.0 ng/ml was demonstrated. The accuracy for bupivacaine ranged from 93.2 to 105.7%, for mepivacaine
from 96.2 to 104.3%, for prilocaine from 94.6 to 105.7% and for ropivacaine from 94.3 to 104.0%, respectively. The limit of quantification
was 1.0 ng/ml for all substances. This method is suitable for pharmacokinetic studies.
© 2005 Elsevier B.V. All rights reserved.
0021-9673/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2005.03.071
A. Koehler et al. / J. Chromatogr. A 1088 (2005) 126–130 127
ously the local anesthetics bupivacaine, mepivacaine, prilo- stein, Germany) with a Chromeleon Chromatography Data
caine and ropivacaine in biological samples, requiring small System (Dionex Softron, Idstein, Germany). The chromato-
sample volumes and enabling a low limit of quantification. graphic separation was performed on a Synergy 4 Polar-RP
80A, 150 mm × 2 mm (Phenomenex, Aschaffenburg, Ger-
many) column with a Security Guard C18, 4 mm × 2 mm i.d.
2. Experimental
(Phenomenex).
2.1. Chemicals The following gradient was applied, with solvent A
(5/95/0.2, v/v/v) and solvent B (95/5/0.2, v/v/v) each being a
Reference substances of the local anesthetics bupivacaine mixture of acetonitrile, 2 mM ammonium acetate and formic
(Carbostesin® 0.5%), mepivacaine (Scandiain® 1%), ropiva- acid:
caine (Naropin® 10 mg/ml) and prilocaine (Xylonest® 1%)
Time (min) 0.0 0.1 2.5 3.0 3.2 7.0
were purchased from AstraZeneca (Wedel, Germany). A (%) 100 100 20 20 100 100
Acetonitrile LiChrosolv (for chromatography), methanol B (%) 0 0 80 80 0 0
LiChrosolv (for chromatography) and ammonium acetate
(analytical reagent grade) were purchased from Merck The flow-rate was 0.5 ml/min, and temperature was 25 ◦ C.
(Darmstadt, Germany). Pure water (18 M) was obtained
using an ion-exchange system RS 40E, SG Ionenaustauscher 2.3. Sample preparation
(Barsbüttel, Germany).
The frozen serum samples (−20 ◦ C) were thawed at room
2.2. LC–MS-MS analysis—apparatus and temperature.
chromatographic conditions The sample volume of 0.4 ml serum was diluted with
0.8 ml of acetonitrile, mixed (Vortex, Genie2) and centrifuged
2.2.1. Mass spectrometer for 10 min. 0.8 ml of the liquid layer was diluted with 1.2 ml
The LC–MS-MS system used was a Quattro micro (Micro- solvent A (mobile phase), removed to a 1 ml autosampler vial
mass, Manchester, UK) equipped with an electrospray inter- and 10 l were injected for LC–MS-MS.
face (ESI). Full scan mass spectrum were acquired by contin-
ual infusion of standard solution (concentration 200.0 ng/ml 2.4. Recovery and ion suppression
with 10 l/min). The product ion mass spectra were obtained
by choosing the molecular ions as the precursor ions and The recovery from serum was evaluated by comparing
scanning product ions from m/z 80–300. For positive ionisa- the peak areas of amounts mixed with assayed blanks and
tion a capillary voltage of 3200 V and ion source temperature injected than directly with those of assayed samples. The ion
of 100 ◦ C were applied. The desolvation gas flow (nitrogen) suppression of the drugs in the mass spectrometer (effect of
was 600 l/h at 300 ◦ C. the matrix) were evaluated by comparing the peak areas of
The multiple reaction monitoring (MRM) was performed amounts injected directly with peak areas of amounts mixed
by monitoring the transitions between m/z 288.9 (parent ion) with assayed blanks and injected than directly.
and m/z 140.2 for bupivacaine, between m/z 247.0 (parent The slope of the quadratic regression line y = ax2 + bx + c
ion) and m/z 98.1 for mepivacaine, between m/z 221.0 (par- of the response x of amounts mixed with assayed blanks (and
ent ion) and m/z 86.0 for prilocaine and between m/z 275.3 than injected directly) and the response of the assayed sam-
(parent ion) and m/z 126.2 for ropivacaine. The collision gas ples y delivers the mean recovery of the concentration range
was argon. The dwell time, collision energy and pressure are investigated. The slope of the quadratic regression line of the
shown in the following table: response x of directly injected amounts and the response of
the amounts mixed with assayed blanks (and than injected
Compound Dwell time (ms) Collision energy (eV) Pressure (mbar) directly) y delivers the mean ion suppression of the concen-
Bupivacaine 200 18 3.20e−3 tration range investigated. The peak areas of local anesthetics
Mepivacaine 200 16 3.20e−3 at seven concentrations in the range of 1.0 up to 200.0 ng/ml
Prilocaine 200 12 3.20e−3 were used. The graphs were evaluated using the “Trend line
Ropivacaine 200 19 3.03e−3
function” of Excel.
The transition was used for the determination of analyte
concentration.
The MassLynx Data System was applied for MS control 3. Results
and QuanLynx for peak area evaluation, regression analysis
of standard curves and calculation of concentrations. 3.1. Mass spectrometry
Fig. 1. MS-MS spectra of bupivacaine, mepivacaine, prilocaine and ropivacaine: the spectrum reveales a base peak at m/z 288.9 and daugther peak m/z 140.2
for bupivacaine, a base peak at m/z 247.0 and daugther peak m/z 98.1 for mepivacaine, a base peak at m/z 221.0 and daugther peak m/z 86.0 for prilocaine and
a base peak at m/z 275.3 and daugther peak m/z 126.2 for ropivacaine.
The fragment ion of bupivacain observed at m/z 140.2 ligible. Because of the low effect of the matrix and the good
corresponds to the butane-1-piperidinyl group, the fragment reproducibility of the sample preparation an internal standard
ion of mepivacain observed at m/z 98.1 corresponds to the was not necessary but it is possible to use one of the drug as
methane-1-piperidinyl group, the fragment ion of prilocain an internal standard for the determination of the other drugs.
observed at m/z 86.0 corresponds to ethylpropylamine and
the fragment ion of ropivacain observed at m/z 126.2 cor- 3.4. Calibration graph
responds to the propane-1-piperidinyl group. Intensities of
other fragments are negligible. The calibration graphs for the local anesthetics were gen-
erated from MRM of increasing amounts of the drug stan-
3.2. Chromatography dards. A quadratic calibration graph was constructed using
least-squares regression of quantities versus peak area. A
Because of the high specificity of the MS-MS method a good response over the range of 1.0–200.0 ng/ml was demon-
complete chromatographic separation of analytes and matrix strated. Samples with a higher concentration of the analytes
is not necessary. To exclude interferences from the biologi- were diluted. The correlation coefficient of regression lines
cal matrix, chromatograms of all local anesthetics were con- was 0.9974 or higher.
trolled separately. No interferences and a low background
noise were found (Fig. 2). 3.5. Reproducibility
MS-MS does not respond to ions originating from impu-
rities of the biological matrices that differ in m/z from the The precision and accuracy of the method were assessed
selected ion. The retention time of bupivacaine was about by determination of seven concentrations in six independent
4.6 min, of mepivacaine about 4.2 min, of prilocaine about series of spiked serum samples (see Table 1). The accuracy
4.2 min and of ropivacaine about 4.4 min (Fig. 2). was calculated to be from 93.2 to 105.7% for bupivacaine,
Fig. 2. Chromatograms of bupivacaine, mepivacaine, prilocaine and ropivacaine: (A) blank samples, (B) standards with 5 ng/ml and (C) patient sample with
56 ng/ml prilocaine and 183 ng/ml ropivacain.
from 96.2 to 104.3% for mepivacaine, from 94.6 to 105.7% In this study an analytical method of local anesthetic deter-
for prilocaine and from 94.3 to 104.0% for ropivacaine, re- mination using LC–MS-MS after simple sample preparation
spectively. The coefficient of variation (CV) ranged from 0.7 by protein precipitation has been developed. Only the small
to 6.6% for bupivacaine, from 0.8 to 9.6% for mepivacaine, sample volume of 0.4 ml serum was necessary to achieve
from 0.9 to 6.5% for prilocaine and from 0.6 to 7.1% for a limit of quantification of 1.0 ng/ml. A number of about
ropivacaine, respectively. The limit of quantification (LOQ) 100 samples a day can be analysed in this way without any
was obtained by comparing the true and measured values for decreasing of the limit of quantification or accumulation of
the lowest standard data points and blanks of the six stan- sample matrix in the ion-source. The method is suitable for
dard curves. The acceptance criterion was a deviation of less routine measurements of local anesthetics in human serum
than ±20%. LOQ of 1.0 ng of local anesthetics/ml could be samples.
accepted. The day-to-day recovery was calculated with three
different quality control samples to be from 85.1 to 111.1%
for bupivacaine, from 89.1 to 113.4% for mepivacaine, from 5. Conclusion
86.2 to 113.7% for prilocaine and from 86.4 to 113.6% for
ropivacaine, respectively. LC–MS-MS is the most sensitive method for a quanti-
tation of local anesthetics in serum. Furthermore, the assay
requires only a rapid and simple sample preparation. To in-
4. Discussion crease the sensitivity it is possible to increase the sample vol-
ume. This method is suitable for pharmacokinetic and clinical
Liquid chromatography tandem mass spectrometry is a studies.
powerful technique for highly specific and quantitative mea-
surements of low levels of analytes in biological matrices.
There have also been several other reports on the use of the Acknowledgement
highly sensitive and specific liquid chromatography–tandem
spectrometry technique to measure local anesthetics in hu- The authors thank Mrs. U. Mann for the technical assis-
man plasma. tance.
130 A. Koehler et al. / J. Chromatogr. A 1088 (2005) 126–130