Development and validation of a chiral LC-MS method for the enantiomeric resolution of (+) and (?)-medetomidine in equine plasma by using polysaccharide-based chiral stationary phases

Article abstract of DOI:10.1002/chir.23166

The detection and separation of medetomidine enantiomers from the complex biological matrices poses a great analytical challenge, especially in the field of forensic toxicology and pharmacology. Couple of researchers reported resolution of medetomidine using protein-based chiral columns, but the reported method is quiet challenging and tedious to be employed for routine analysis. This research paper reported a method that enables the enantio-separation of medetomidine by using polysaccharide cellulose chiral column. The use of chiralcel OJ-3R column was found to have the highest potential for successful chiral resolution. Ammonium hydrogen carbonate was the ideal buffer salt for chiral liquid chromatography (LC) with electrospray ionization (ESI)+ mass spectrometry (MS) detection for the successful separation and detection of racemic compound. The method was linear over the range of 0 to 20 ng/mL in equine plasma and the inter-day precisions of levomedetomidine, dexmedetomidine were 1.36% and 1.89%, respectively. The accuracy of levomedetomidine was in the range of 99.25% to 101.57% and that for dexmedetomidine was 99.17% to 100.99%. The limits of quantification for both isomers were 0.2 ng/mL. Recovery and matrix effect on the analytes were also evaluated. Under the optimized conditions, the validated method can be adapted for the identification and resolution of the medetomidine enantiomers in different matrices used for drug testing and analysis.

Full text of DOI:10.1002/chir.23166

Received: 22 October 2019
DOI: 10.1002/chir.23166
Revised: 17 November 2019
Accepted: 17 December 2019
S H O R T C O M M U N I C A T I O N
Development and validation of a chiral LC-MS method for
the enantiomeric resolution of (+) and (−)-medetomidine
in equine plasma by using polysaccharide-based chiral
stationary phases
Abdul Khader Karakka Kal
| Jahfar Nalakath | Tajudheen Kunhamu Karatt |
Zubair Perwad | Binoy Mathew | Michael Subhahar
Equine Forensic Unit, Central Veterinary
Abstract
Research Laboratory, Dubai, United Arab
Emirates
The detection and separation of medetomidine enantiomers from the complex
biological matrices poses a great analytical challenge, especially in the field of
forensic toxicology and pharmacology. Couple of researchers reported resolu-
tion of medetomidine using protein-based chiral columns, but the reported
method is quiet challenging and tedious to be employed for routine analysis.
This research paper reported a method that enables the enantio-separation of
medetomidine by using polysaccharide cellulose chiral column. The use of
chiralcel OJ-3R column was found to have the highest potential for successful
chiral resolution. Ammonium hydrogen carbonate was the ideal buffer salt for
chiral liquid chromatography (LC) with electrospray ionization (ESI)+ mass
spectrometry (MS) detection for the successful separation and detection of
racemic compound. The method was linear over the range of 0 to 20 ng/mL in
equine plasma and the inter-day precisions of levomedetomidine,
dexmedetomidine were 1.36% and 1.89%, respectively. The accuracy of
levomedetomidine was in the range of 99.25% to 101.57% and that for
dexmedetomidine was 99.17% to 100.99%. The limits of quantification for both
isomers were 0.2 ng/mL. Recovery and matrix effect on the analytes were also
evaluated. Under the optimized conditions, the validated method can be
adapted for the identification and resolution of the medetomidine enantiomers
in different matrices used for drug testing and analysis.
Correspondence
Abdul Khader Karakka Kal and Jahfar
Nalakath, Equine Forensic Unit, Central
Veterinary Research Laboratory, PO Box
597, Dubai, United Arab Emirates.
Email: a.khader@efudubai.com;
j.nalakath@efudubai.com
K E Y W O R D S
chirality, medetomidine, levomedetomidine, dexmedetomidine, polysaccharide chiral column,
equine plasma
1 | INTRODUCTION
massive abuse. The scenario is not different in sports
as misuse of drugs occurs in every stage of competi-
As rightly said, everything has its own pros and cons.
Even therapeutic drugs, which are unavoidable part of
life, have now become a social evil owing to their
tion. Many clandestine laboratories produce drugs that
often beat testing or next-generation designer drugs,
which
necessitates
an
intense
research
and
Chirality. 2020;1–10.
wileyonlinelibrary.com/journal/chir
© 2020 Wiley Periodicals, Inc.
1

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KAL ET AL.
development of highly robust analytical methods.
Alpha-2 adrenoceptors agonists have been widely used
for their sedative effects in veterinary medicine 1,2. It
has been estimated that over 7 million veterinary
patients receive an alpha-2 adrenoceptor agonist as
part of their anesthetic protocol each year 3,4. These
agents reliably produce not only sedation but also
potentiate most anesthetic and analgesic drugs.
Medetomidine,
(+/−)-4-[1-(2,3-dimethylphenyl)ethyl]-
FIGURE 2 Structures of medetomidine and its enantiomers
1H-imidazole, is a very potent, selective, and specific
full agonist at both pre-synaptic and postsynaptic
alpha-2 adrenoceptors as demonstrated in several
models 5-8. More recently, medetomidine has been
used as a replacement for xylazine, as its α2/α1-
receptor selectivity binding ratio is 10-fold higher, lead-
ing to fewer side effects. Medetomidine is frequently
used alone or in combination with opioids for minor
procedures or as a pre-medicant before a general anes-
thetic; facilitate minor surgical and diagnostic proce-
dures 9-12. Alpha-2 adrenoceptor agonists may cause
dose-dependent hypertension, bradycardia, cardiopul-
monary depression, and peripheral vasoconstriction,
A significant number of research works have been
published related to the chiral resolution of racemates on
a large scale using kinetic resolution by crystallization
and enzymatic resolution methods 26-29. Liquid chroma-
tography (LC) evolved as a key technique for chiral sepa-
rations due to the availability of large number of chiral
stationary phases and lesser time to establish a method
30-39. Despite the recent advances in the development of
chiral stationary phases, the enantiomeric analysis of
underivatized medetomidine has invited only very little
attention. It is possible to separate the two enantiomers
by forming carbamate derivatives with ethyl
chloroformate and to separate them by high-performance
LC using a Baker-bond Chiral Phase DNBPG covalent
column. Even though the method is very sensitive but is
time consuming as the reaction time between med-
etomidine hydrochloride and ethyl chloroformate is fairly
long, and the reaction also has minor side-products. Cou-
ple of methods have been described based on LC and cap-
illary electrophoresis 40. G. Orn et al reported direct high-
performance liquid chromatographic (HPLC) separation
of (+)- and (−)-medetomidine hydrochloride with an
α1—acid glycoprotein chiral column 41, but the method
employs UV detector at 210 nm, and the limited informa-
tion was provided in the report, especially limit of detec-
tion (LOD) is not reported. A highly robust, fast, and
accurate enantiomeric resolution method is required to
determine the purity of one enantiomer in order to
address the challenges posed by low dose administration
of medetomidine.
but these effects might be reduced if lower doses of
13-15
medetomidine are used
.
Medetomidine is eliminated principally by biotrans-
formation in liver 16,17. Hydroxylation of a methyl group
is the primary metabolic event reported for med-
etomidine in rats, which provided 30-hydroxy-
medetomidine and medetomidine 30-carboxylic acid as
18,19
the major metabolites
(Figure 1). There is an
increased trend in the usage of medetomidine and related
drugs in racing sport as a doping agent, and hence need
to be controlled or regulated 20-23. Medetomidine is
the racemic mixture of two enantiomers, ie,
dexmedetomidine and levomedetomidine (Figure 2).
Intensive pharmacological studies with both enantiomers
have been carried out over the years, and the results have
shown that only the (+)—form, ie, dexmedetomidine is
pharmacologically active. Also the α2-adrenoceptor activ-
ity of medetomidine resides predominantly in its d-
enantiomer 24,25
.
FIGURE 1 Reported metabolic scheme for
dexmedetomidine

KAL ET AL.
3
2 | MATERIALS AND METHODS
The sample was then dried under nitrogen at 60 ꢀC and
reconstituted with 1:1 methanol-water mixture (30 μL). It
was then transferred to HPLC auto-sampler vial and
injected (10 μL) into LC-MS system for analysis.
Medetomidine, dexmedetomidine, levomedetomidine,
and d3-morphine were procured from Sigma (St Louis,
MO, USA). Acetonitrile (LiChrosolv hyper grade for
liquid chromatography-mass spectrometry (LC-MS)
≥99.9%), methanol (LiChrosolv, gradient grade, for LC,
≥99.9%), methyl t-butyl ether (≥99%), chloroform
(≥99%), ammonium hydrogen carbonate (≥99%),
trifluoroacetic acid (≥99%), ammonium acetate (≥99%),
formic acid (reagent grade, ≥99%), acetic acid (reagent
grade, ≥99%), and ammonium hydroxide solution (ACS
reagent, 28%-30% NH3 basis) were supplied by Merck
KGaA (Darmstadt, Germany). The deionized water was
purified by a Milli-Q water purification system from
Millipore (Bedford, USA). The columns used for the
separation was bought from different suppliers. Chiralcel
OJ-3R and Chiralcel OD-RH, from DAICEL, Osaka,
Japan; Lux cellulose-1, Lux cellulose-2, Lux i-cellulose-5,
Lux amylose-1, Lux i-amylose-1, and Lux amylose-2 were
obtained from Phenomenex, Torrance, CA, USA.
2.1.3 | Mobile phases
The LC mobile phase consist of ammonium acetate,
ammonium hydrogen carbonate, formic acid or
trifluoroacetic acid in water (solvent A), and acetonitrile,
methanol or tetrahydrofuran (solvent B). Small amounts
of organic acids or bases also tried as mobile phase addi-
tives for optimizing separations and improving peak
shape. The acidic buffer [0.01%-0.1% (v/v)] was prepared
by dissolving formic acid or trifluoroacetic acid in mobile
phase A or B. While the neutral or basic buffer
(1-10mM), were prepared by dissolving ammonium ace-
tate, ammonium bicarbonate or diethylamine in mobile
phase A or B. All solutions were filtered through 0.45 μm
nylon membranes (Teknokroma, Barcelona, Spain) and
degassed before use.
This method was validated under the guidelines
42
suggested by Shah et al.
Parameters such as inter-
day/intraday accuracy and precision, linearity, recovery,
LOD, and limit of quantitation (LOQ) were determined.
2.2 | Instrumentation
2.2.1 | Liquid chromatography
2.1 | Sample preparation
2.1.1 | Control sample
The chromatographic separation was performed using
Dionex UltiMate 3000 UPLC+ system. It consists of a
quaternary pump, an autosampler, and a thermostatted
column compartment. Chiral separation was attempted
by using various chiral HPLC columns, different set of
buffers, and additives. Varied temperature ranges of 25ꢀC
to 50ꢀC and the flow rates of 0.6 to1.0 mL/min were
also used.
Either commercially available medetomidine or racemic
mixture of isomers was prepared by mixing equal
volumes of the stock solutions of the individual isomer.
Stock standard solutions of each isomer were prepared in
methanol, and the working solutions of enantiomers
were prepared by further dilution of the stock solutions
with methanol. They were further diluted with methanol
to get a final concentration of 500 ng/mL. All the prepa-
rations were stored in the dark at 2ꢀC.
2.2.2 | Mass spectrometry
The MS analysis was carried out on QExactive high reso-
lution accurate MS (Thermo Scientific, Waltham, MA,
USA) coupled with Dionex UltiMate 3000 UHPLC+
(Dionex, Sunnyvale, CA, USA). The MS data were
acquired at 70 000 resolutions over the mass range m/z
50 to 750. The capillary temperature was set to 320ꢀC,
capillary voltage to 4 kV, and the S-lens value to 50. The
sheath and auxiliary gas were set to 45 and 10 units,
respectively. The instrument was operated in the positive
ionization mode, and the precursor ion detection was car-
ried out using full scan MS experiment while the product
ion detection was achieved using data independent acqui-
sition experiment.
2.1.2 | Analyte extraction from plasma
The liquid-liquid extraction technique was used for the
extraction of plasma samples. Solutions (3 mL each) of
bio-blank, sample, and spiked control were dispensed
into a labeled Kimble tube. The internal standard (d3-
morphine; 30 μL; 1.0 μg/mL) was added to plasma
sample, and pH was adjusted to 9.0 by using 2M NaOH
solution and vortexed. The sample was extracted with 3:1
mixture of methyl t-butyl ether: chloroform (2 × 5 mL)
and the organic layer were transferred to a 10 mL tube.

4
KAL ET AL.
3 | RESULTS AND DISCUSSION
with MS detectors. The addition of DEA mobile phase
additive can visibly improve the enantio resolution; how-
ever, it can also severely suppress analyte response in
positive mode electrospray (ESI+) MS/MS. From the ini-
tial mobile phase additive screening, it was clear that the
ammonium hydrogen carbonate buffer is necessarily
required for the resolution of the enantiomers (Figure 3).
Based on the above observation, further testing on vari-
ous columns using the same chromatographic conditions
were attempted and the results are tabulated (Table 1).
Chiralcel OJ-3R proved be the most effective, giving
good separation of the medetomidine enantiomers.
The initial attempt was to optimize parameters such as
selection of column, elution gradient, mobile phase
buffer, flow rate, and column oven temperature. The elu-
tion gradient, which gives a baseline separation within a
relatively shorter analysis time, was selected as the opti-
mized elution condition. Higher pH mobile phase buff-
ered with ammonium salts such as acetate or hydrogen
carbonate can be effective in the chiral separation of
basic (and neutral) racemic compounds. Ammonium
salts are also thermally labile, hence fully compatible
FIGURE 3 The separation of medetomidine enantiomers

KAL ET AL.
5
TABLE 1 Liquid chromatography (LC) conditions
optimization by using chiral columns
An isocratic method was applied, using mobile phase
A (10mM ammonium bicarbonate in water) and mobile
phase B (acetonitrile) in a 70:30 ratio. A flow-rate of 0.7
mL/min was chosen, as higher flow-rates permit rapid
analysis. This outweighs the fact that higher flow-rates
give a slightly lower resolution and high column pressure.
Ambient temperature (25ꢀC) was used for analysis, as
higher temperatures had a slight adverse effect on resolu-
tion. The total run time was 14 minutes, and the results
were found to be reproducible in the varied concentra-
tion. Dexmedetomidine eluted at 9.84 minutes, and
levomedetomidine eluted at 10.47 minutes, as shown in
Figures 4–6.
No.
Columns
Additives
NH4CH3CO2
HCOOH
R
X
1
Lux amylose-1
X
(NH4)HCO3
NH4CH3CO2
HCOOH
X
2
3
4
5
6
7
8
Lux i-amylose-1
Lux amylose-2
Lux cellulose-1
Lux cellulose-2
Lux i-cellulose-5
Chiralcel OD-RH
Chiralcel OJ-3R
X
X
(NH4)HCO3
NH4CH3CO2
HCOOH
0.3
X
X
(NH4)HCO3
NH4CH3CO2
HCOOH
X
X
4 | METHOD VALIDATION
X
(NH4)HCO3
NH4CH3CO2
HCOOH
X
The proposed method was fully validated by evaluating
the parameters such as accuracy, precision, linearity,
LOD, and LOQ.
X
X
(NH4)HCO3
NH4CH3CO2
HCOOH
0.5
X
X
4.1 | Linearity
(NH4)HCO3
NH4CH3CO2
HCOOH
0.3
X
Linearity was examined by injections (in triplicate) at six
concentration levels of racemic medetomidine (from 0 to
20 ng/mL). Calibration curves of each enantiomer were
linear over the tested concentration range. Regression
equations (the peak area versus the concentration of each
enantiomer in ng/mL) showed fairly good linearity
with regression coefficients (r2) of.998 for both
levomedetomidine and dexmedetomidine being pres-
ented as in Table 2.
X
(NH4)HCO3
NH4CH3CO2
HCOOH
1.1
X
X
(NH4)HCO3
1.6
Note. Method: isocratic; mobile phase A: NH4CH3CO2, NH4HCO3 or
HCOOH in water; mobile phase B: acetonitrile. NH4CH3CO2, ammonium
acetate; NH4HCO3, ammonium hydrogen carbonate; HCOOH, formic acid;
X, no indication of enantio-separation; R, resolution.
4.2 | Accuracy and precision
Once the Chiralcel OJ-3R (3 μ, 150 × 4.6 mm) was
selected, the method was optimized for other parame-
ters such as column temperature, flow rate, ionic
strength of the buffer, and pH of the buffer. The
results show that changing the column temperature,
flow-rate, and ionic strength of the buffer are having
only a minimal effect on the resolution. However, pH
of the buffer has a significant effect. The enantio selec-
tivity of chiralcel OJ-3R was evaluated in ammonium
hydrogen carbonate-containing mobile phase (with ace-
tonitrile as organic modifier). Ammonium hydrogen
carbonate (buffer of pH 8.0), afford better peak shapes,
therefore higher resolution of the enantiomers. As the
prolonged use of buffer with pH higher than 7.5 may
decrease the column longevity (owing to decomposition
of the silica), a pH of 7.0 was finally chosen for rou-
tine analysis.
A detailed analysis was conducted to establish the pre-
cision and accuracy of the results. Precision of the
method was expressed as relative standard deviation
(RSD). A set of 18 medetomidine fortified plasma sam-
ples (10 ng/mL) were assessed on three validation days
to verify precision and accuracy. The inter-day and
intraday precision values for the dexmedetomidine and
levomedetomidine were satisfactory and are demon-
strated in Table 3.
%RSD = ðsample standard deviation=sample meanÞ*100: ð1Þ
4.3 | Recovery
To determine the recovery of isomers in plasma samples,
spiked samples 1.0, 5.0, 10, and 20 ng/mL levels in

6
KAL ET AL.
FIGURE 4 Liquid chromatography
(LC) chromatogram of individual
levomedetomidine by developed chiral
separation technique
FIGURE 5 Liquid chromatography
(LC) chromatogram of individual
dexmedetomidine by developed chiral
separation technique
duplicate were extracted and analyzed. They were com-
pared with standards prepares in blank matrix extracts
(Table 3). Recovery was calculated by using below simple
equation.
4.4 | LOD and LOQ
The detection limit was determined using signal to
noise ratio. A series of matrix spikes were used for this
experiment. The concentration, which gives
a
RE ð%Þ = ðC=BÞ*100:
ð2Þ
chromatographic peak at least three times greater than
the noise level, is established as LOD. In this method,
the LOD value was 0.02 ng/mL for both isomers. LOQ
was the concentration, which gives a chromatographic
peak area that is at least 10 times greater than
the noise level. The LOQ was 0.2 ng/mL for both
isomers.
C
= Peak area of the dexmedetomidine and
levomedetomidine in the plasma spike.
Peak area of the dexmedetomidine and
levomedetomidine in standard prepared in blank matrix
extract.
B
=

KAL ET AL.
7
FIGURE 6 Chromatogram depicting the resolution (top) and MSMS spectra (middle and bottom) of
levomedetomidine and dexmedetomidine
TABLE 2 Method linearity—levomedetomidine and dexmedetomidine in horse plasma
Isomer
r2
Intercept
Slope
Range (ng/mL)
Levomedetomidine
Dexmedetomidine
.998
.998
0
0
3926405
3847169
0-20
0-20
Note. r2 = correlation coefficient, n = 6.
TABLE 3 Accuracy, precision and recovery of levomedetomidine and dexmedetomidine in horse plasma
Precision (RSD %)
Isomer
Batch
Day 1
Day 2
Day 3
Day 1
Day 2
Day 3
Accuracy, %
100.10
99.25
Intraday
0.38
Inter-day
Recovery, %
Levomedetomidine
1.36
83
0.841
1.108
0.799
0.951
1.490
101.57
99.78
Dexmedetomidine
1.89
89
100.99
99.17
Note. n = 6; Conc. 10 ng/mL.
Abbreviation: RSD, relative standard deviation.

8
KAL ET AL.
TABLE 4 Matrix effect on dexmedetomidine and levomedetomidine detection in horse plasma
A
B
Matrix Effect, %
Conc., ng/mL
Dex
0.97
9.58
19.68
Levo
0.92
Dex
1.05
9.76
19.92
Levo
0.88
9.25
19.5
Dex
Levo
4.35
3.37
1.71
1.0
10
20
−8.25
−1.88
−1.22
9.78
19.84
Note. Dex = dexmedetomidine; Levo = levomedetomidine, A = standard, B = spiked in extracted matrix, n = 6.
4.5 | Matrix effect
confirmatory analysis of samples in anti-doping
laboratories.
To evaluate the matrix effect, both the isomers at three
different concentrations (1, 10, and 20 ng/mL) were
used. The chromatographic peak areas averaged from
five sample duplicates for the analyte standard in
sample solvent and for the standards added to plasma
extracts and reconstituted were used for estimation of
the contribution of sample matrix to ion suppression or
enhancement in analyzing the analytes. Matrix effect
on the analytes of interest was also evaluated in this
study and was calculated by using below equation.
ACKNOWLEDGMENTS
The authors are thankful to Dr. Ali Ridha, Director Gen-
eral (Central Veterinary Research Laboratory), for the
valuable suggestions and support. The authors are
acknowledging the support and assistance of the Priv.
Doz. Dr. Dr. habil. U. Wernery (Scientific Director, Cen-
tral Veterinary Research Laboratory), Ms. Marina
Rodriguez Caveney, and Equine Forensic Unit staff in
this project.
Matrix effect ð%Þ = ½1−ðB=AÞꢁ*100:
ð3Þ
CONFLICT OF INTEREST
The authors declare that they have no conflict of interest.
A
= Peak area of the dexmedetomidine and
levomedetomidine standard in neat solvent.
Peak area of the dexmedetomidine and
levomedetomidine in standard prepared in blank matrix
extracts.
ETHICAL APPROVAL
This article does not contain any studies with human par-
ticipants or animals performed by any of the authors.
B
=
A positive value indicates ion suppression effect,
whereas negative value represents ion enhancement
effect (Table 4). The results obtained are within the stan-
dard experimental error and, thus, matrix effect attribut-
able to plasma on the analysis of dexmedetomidine and
levomedetomidine are negligible.
ORCID
Abdul Khader Karakka Kal https://orcid.org/0000-
0002-6928-5513
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