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We report the evaluation of several mass spectrometry-based methods for the determination of carisoprodol and meprobamate in samples obtained from the rat brain by in vivo intracranial microdialyis. Among the techniques that aspire to perform analyses without chromatographic separation and thereby increase throughput, chip-based nanoelectrospray ionization and the use of an atmospheric pressure solids analysis probe fell short of requirements because of insufficient detection sensitivity and hard ionization, respectively. Although direct analysis in real time provided the required soft ionization, shortcomings of a tandem mass spectrometry-based assay also included inadequate detection sensitivity and, in addition, poor quantitative reproducibility. Therefore, liquid chromatography coupled with atmospheric pressure chemical ionization tandem mass spectrometry was developed to determine carisoprodol and meprobamate from artificial cerebrospinal fluid as the medium. No desalting and/or extraction of the samples was necessary. The assay, combined with in vivo sampling via intracranial microdialyis, afforded time-resolved concentration profiles for the drug and its major metabolite from the nucleus accumbens region of the brain in rats after systemic administration of carisoprodol. Copyright
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Mass spectrometric analysis of carisoprodol and meprobamate
in rat brain microdialysates
Laszlo Prokai*, Petr Fryčák1, Vien Nguyen, and Michael J. Forster
Center for Neuroscience Discovery, Institute for Healthy Aging, University of North Texas Health
Science Center, Fort Worth, Texas, USA
Abstract
We report the evaluation of several mass spectrometry-based methods for the determination of
carisoprodol and meprobamate in samples obtained from the rat brain by
in vivo
intracranial
microdialyis. Among the techniques that aspire to perform analyses without chromatographic
separation and thereby increase throughput, chip-based nanoelectrospray ionization and the use of
an atmospheric pressure solids analysis probe fell short of requirements because of insufficient
detection sensitivity and hard ionization, respectively. Although direct analysis in real time
(DART) provided the required soft ionization, shortcomings of a tandem mass spectrometry-based
assay also included inadequate detection sensitivity and, in addition, poor quantitative
reproducibility. Therefore, liquid chromatography coupled with atmospheric-pressure chemical
ionization tandem mass spectrometry was developed to determine carisoprodol and meprobamate
from artificial cerebrospinal fluid as the medium. No desalting and/or extraction of the samples
were necessary. The assay, combined with
in vivo
sampling via intracranial microdialyis, afforded
time-resolved concentration profiles for the drug and its major metabolite from the nucleus
accumbens region of the brain in rats after systemic administration of carisoprodol.
Keywords
In vivo
intracranial microdialysis; carisoprodol; meprobamate; nanoelectrospray ionization; direct
analysis in real time; atmospheric pressure solids analysis probe; LC–MS/MS; atmospheric-
pressure chemical ionization
Introduction
Carisoprodol (N-isopropylmeprobamate, Soma®) is a centrally-acting skeletal muscle
relaxant frequently indicated for the alleviation of lower back pain and short-term treatment
of acute musculoskeletal conditions.[1,2] Carisoprodol is metabolized relatively rapidly in the
liver to the barbiturate sedative hypnotic, meprobamate,[3] and the latter would seem to
account for both its therapeutic effects and abuse potential.[4] Meprobamate (Miltown®,
*Correspondence to: Laszlo Prokai, Center for Neuroscience Discovery, Institute for Healthy Aging, University of North Texas Health
Science Center, 3500 Camp Bowie Boulevard, Fort Worth, Texas 76107, USA. Tel.: +1-817-735-2206. Laszlo.Prokai@unthsc.edu.
1Present address: Regional Centre of Advanced Technologies and Materials, Department of Analytical Chemistry, Palacky University
in Olomouc, Olomouc, Czech Republic
Supporting Information
Additional supporting information may be found in the online version of this article at the publisher’s web site.
HHS Public Access
Author manuscript
J Mass Spectrom
. Author manuscript; available in PMC 2017 October 01.
Published in final edited form as:
J Mass Spectrom
. 2016 October ; 51(10): 900–907. doi:10.1002/jms.3799.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
Equanil®) was commonly used in the treatment of anxiety before its classification as a
Schedule IV controlled substance in the USA.[5] Based on a propensity to cause abuse,
tolerance and physical dependence similar to meprobamate,[6] carisoprodol also was placed
more recently to Schedule IV.[7] While it has been widely accepted that the therapeutic
actions and abuse potential of carisoprodol can be attributed to its metabolism to
meprobamate,[5] recent studies have confirmed that carisoprodol has meprobamate-
independent intrinsic effects,[8–10] and is itself is an active compound with significant
barbiturate-like actions in the central nervous system (CNS).[11]
One approach to discern how carisoprodol itself and meprobamate contribute to rewarding
effects that confer abuse, and dependence, would be to compare the time course of each at
relevant sites in the CNS. Sampling via
in vivo
intracerebral microdialysis has been an
attractive approach to gain insight in distribution of drugs and drug metabolites to the
brain.[12,13] The microdialysis technique offers the possibility to obtain a large number of
sample fractions from a single animal with temporal resolution of a few minutes and study
duration of several hours. Accordingly, it is desirable to have an analytical method with
corresponding throughput available to match the sampling rate of the experiment. Mass
spectrometric detection appears to be attractive for this purpose due to its speed, sensitivity,
selectivity and ease of use. Due to minimal or no sample preparation, methods that could
eliminate online chromatographic separations and thereby increase sample throughput by
using ambient ionization and flow-injection analysis[14,15] have been proposed to increase
productivity. For brain microdialysates obtained from pharmacokinetics and drug
metabolism studies, this approach has appeared to be especially promising because the
protein-free matrix could be considered “simple” compared to biological fluids such as
plasma.[16] Here, we report the evaluation of three sample introduction and ionization
methods for the prospective analysis of carisoprodol and meprobamate in rat brain
microdialysates without chromatographic separation: chip-based nanoelectrospray ionization
(nanoESI),[17] as well as contact surface desorption/ionization using direct analysis in real
time (DART)[18] and atmospheric pressure solids analysis probe (ASAP).[19]
Carisoprodol and meprobamate concentrations have also been determined by
chromatographic methods combined with mass spectrometric (MS) detection. Gas
chromatography–mass spectrometry (GC–MS) without derivatization has a rather high limit
of detection (2 μg/mL)[20] and, therefore, derivatization has been employed to increase
volatility and assay sensitivity for these analytes after their extraction from clinical
samples.[21] Carisoprodol and meprobamate can be conveniently separated by reversed-
phase (RP) liquid chromatography (LC), and LC-MS methods published using electrospray
ionization (ESI) for their determination in human urine and plasma,[22] bovine serum,[23] as
well as equine urine and serum[24] have reached limit of quantitation (LOQ) from 2 μg/mL
on a single quadrupole instrument[22] down to 0.25 – 5.0 ng/mL on a triple quadrupole
tandem mass spectrometer.[23] Due to the option of avoiding tedious offline extraction from
small volumes followed by derivatization and, instead, simply using online desalting on an
RP column, LC appears to be an ideal choice to the hyphenated MS analyses of carisoprodol
and meprobamate in samples obtained through
in vivo
intracranial microdialysis. In our
studies, a linear ion trap mass spectrometer was used to achieve the necessary sensitivity and
selectivity for the reported measurements.
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Experimental
Chemicals
Artificial cerebrospinal fluid (aCSF) was obtained from Harvard Apparatus (Holliston, MA,
USA). All other drugs, chemicals, analytical standards, reagents and solvents were
purchased from Sigma-Aldrich (St. Louis, MO, USA).
Mass spectrometry
Mass spectra were recorded using a linear ion trap (LTQ, Thermo, San Jose, CA, USA) in
the
m/z
range of 50–300. To perform tandem mass spectrometry (MS/MS), the precursor
ions were isolated for collision-induced dissociation (CID) in the LTQ. Specific MS
conditions and, if applicable, set-up for MS/MS acquisitions are detailed in the sections
below.
Chip-based nanoESI-MS
Nanoflow infusion was performed by a TriVersa Nanomate (Advion, Ithaca, NY, USA) chip-
based nanoelectrospray system operated at 0.5-psi nitrogen pressure and 1.5-kV nanoESI
voltage in positive ion mode using an ESI Chip with 400 microfabricated nanoelectrospray
emitters.[25] Samples were prepared in water/methanol (1:1, v/v) solutions containing 0.25%
acetic acid, and 5 μL aliquots were deposited into the wells of the microtiter plate placed in
the Nanomate. NanoESI and MS data acquisition were carried out by ChipSoft (Advion,
version 8.1.0) and the Xcalibur (Thermo, version 2.0) software, respectively. Ion transfer
capillary temperature was maintained at 200 °C.
DART
The DART source was a model 100 device (IonSense, Saugus, MA, USA) operated with
helium as working gas at a flow rate of 12 L/min and temperature of 300 °C. The discharge
needle voltage was set to 3800 V and the voltages of electrode 1 and electrode 2 to +400 V
and +500 V, respectively. Samples were prepared by depositing 3 μL from methanolic
solution onto the outer surface of sealed end of glass melting point determination tubes (100
mm × 1.5 mm o.d., Sigma-Aldrich) followed by air drying. Mass spectra were acquired by
bringing the sealed end of the tube with the deposited sample into the stream of helium gas
and thereby sweeping the desorbed and ionized molecules into the heated capillary of the
atmospheric pressure interface of the instrument.
The [M+H]+ ions of carisoprodol (
m/z
261.2), meprobamate (
m/z
219.1) and diethyl
acetamidomalonate internal standard (ISTD,
m/z
218.1) were subjected to collision-induced
dissociation (CID) in the MS/MS-based experiments. Meprobamate and the ISTD were
fragmented using a common precursor isolation window of
m/z
218.6 ± 1.5 Th, collision
energy of 20%, and activation time of 30 ms, but using
m/z
158.1 and
m/z
176.1 as product
ions, respectively, in selected reaction monitoring (SRM). Carisoprodol was detected with
precursor isolation window of
m/z
261.2 ± 1.0 Th, collision energy of 20%, activation time
of 30 ms, and relying on
m/z
176.1 as product ion in the SRM.
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ASAP
The ASAP probe (M&M Mass Spec Consulting, Newark, DE, USA) was mounted on the
side of the atmospheric-pressure ionization source fitted with the APCI vaporizer and
discharge electrode (needle). The vaporizer and heated capillary temperatures were set to
350 °C and 175 °C, respectively, the discharge current to 4 μA, sheath gas (nitrogen) flow
rate to 25 units and auxiliary and sweep gas flow rates to 5 units. As described in the DART
section, analytes were deposited onto the outer surface of sealed end of glass melting point
determination tubes from methanolic solutions. Then, the air-dried samples were introduced
into the corona discharge zone of the APCI source adjacent to the orifice of the heated
capillary using the ASAP probe.
LC–APCI-MS/MS
The study was conducted using a Surveyor HPLC gradient pump with a Micro AS
autosampler (Thermo, San Jose, CA, USA). The separation was run on a Discovery HS C18
50 mm × 2.1 mm column with 5 μm sorbent particles (Supelco, Bellefonte, PA, USA) under
isocratic conditions with water/acetonitrile/acetic acid 68:32:0.5 (v/v/v) as a mobile phase at
flow rate of 0.25 mL/min. The injection volume was 5 μL. Online desalting was used with
the divert valve mounted on the LTQ directing the effluent to waste for 0.7 min after
injection.
Again, the [M+H]+ ions of carisoprodol (
m/z
261.2), meprobamate (
m/z
219.1) and the
ISTD (
m/z
218.1) were used as precursor ions of MS/MS scans. Meprobamate and the ISTD
were fragmented in a single scan event (fragmentation window
m/z
218.6 ± 1.5 Th, collision
energy 20%, activation time 30 ms) using
m/z
158.1 and
m/z
176.1 as product ions for
meprobamate and the ISTD, respectively. Carisoprodol was monitored through its own SRM
scan (precursor-ion isolation window of
m/z
261.2 ± 1.0 Th, collision energy of 20%,
activation time of 30 ms, detecting the analyte using the major fragment of
m/z
176.1 in the
SRM chromatograms.
The LC/APCI/MS/MS method was validated in terms of sensitivity, accuracy, precision and
linearity using 5 μL injections of solutions containing carisoprodol and meprobamate
standards and 10 ng/mL of ISTD. Limit of detection (LOD) and LOQ were assessed as a
concentration that yielded peaks with signal-to-noise ratio (S/N) of ≥3 and ≥10, respectively.
Calibration was performed for both analytes dissolved in artificial cerebrospinal fluid and in
the 1 ng/mL to 1000 ng/mL concentration range. To determine precision and accuracy of the
method, solutions containing 10 ng/mL of both analytes and the ISTD were injected in
groups of six replicates five times within one day in two-hour intervals (intraday
reproducibility) and in six consecutive days (day-to-day reproducibility). The observed
relative errors and relative standard deviations (RSD) were calculated from arithmetic means
of the six-replicate groups.
In vivo intracerebral microdialysis in rats
Samples were collected from four male Sprague-Dawley rats (Hsd:Sprague Dawley®SD®,
250–300 g body weight, purchased from Harlan Laboratories, Indianapolis, IN) using
CMA/12 probes (CMA Microdialysis, Torshamnsgatan, Sweden) according to a protocol
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described earlier in detail[26,27] and modified to sample from the nucleus accumbens region
of their brain.[28] Carisoprodol was administered intraperitoneally (i.p.) at a single dose of
100 mg/kg body weight. At a probe perfusion rate of 2 μL/min using aCSF, microdialysates
were collected every 5 min in the first hour after carisoprodol administration, then every 20
min for 2 hours. The procedures were reviewed and approved by the Institutional Animal
Care and Use Committee at the University of North Texas Health Science Center before the
initiation of the study. The samples were prepared by mixing with equal volume of 20
ng/mL aqueous ISTD solution to achieve 10 ng/mL concentration of ISTD in the solutions
to be analyzed.
A separate in vitro experiment was carried out to determine the microdialysis recovery. A
solution containing 200 ng/mL of meprobamate and 20 ng/mL of carisoprodol in aCSF was
dialysed at 2 μL/min to collect four 20-min samples in succession after 1-h probe
equilibration, and the concentrations of analytes were then determined in the dialyzed
solution and in the collected microdialysates. The recovery was defined as the ratio or
percentage of these concentrations (microdialysate/dialysed solution).
Results and discussion
After systemic administration, carisoprodol enters the bloodstream and is distributed
throughout the body, including the brain.[29,30] N-Dealkylation of carisoprodol catalyzed by
the cytochrome P450 (CYP) isoform CYP2C19 in the liver results in meprobamate,[3,30]
which also has the ability to enter the central nervous system.[31] As the midbrain-forebrain-
extrapyramidal circuit with its focus in the nucleus accumbens has been linked
mechanistically to substance abuse,[32] psychoactive drug and metabolite concentrations
measured from this site were expected to yield the most relevant information regarding
mechanistic and behavioral correlates studied in experimental animals.[10,11]
NanoESI, DART and ASAP were evaluated regarding their applicability of chromatography-
free MS to carisoprodol and meprobamate analyses in microdialysis samples. Desalting of
brain microdialysates is essential for nanoESI; however, it can be done rapidly as part of
sample preparation by using ZipTips.[17] DART and ASAP may tolerate inorganic salts as
sample constituents without significant adverse impact on MS analyses of small organic
compounds.[33,34] Nevertheless, desalting will prevent source contamination, which could
reduce frequency of cleaning due to formation of nonvolatile inorganic deposits affecting
assay performance. Therefore, we also employed acidified aqueous/methanolic sample
solutions (containing 10 μg/mL of the analyte) for the initial “go/no go” evaluation of these
methods to determine carisoprodol and meprobamate in rat brain microdialysates.
In addition to focusing on signal intensity attributable to the analytes in the initial
evaluations, soft ionization was also considered an important criterion to enable mass
spectrometric analyses without chromatographic separation. Specifically, the lack of in-
source fragmentation of the molecular ions simplifies mass spectra and facilitates the
determination of analytes present at low concentration through MS/MS-based methods.[35]
As demonstrated in Fig. 1, only DART was worth further consideration based on these
requirements. Chip-based nanoESI performed poorly for carisoprodol, because the recorded
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mass spectrum featured ions unrelated to the analyte (emanating from contaminants of the
solvents and/or sample). ASAP was found to be a relatively “hard” (energetic) method of
ionization, because extensive fragmentation diminished the relative abundance of the
molecular ions ([M+H]+ at
m/z
261 and 219) to merely 0.4 and 0.1 percent, respectively,
with a small fragment ion at
m/z
55 (plausibly C4H7+) representing the base peak in the
recorded ASAP mass spectra of both carisoprodol and meprobamate.
For a pilot evaluation of an MS/MS-based quantification without chromatography, DART
was first optimized by varying parameters of the ion source while ionizing same amounts (9
ng) of carisoprodol and meprobamate to ensure that the system was tuned for the best
possible response. Considering the features of the linear ion trap used in our experiments,
diethyl acetamidomalonate ([M+H]+ at
m/z
218.1) was evaluated as an ISTD despite the
mere 1.0 Th difference between the
m/z
of its precursor ions from that of meprobamate ([M
+H]+ at
m/z
219.1). The first 13C-isotopic peak of diethyl acetamidomalonate is isobaric
with meprobamate; hence, this overlap is non-ideal and should be avoided generally when
choosing an ISTD. However, meprobamate and diethyl acetamidomalonate could be isolated
together for CID-MS/MS, while the resulting series of product-ion scans could be
transformed into two separate SRM traces thanks to the distinct masses of corresponding
major fragments (
m/z
158.1 for meprobamate and m/z 176.1 for diethyl
acetamidomalonate). This latter observation supported our consideration of diethyl
acetamidomalonate as an acceptable ISTD even for meprobamate. Figure 2 shows three
replicates of desorption/ionization followed by CID-MS/MS after depositing 3 ng of
carisoprodol and meprobamate to the glass probes. These were the lowest quantities of the
analytes that yielded reliable signal. Nevertheless, it was apparent from the SRM peak areas
that the repeatability was not satisfactory for accurate quantification, even without evaluating
the coefficient of variation (CV) of the responses, at ≤1 μg/mL analyte levels in sample
solutions without preconcentration. Therefore, our implementation of DART-MS/MS was
not suitable to determine carisoprodol and meprobamate in rat brain microdialysates.
Possible improvement could consider additional pumping applied to the atmospheric
pressure inlet on the mass spectrometer to compensate for the increased vacuum load
because of the use of high-flow helium, which has been reported to increase detection
sensitivity by 10 to 100 times.[36] Automated sample introduction into the DART beam with
high precision may also improve reproducibility of the method.[36] The application of these
custom measures has been beyond the scope of our work; therefore, we decided to develop
an LC–MS/MS assay for the analysis of carisoprodol and meprobamate in rat brain
microdialysates.
Compared to ESI,[37] we found that APCI afforded higher detection sensitivity for LC–MS
analyses of carisoprodol and meprobamate. APCI also is less prone to matrix effect for
dilute samples than ESI.[38] Full-scan APCI mass spectra of the analytes and product-ion
spectra of their protonated molecules are shown in Fig. 3. Meprobamate and carisoprodol
were separated well on a short octadecylsilica reversed-phase column under isocratic
conditions with an acidified water/acetonitrile mobile phase. Similarly to other assays we
developed,[27,39] online desalting also could be done conveniently by diverting the effluent
to waste and thereby avoiding laborious sample preparation such as liquid-liquid extraction
also applied to
in vivo
microdialysates.[40] Meprobamate and diethyl acetamidomalonate
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eluted with retention time difference of about 0.1 min. Again, the resulting series of product-
ion scans could be transformed into two separate SRM chromatograms thanks to the distinct
masses of corresponding major fragments (
m/z
158.1 for meprobamate and
m/z
176.1 for
diethyl acetamidomalonate). Therefore, we developed MS/MS-based quantitation using
diethyl acetamidomalonate as an ISTD added to the microdialysates at 10 ng/mL
concentration. In Figure 4, SRMs resulting from three replicate 5-μL injections of
meprobamate and carisoprodol solutions, 1 ng/mL each in aCSF, with ISTD concentration of
10 ng/mL are displayed. These analyte concentrations were equal or close to the LOQs
based on the S/N values obtained. Therefore, the sensitivity and reproducibility of the
measurements demonstrated that LC–APCI-MS/MS was suitable for an accurate
determination of carisoprodol and meprobamate concentrations in aCSF as a matrix.
Performance of the developed assay is summarized in Table 1. Overall, LOD/LOQ,
reproducibility and accuracy met requirements of experimental studies for samples collected
by
in vivo
microdialysis from the rat brain. Calibration plots are shown in Figure S-1 in the
online Supporting Information. Higher detection sensitivity for meprobamate was due to its
higher response factor (approximately 50% higher than that of carisoprodol), partly because
it eluted earlier than carisoprodol and, thus, afforded narrower peaks upon isocratic elution,
as shown in Fig. 4. Further improvement of the method may include the application of
ultrahigh performance liquid chromatography (UPLC), which has been reported to reduce an
assay’s cycle time to one minute upon measuring heroin and its metabolite from the
extracellular fluid of the rat brain using
in vivo
microdialysis sampling.[41]
Sampling by microdialysis from the extracellular fluid of the rat brain represents a dynamic
non-equilibrium system that is not characterized by an explicit link between the
concentration of analytes in the tissue and that in the microdialysate collected.[12] Therefore,
a separate
in vitro
experiment was carried out to estimate a “recovery” percentage for
sampling, which turned out to be 20% for both carisoprodol and meprobamate. Figure 5
summarizes the concentration–time profile of the drug and its metabolite based on
quantitative LC–APCI-MS/MS assays of in vivo microdialysates from the nucleus
accumbens of rats administered with 100 mg/kg i.p. carisoprodol and considering these
recovery estimates. Our preliminary results obtained by using four experimental animals
have shown that carisoprodol reached its highest concentration (cmax) 20 min after injection
(tmax), while meprobamate afforded tmax of about 2 h. For about 40 min, carisoprodol
concentrations in the extracellular fluid of the nucleus accumbens were higher than or equal
to those of meprobamate. On the other hand, cmax of the metabolite was about 5 times higher
than that of the parent drug.
Conclusions
Chip-based nanoESI and ASAP fell short of requirements because of insufficient detection
sensitivity and hard ionization, respectively, for the development of rapid mass
spectrometry-based quantitation without chromatographic separation for the analyses of
carisoprodol and meprobamate in rat brain microdialysates. Although DART was promising,
subsequent evaluation of an MS/MS-based assay also revealed shortcomings of inadequate
detection sensitivity and poor quantitative reproducibility. On the other hand, LOD and LOQ
of an LC–APCI-MS/MS method we developed for both analytes were found to be equivalent
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to or improved substantially with a concomitant decrease in the assay’s cycle time compared
to previously published methods. Owing to the relative cleanliness of aCSF as a medium,
extraction step(s) could be avoided, and the samples were injected directly on the column
with desalting performed online. Combined with
in vivo
intracerebral microdialysis
sampling, the method was applicable to obtain time-resolved concentration profiles for
carisoprodol and its major metabolite from the nucleus accumbens region of the brain in
rats.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
The contributions of Drs. Szabolcs Szarka and Jia Guo to running LC-MS/MS assays and recording the nanoESI
mass spectra are kindly acknowledged. This work has been supported in part by a grant (DA022370) from the
National Institute on Drug Abuse (Bethesda, Maryland, USA) and by The Welch Foundation (endowment
BK-0031). P.F. also thanks the Czech Science Foundation (P206/12/1150).
References
1. Toth PE, Urtis J. Commonly used muscle relaxant therapies for acute low back pain: A review of
carisoprodol, cyclobenzaprine hydrochloride, and metaxalone. Clin Ther. 2004; 26:1355.doi:
10.1016/j.clinthera.2004.09.008 [PubMed: 15530999]
2. Chou R, Peterson K, Helfand M. Comparative efficacy and safety of skeletal muscle relaxants for
spasticity and musculoskeletal conditions: a systematic review. J Pain Symptom Manag. 2004;
28:140.doi: 10.1016/j.jpainsymman.2004.05.002
3. Dalen P, Alvan G, Wakelkamp M, Olsen H. Formation of meprobamate from carisoprodol is
catalysed by CYP2C19. Pharmacogenetics. 1996; 6:387–394. [PubMed: 8946470]
4. Haizlip TM, Ewing JA. Meprobamate habituation: a controlled clinical study. N Eng J Med. 1958;
258:1181.
5. US Code of Federal Regulations, Title 21, Section 1308.14.
6. Reeves RR, Carter OS, Pinkofsky HB, Struve FA, Bennett DM. Carisoprodol (Soma): Abuse
potential and physician unawareness. J Addict Dis. 1999; 18:51.
7. US Department of Justice. Schedules of Controlled Substances: Placement of Carisoprodol into
Schedule IV” (PDF). Federal Register. 2011; 76:77330–77360. [accessed on August 25, 2015]
http://www.gpo.gov/fdsys/pkg/FR-2011-12-12/pdf/2011-31542.pdf.
8. Roth BA, Vinson DR, Kim S. Carisoprodol-induced myoclonic encephalopathy. J Toxicol Clin
Toxicol. 1998; 36:609. [PubMed: 9776967]
9. Bramness JG, Skurtveit S, Mørland J. Impairment due to intake of carisoprodol. Drug Alcohol
Depend. 2004; 74:311.doi: 10.1016/j.drugalcdep.2004.01.007 [PubMed: 15194209]
10. Gatch MB, Nguyen JD, Carbonaro T, Forster MJ. Carisoprodol tolerance and precipitated
withdrawal. Drug Alcohol Depend. 2012; 123:29.doi: 10.1016/j.drugalcdep.2011.10.010
[PubMed: 22055010]
11. Gonzalez LA, Gatch MB, Taylor CM, Bell-Horner CL, Forster MJ, Dillon GH. Carisoprodol-
mediated modulation of GABAA receptors: In vitro and in vivo studies. J Pharmacol Exp Ther.
2009; 329:827–837. DOI: 10.1124/jpet.109.151142 [PubMed: 19244096]
12. Darvesh AS, Carroll RT, Geldenhuys WJ, Gudelsky GA, Klein J, Meshul CK, Van der Schyf CJ. In
vivo brain microdialysis: advances in neuropsychopharmacology and drug discovery. Expert Opin
Drug Discov. 2011; 6:109.doi: 10.1517/17460441.2011.547189 [PubMed: 21532928]
Prokai et al. Page 8
J Mass Spectrom
. Author manuscript; available in PMC 2017 October 01.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
13. Anderzhanova E, Wotjak CT. Brain microdialysis and its applications in experimental
neurochemistry. Cell Tissue Res. 2013; 354:27–39. DOI: 10.1007/s00441-013-1709-4 [PubMed:
24022232]
14. Venter A, Nefliu M, Cooks RG. Ambient desorption ionization mass spectrometry. TRAC-Trends
Anal Chem. 2008; 27:284.doi: 10.1016/j.trac.2008.01.010
15. Nanita SC, Kaldon LG. Emerging flow injection mass spectrometry methods for high-throughput
quantitative analysis. Anal Bioanal Chem. 2016; 408:23.doi: 10.1007/s00216-015-9193-1
[PubMed: 26670771]
16. Yeniceli D, Sener E, Korkmaz OT, Dogrukol-Ak D, Tuncel N. A simple and sensitive LC–ESI-MS
(ion trap) method for the determination of bupropion and its major metabolite, hydroxybupropion
in rat plasma and brain microdialysates. Talanta. 2011; 84:19.doi: 10.1016/j.talanta.2010.11.063
[PubMed: 21315892]
17. Erve JCL, Beyer CE, Manzino L, Talaat RE. Metabolite identification in rat brain microdialysates
by direct infusion nanoelectrospray ionization after desalting on a ZipTip and LTQ/Orbitrap mass
spectrometry. Rapid Commun Mass Spectrom. 2009; 23:4003.doi: 10.1002/rcm.4341 [PubMed:
19918933]
18. Cody RB, Laramee JA, Durst HD. Versatile new ion source for the analysis of materials in open air
under ambient conditions. Anal Chem. 2005; 77:2297.doi: 10.1021/ac050162j [PubMed:
15828760]
19. McEwen CN, McKay RG, Larsen BS. Analysis of solids, liquids, and biological tissues using
solids probe introduction at atmospheric pressure on commercial LC/MS instruments. Anal Chem.
2005; 77:7826.doi: 10.1021/ac051470k [PubMed: 16316194]
20. Tsushima J, DeSouza M, Batra K. Carisoprodol and meprobamate in plasma by quantitative
GC/MS. Clin Chem. 1992; 38:1004.
21. Kim JY, In MK, Paeng KJ, Chung BC. Simultaneous determination of carisoprodol and
meprobamate in human hair using solid-phase extraction and gas chromatography/mass
spectrometry of the trimethylsilyl derivatives. Rapid Commun Mass Spectrom. 2005; 19:3056.doi:
10.1002/rcm.2173 [PubMed: 16200657]
22. Matsumoto T, Sano T, Matsuoka T, Aoki M, Maeno Y, Nagao M. Simultaneous determination of
carisoprodol and acetaminophen in an attempted suicide by liquid chromatography-mass
spectrometry with positive electrospray ionization. J Anal Toxicol. 2003; 27:118.doi: 10.1093/jat/
27.2.118 [PubMed: 12670008]
23. Miksa IR, Poppenga RHJ. Direct and rapid determination of baclofen (Lioresal®) and carisoprodol
(Soma®) in bovine serum by liquid chromatography-mass spectrometry. Anal Toxicol. 2003;
27:275.doi: 10.1093/jat/27.5.275
24. Skinner W, McKemie D, Stanley S. Ouantitative determination of carisoprodol and its metabolites
in equine urine and serum by liquid chromatography-tandem mass spectrometry.
Chromatographia. 2004; 59:S61.doi: 10.1365/s10337-004-0244-6
25. Szarka S, Prokai L. Chip-based nanoelectrospray ionization with Fourier transform mass
spectrometric detection to screen for local anesthetics intended to mask limb sore in walking
horses. J Mass Spectrom. 2015; 50:533.doi: 10.1002/jms.3558 [PubMed: 25800188]
26. Prokai L, Zharikova AD, Janáky T, Li X, Braddy AC, Perjési P, Matveeva L, Powell DH, Prokai-
Tatrai K. Integration of mass spectrometry into early-phase discovery and development of central
nervous system agents. J Mass Spectrom. 2001; 36:1211.doi: 10.1002/jms.227 [PubMed:
11747117]
27. Prokai L, Prokai-Tatrai K, Zharikova AD, Nguyen V, Perjesi P, Stevens SM Jr. Centrally-acting and
metabolically stable thyrotropin-releasing hormone analogues upon replacement of histidine with
substituted pyridinium. J Med Chem. 2004; 47:6025.doi: 10.1021/jm020531t [PubMed:
15537357]
28. Paxinos, G., Watson, C. The Rat Brain in Stereotaxic Coordinates. 2. Academic Press; San Diego:
1986.
29. Kato R, Bolego A, Fontino G, Vassanel P. Metabolism and distribution of carisoprodol in tissues
and organs of rats. Med Exp. 1962; 6:149. [PubMed: 14454289]
Prokai et al. Page 9
J Mass Spectrom
. Author manuscript; available in PMC 2017 October 01.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
30. Simon S, D’Andrea C, Wheeler WJ, Sacks H. Bioavailability of oral carisoprodol 250 and 350 mg
and metabolism to meprobamate: A single-dose crossover study. Curr Ther Res. 2010; 71:50.doi:
10.1016/j.curtheres.2010.02.003 [PubMed: 24683250]
31. Agranoff BW, Bradley RM, Axelrod J. Determination and physiologic disposition of meprobamate.
Proc Soc Exp Biol Med. 1957; 96:261. [PubMed: 13485074]
32. Koob GF. Drugs of abuse - Anatomy, pharmacology and function of reward pathways. Trends
Pharmacol Sci. 1992; 13:177.doi: 10.1016/0165-6147(92)90060-J [PubMed: 1604710]
33. Yu S, Crawford E, Tice J, Musselman B, Wu JT. Bioanalysis without sample cleanup or
chromatography: The evaluation and initial implementation of direct analysis in real time
ionization mass spectrometry for the quantification of drugs in biological matrixes. Anal Chem.
2009; 81:193.doi: 10.1021/ac801734t [PubMed: 19117450]
34. Eberherr W, Buchberger W, Hertsens R, Klampfl CW. Investigations on the coupling of high-
performance liquid chromatography to direct analysis in real time mass spectrometry. Anal Chem.
2011; 82:5792.doi: 10.1021/ac1008496
35. Wu, JT. Using Mass Spectrometry for Drug Metabolism Studies, Second Edition. Korfmacher,
WA., editor. CRC Press; Boca Raton: 2009. p. 377-389.
36. Yu SX, Crawford E, Tice J, Musselman B, Wu JT. Bioanalysis without sample cleanup or
chromatography: the evaluation and initial implementation of direct analysis in real time ionization
mass spectrometry for the quantification of drugs in biological matrixes. Anal Chem. 2009;
81:193.doi: 10.1021/ac801734t [PubMed: 19117450]
37. Esslera S, Brunsa K, Frontzb M, McCutcheon JR. A rapid quantitative method of carisoprodol and
meprobamate by liquid chromatography–tandem mass spectrometry. J Chromatogr B. 2012;
908:155.doi: 10.1016/j.jchromb.2012.09.001
38. Schuhmacher J, Zimmer D, Tesche F, Pickard V. Matrix effects during analysis of plasma samples
by electrospray and atmospheric pressure chemical ionization mass spectrometry: Practical
approaches to their elimination. Rapid Commun Mass Spectrom. 2003; 17:1950.doi: 10.1002/rcm.
1139 [PubMed: 12913858]
39. Prokai L, Fryčák P, Stevens SM Jr, Nguyen V. Measurement of acetylcholine in rat brain
microdialysates by LC-isotope dilution tandem MS. Chromatographia. 2008; 68:S101.doi:
10.1365/s10337-008-0697-0 [PubMed: 19802332]
40. Weia J, Lai Q, Shumyak SP, Xu L, Zhang C, Ling J, Yua Y. An LC/MS quantitative and
microdialysis method for cyclovirobuxine D pharmacokinetics in rat plasma and brain: The
pharmacokinetic comparison of three different drug delivery routes. J Chromatogr B. 2015;
1002:185.doi: 10.1016/j.jchromb.2015.08.022
41. Gottasa A, Oiestada EL, Boixa F, Ripela A, Thaulowa CH, Pettersena BS, Vindenesa V, Morlanda
J. Simultaneous measurement of heroin and its metabolites in brain extracellular fluid by
microdialysis and ultra performance liquid chromatography tandem mass spectrometry. J
Pharmacol Toxicol Methods. 2012; 66:14.doi: 10.1016/j.vascn.2012.04.009 [PubMed: 22561414]
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Figure 1.
Chip-based nanoESI, DART and ASAP mass spectra of (A) carisoprodol and (B)
meprobamate.
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Figure 2.
DART-MS/MS analysis of meprobamate (3 ng, upper trace) and carisorprodol (3 ng, middle
trace) in triplicate using diethyl acetamidomalonate as an ISTD (1.5 ng, lower trace). The
insets indicate the SRMs and the fragmentation of [M+H]+ based on which the compound
was detected (
m/z
given as nominal mass). Carisoprodol-to-ISTD peak area ratio:
0.075±0.032 (CV: 42.1%); meprobamate-to-ISTD peak area ratio: 0.076±0.060.96 (CV:
78.5%); carisoprodol-to-meprobamate peak area ratio: 1.525±1.360.96 (CV: 89.2%).
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Figure 3.
APCI mass spectra and CID-MS/MS product ion spectra of the [M+H]+ ions of (A)
carisoprodol and (B) meprobamate.
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Figure 4.
Three replicate 5-μL injections of meprobamate (1 ng/mL, upper traces), carisoprodol (1
ng/mL, middle traces) for LC–APCI-MS/MS analysis using diethyl acetamidomalonate as
ISTD (10 ng/mL, lower traces) and SRMs indicated (
m/z
given as nominal mass).
Carisoprodol-to-ISTD peak area ratio: 1.43·10−2±1.03·10−3 (CV: 9.8%); meprobamate-to-
ISTD peak area ratio: 2.60·10−2±1.89·10−3 (CV: 7.3%).
Prokai et al. Page 14
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Figure 5.
Carisoprodol and meprobamate concentrations in the extracellular fluid of the nucleus
accumbens of rats determined by
in vivo
microdialysis followed by LC–APCI-MS/MS assay
after administration of 100 mg/kg i.p. carisoprodol.
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Author Manuscript Author Manuscript Author Manuscript Author Manuscript
Prokai et al. Page 16
Table 1
Figures of merit summary for the determination of carisoprodol and meprobamate in aCSF solutions by LC–
APCI-MS/MS.
carisoprodol meprobamate
LOD (ng/mL) 0.25 0.15
LOQ (ng/mL) 1 0.25
Intraday reproducibility (RSD, %) 2.3 4.3
Day-to-day reproducibility (RSD, %) 7.2 12.7
Accuracy (relative error, %) −16.0 11.6
J Mass Spectrom
. Author manuscript; available in PMC 2017 October 01.
... CCP chemically known as [2-(carbamoyloxymethyl)-2-methylpentyl] N-propan-2-ylcarbamate ( Fig. 1). The detailed survey of literature revealed that few methods have been reported for the estimation of CCP by liquid chromatography-tandem mass spectrophotometry [5], gas chromatography [6,7], homogeneous immunoassay [8], highperformance thin-layer chromatography [9], liquid chromatography/ mass spectrophotometry [10], and ultraviolet (UV)-high-performance liquid chromatography [11]. The above reported chromatographic methods employed sophisticated and expensive instrumentation. ...
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