Miroshnichenko et al.

, J Sports Med Doping Stud 2013, 3:1

Sports Medicine & Doping Studies http://dx.doi.org/10.4172/2161-0673.1000120

Research Article Open Access

A Rapid and Sensitive LC–MS/MS Assay for the Quantitation of

Bromantane in Human Plasma
Igor I Miroshnichenko, Svetlana A Sergeeva, Angelina I Platova* and Lyudmila M Krasnykh
Mental Health Research Center of RAMS, Moscow, Russia

Abstract
The purpose of this study was to develop and validate a high-performance liquid chromatographic–tandem
mass spectrometric (LC–MS/MS) method for analysis of the bromantane in human plasma. The analyte and Internal
Standard (IS), selenox, were extracted from human plasma by solid-phase extraction and separated on a Zorbax
SB-C18 column using methanol–0.2% formic acid as mobile phase. Detection was performed using an atmospheric
pressure chemical ionization source and mass spectrometric positive Multi-Reaction-Monitoring-Mode (+MRM) at an
ion voltage of +2000 V. The assay was linear over the concentration range 1–500 ng/mL with the Lowest Limit of
Quantification (LLOQ) of 1 ng/mL. The method also afforded satisfactory results in terms of the sensitivity, specificity,
precision (intra- and inter-day, CV<10%), accuracy, recovery as well as the stability of the analyte under various
conditions. The method can be applied to pharmacokinetic and toxicological studies.

Keywords: Bromantane; Liquid Chromatography–Mass Spectrom- over LLE, including better specificity, the ability to obtain cleaner
etry (LC–MS/MS); APCI; Plasma samples, good reproducibility, and a substantial reduction in the
volume of solvent required [13].
Introduction
However, we do not find HPLC–MS/MS methods reported in the
Bromantane, N-(4-bromophenyl) adamantan-2-amine, is a drug literature for quantification of bromantane in biological samples. This
with anxiolytic and immunostimulatory actions and elements of
paper reports on a rapid and sensitive LC–MS/MS method for the
actoprotective activity [1-3]. Bromantane is not potentially addictive
determination of bromantane in human plasma using SOLA cartridges.
and does not exert effects of hypnosedation or neuromuscular
The method employs using 9-phenyl-sym-octahydroselenoxanthene
relaxation. The use of the drug, in contrast to the action of a typical
psycho stimulant, is not associated with the phenomenon of hyper (selenox) as internal standard and is completed in a run time of 6 min.
stimulation and causes no consequences such as functional exhaustion Experimental
of the body [4]. These properties of drug stipulated its using as a doping
agent [5]. According to World Anti-Doping Agency classification, Chemicals and reagents
Bromantane belongs to S6; a class of prohibited substances [6].
Bromantane was kindly supplied by the Pharmacology Institute of
The therapeutic action of Bromantane in patients with asthenic and RAMS, Moscow, 9-phenyl-sym-octahydroselenoxanthene synthesized
anxiety-asthenic disorders is exhibited from first day of application, by “Medbiopharm”, Obninsk, and Kaluga region. Formic acid (Sigma
which is expressed by a marked reduction of asthenic symptoms, Inc., USA), methanol and acetonitrile (Lab-Scan, Poland) were HPLC-
signs of emotional tension, and somatoautonomic manifestations; the grade. Deionized water was prepared from distilled water using
drug works by restoring performance and enhancing the endurance Simplicity UV System (Millipore, USA). All other chemicals and
of the body. Its mechanism of action is associated with the release of solvents were analytical grade and used as received. The SOLA reversed
reinforcing dopamine from the presynaptic terminal [7], blockade of phase extraction cartridges were purchased from Thermo Fisher
its reuptake, and enhancement of biosynthesis and induced tyrosine Scientific Inc. (USA).
hydroxylase gene expression, as well as with a modulatory effect on the
Benzodiazepine-GABA-receptor-chloride ion channel complex, up- LC–MS/MS conditions
regulating the stress-induced decrease of benzodiazepine reception.
The HPLC–MS/MS system consisted of an Agilent 1200 series
Bromantane increases GABAergic transmission, reduces gene
binary pump, an auto sampler connected to Agilent 6410-2K Triple
expression, and regulates the synthesis of GABA-mediators, which act
as reuptake transmitters. Bromantane has low toxicity (LD50 in rats is Quad LC-MS (Agilent Technologies, Palo Alto, CA, USA) using a
higher than 10000 mg/kg, exceeding the effective dose by 100 times). atmospheric pressure chemical ionization (APCI). The instrument
was interfaced to a computer running Agilent Mass Hunter B.01.04.
Appropriate analytical method is an urgent need to study bromantane Software.
pharmacokinetic properties. The gas chromatography approach for
content determination has been applied to a pharmacokinetic study of
Bromantane [8]. However, this method was not quite appropriate for *Corresponding author: Angelina I Platova, Mental Health Research Center of
clinic pharmacokinetic studies of bromantane. LC/MS spectrometry RAMS, Moscow, Russia, E-mail: [email protected]
based techniques are now the mainstay for such pharmacokinetic Received January 20, 2013; Accepted February 21, 2013; Published February
studies because of sensitivity, selectivity, speed and cost effectiveness [9]. 25, 2013
Recently some papers devoted LC-MS/MS method for the quantitative Citation: Miroshnichenko II, Sergeeva SA, Platova AI, Krasnykh LM (2013) A Rapid
estimation of amantadine (1-adamantylamine) [10] and memantine and Sensitive LC–MS/MS Assay for the Quantitation of Bromantane in Human
appeared in press [11,12]. Plasma. J Sports Med Doping Stud 3: 120. doi:10.4172/2161-0673.1000120

The most commonly used sample preparation methods for blood Copyright: © 2013 Miroshnichenko II, et al. This is an open-access article
distributed under the terms of the Creative Commons Attribution License, which
plasma samples are Liquid–Liquid Extraction (LLE) and Solid-Phase permits unrestricted use, distribution, and reproduction in any medium, provided
Extraction (SPE). SPE is growing in use, and it offers certain advantages the original author and source are credited.

J Sports Med Doping Stud Volume 3 • Issue 1 • 1000120

ISSN: 2161-0673 JSMDS, an open access journal
Citation: Miroshnichenko II, Sergeeva SA, Platova AI, Krasnykh LM (2013) A Rapid and Sensitive LC–MS/MS Assay for the Quantitation of Bromantane
in Human Plasma. J Sports Med Doping Stud 3: 120. doi:10.4172/2161-0673.1000120

Page 2 of 6

Bromantane and IS were separated a Zorbax SB-C18 column comparing the peak area of analytes resolved in blank sample (the final
(3.5  μm, 100  mm  ×  4.6  mm I.D., Agilent, USA) through a solution of blank plasma after extraction and reconstitution) with that
12.5 mm × 4.6 mm Zorbax SB-C18 precolumn maintained 20°C. The resolved in mobile phase.
mobile phase consisted of methanol–0.2% formic acid (95:5, v/v). The
Three different concentration levels of bromantane (4, 45 and
flow rate throughout the chromatographic analysis was 0.8  mL/min,
125 ng/mL) were evaluated by analyzing five samples at each level. The
and the total run time was 6 minutes. The injection volume was 5 μL.
blank plasma used in this study was five different batches of healthy
The retention times were 3.2 min for bromantane and 5.1 min for IS.
human blank plasma. If the ratio <85 or >115%, an exogenous matrix
The detector was operated at unit resolution in the positive Multiple- effect was implied.
Reaction Monitoring-Mode (+MRM) mode using the transitions of
For the calibration standards, peak area ratios (the analyte/IS) were
the protonated molecular ions of bromantane (308.1 → 135.2 m/z), IS
plotted against nominal plasma concentrations, and fitted by weighted
(331.0 → 250.0 m/z). MS parameters were optimized by syringe pump
(1/y2) least-squares linear regression. Plasma calibration curves were
infusing of standard solution containing analyte and IS. The spray
prepared and assayed in triplicate on three separate days. In addition,
voltage was set at 2000 V. Corona discharge was 4 μA. Nitrogen was
blank plasma was also analyzed to confirm absence of interferences.
used as nebulizer gas and pressure was set at 20 psi. Desolvation gas
(nitrogen) was heated to 350°C and delivered at a flow rate of 5 L/min. The intra-day precision and accuracy of the assay was measured by
For Collision-Induced Dissociation (CID), high purity nitrogen was analyzing five spiked samples of bromantane at each QC level (4, 45 and
used at a pressure of 0.15 MPa. During the data acquisition, dwell time 125 ng/mL). The inter-day precision and accuracy was determined over
was 200 ms. The fragmentation energy of Q1 and collision energy were three consecutive days by analyzing 15 QC samples. The acceptance
135 V and 25 V (bromantane), 90 V and 5 V (IS), respectively. criteria for precision and accuracy deviation values should be within
15% of the actual values. The extraction yield (or absolute recovery)
Preparation of calibration standards and quality control was determined by comparing the bromantane/IS peak area ratios
samples obtained following the outlined extraction procedure (the procedure
A stock solution of bromantane was prepared in methanol at a was a little different from the outlined extraction procedure for QC,
concentration of 1.0 mg/mL. Standard solutions (10, 25, 50, 100, 500, calibration curve and clinical plasma samples, that is IS was added to
1000, 5000 ng/mL) were prepared by serial dilution of the stock solution the organic layer after the extraction of bromantane) and the result
with methanol. Low, medium and high concentration Quality Control compared with those obtained from samples which contained the same
(QC) solutions (4, 45, 125  ng/mL) were prepared in the similar way. amount of bromantane in extracted plasma but not be extracted after
The stock solution of I.S. (1.0 mg/mL) was also prepared in methanol addition of the drug. This procedure was repeated for the three different
and then diluted with methanol to a final concentration of 10  μg/ concentrations of bromantane added, namely 4, 45 and 125 ng/mL.
mL. All solutions were stored at 4°C and used within one month after For sensitivity determination, the LLOQ was defined as the lowest
preparation. Calibration standards and QC samples were prepared by concentration in the calibration curve at which both precision and
adding 50 μL standard and 50 μL IS or QC solution to 500 μL blank accuracy were less than or equal to 20%, and signal/noise (S/N) >10.
human plasma. The 1 ng/mL concentration was investigated as the lower limit of
Sample preparation quantification. Reproducibility and precision were also determined.

A sample (500 μL) of human plasma was transferred to a 5.0 mL To evaluate stability on repeat analysis of samples, freeze-thaw
glass tube, 50 μL methanol or 50 μL standard of bromantane and 50 μL stability was determined for three concentrations of bromantane in
IS solutions were added. If needed (sample not transparent), the mixture plasma. QC plasma samples were tested after three freeze (−20°C) and
was ultra-sonicated. SPE was conducted by using SOLA RP extraction thaw (room temperature) cycles.
cartridges. The cartridges were conditioned sequentially with 500 μL of Results and Discussion
ethanol and with 500 μL of water, and 500 µL of plasma sample diluted
equal volume of water was then loaded. The loaded cartridges were LC/MS/MS conditions
washed with 500 μL 5% ethanol in water, and subsequently the analyte
Certain classes of compounds are traditionally very difficult to ionize
was eluted with ethanol. The elution was then evaporated to dryness
or tend to show low sensitivity in LC/MS/MS techniques. Although
under N2 stream at 50°C, reconstituted with 200 µL of methanol,
electrospray ionization is often used as an LC-MS interface, APCI,
and vortex mixed for 10 s. Finally, the solution was transferred to the
another type of atmospheric pressure ionization, is more applicable
autosampler vials, and 5 µL was injected into HPLC/MS/MS system.
to low polarity compounds [15]. In APCI, the corona discharge can
Method validation provide a source of electrons in the gas-phase. This can be an advantage
for such non polar compounds as adamantanes.
The method validation assays were carried out according to
the currently accepted US Food and Drug Administration (FDA) The LC–MS/MS operation parameters for determination of
bioanalytical method validation guidance [14]. bromantane and IS were carefully optimized. Selenox was selected as the
IS because its chromatographic behavior and extraction efficiency were
The method’s specificity was tested by screening six different similar to those of analyte. Both analyte and IS responded best to the
batches of healthy human blank plasma. Each blank sample was positive ionization mode, with the protonated molecular ions [M+H] + as
tested for interference using the proposed extraction procedure and the major species. Full scan ion spectrum of Bromantane was shown in
chromatographic/spectroscopic conditions and compared with those figure 1. The MRM acquisitions were performed at unit resolution using
obtained with an aqueous solution of the analyte at a concentration the transition 308.1 → 135.2 m/z for bromantane and 331.0 → 250.0 m/z
near to the LLOQ. for IS respectively (Figure 2). The transition 308.1 → 135.2 m/z was used
The matrix effect on the ionization of analytes was evaluated by for quantification of bromantane because of its stabilized ions response

J Sports Med Doping Stud Volume 3 • Issue 1 • 1000120

ISSN: 2161-0673 JSMDS, an open access journal
Citation: Miroshnichenko II, Sergeeva SA, Platova AI, Krasnykh LM (2013) A Rapid and Sensitive LC–MS/MS Assay for the Quantitation of Bromantane
in Human Plasma. J Sports Med Doping Stud 3: 120. doi:10.4172/2161-0673.1000120

Page 3 of 6

x106 + Scan (3.237-3.280 min. 6 scans) scan 100st.d

8
226.20000
7

4
308.10000
3

2 135.20000

0
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
Counts vs. Mass-to-Charge [m/z]

Figure 1: Full scan positive ion turboIonspray Q1 mass spectrum of Bromantane.

x105 + Product Ion (3.223.301 min. 10 scans) (308.10001

xx
pr10.20.d
135.20000
3
2.75
2.5
2.25
2
1.75
1.5
1.25
1
0.75
0.5 308.00000
0.25
0
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400
Counts vs. Mass-to-Charge [m/z]

Figure 2: MS/MS spectrum of the m/z = 308.1 parent ion, exhibiting the product ions of Bromantane at m/z =135.2.

Bromantan - 7 Levels, 7 Levels Used, 7 Points, 7 Points Used, 0 QCs

x
x10 1 y =∧ 0.0255 x + 0.0081
Relative Responses

1.4 R 2 = 0.99533769

1.3

1.2

1.1

0.9

0.8

0.7

0.6

0.5

0.4

0.3
0.2

0.1

-0.1
-40 -20 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540
Concentration (ng/ml)
Figure 3: Regression line for the determination of Bromantane in human plasma.

and no endogenous interference. The mass parameters were optimized voltages of 90, 100, 120, 130, 135, 140 and 150 V in order to determine the
by observing the maximum response obtained for the product ions. The optimal collision energy. The results showed that the highest sensitivities
intensities of its protonated molecules were compared at fragmentor could be obtained by using a 135 V fragmentor voltage.

J Sports Med Doping Stud Volume 3 • Issue 1 • 1000120

ISSN: 2161-0673 JSMDS, an open access journal
Citation: Miroshnichenko II, Sergeeva SA, Platova AI, Krasnykh LM (2013) A Rapid and Sensitive LC–MS/MS Assay for the Quantitation of Bromantane
in Human Plasma. J Sports Med Doping Stud 3: 120. doi:10.4172/2161-0673.1000120

Page 4 of 6

Furthermore, the mobile phase system was optimized through endogenous substances hardly inhibited the ionization of bromantane,
several trials to achieve satisfactory chromatographic behavior and and the ion suppression from human plasma matrix was consistent for
the ionization responses of bromantane and IS. The ionization of this analytical method and would not interfere with the measurement
bromantane and IS was affected by the composition of mobile phase. of analyte. Thus, no ion suppression was observed.
In the preliminary experiments, methanol, acetonitrile, ammonium
The extraction recovery determined for bromantane was shown
acetate and formic acid in various proportions were tested. Methanol,
to be consistent, precise and reproducible. The absolute recoveries of
rather than acetonitrile, was chosen as the organic modifier because it
bromantane from the rat plasma at the three concentrations were about
leads to the good peak shape of bromantane and satisfactory retention
time. When ammonium acetate was added in the mobile phase, the 89%. There was no significant difference in the extraction efficacy of the
response of the analyte was distinctly decreased. Both the analyte and present assay over the range of concentrations studied.
IS were found to have higher response and better peak shapes in the The intra-day and inter-day precision (CV, n = 5) for bromantane
mobile phase containing 0.5% formic acid. Finally, in terms of peak were satisfactory at the three concentrations studied. Data on precision,
shape, retention time and sensitivity, we employed methanol –0.5% accuracy are shown in table 2. The results of freeze–thaw stability
formic acid (95:5, v/v), as the mobile phase. indicated that the analyte is stable in plasma for three freeze–thaw cycles,
when stored at−20°C and thawed to room temperature. Long-term
Assay validation
stability indicates that storage of bromantane plasma samples at −20°C
The linearity for bromantane was investigated by linear regression is adequate when stored for 30 days and no stability related problems
of peak area ratios against concentrations (Figure 3). The regression would be expected during routine analysis for the pharmacokinetic or
equation for the calibration plot was y=0.081+0.0255*x, with a abuse control studies.
coefficient of correlation (r) of 0.995 (where y – the ratio of the
peak areas of bromantane to the peak area of the internal standard; Conclusion
x – bromantane concentration, ng/mL). The calibration curves of The proposed method of analysis provided a sensitive and specific
bromantane showed good linearity in the ranges of 1–500 ng/mL assay for bromantane determination in human plasma. No significant
(Table 1). LLOQ for bromantane in plasma was proved to be 1 ng/mL interference caused by endogenous compounds was observed. The
(S/N=9.5) (Figure 4). method was rapid, selective and highly sensitive with a LLOQ of 1 ng/
Our investigations have confirmed the fact of negligible matrix mL for bromantane. Simple liquid–liquid extraction procedure and
effects coupled to APCI [16]: the ratios of the peak responses for short run time can provide a short analysis time that is important for
bromantane were 99.0%, 98.9%, and 100.3% at 4, 45 and 135  ng/ large sample batches. The developed method was completely validated
mL, respectively (Figure 5). The results indicated that co-eluting showing satisfactory data for all the method validation parameters

Spike Conc. RT Final Conc. Accuracy Area S/N Area IS

1 3.21 1.02 102.3 661 9.5 19337
2.5 3.22 2.53 101.4 1321 16.0 18147
5 3.22 4.67 93.4 2305 26.7 18105
10 3.22 9.63 96.3 3841 16.3 15123
50 3.22 47.52 95.0 19068 82.6 15611
100 3.22 109.02 109.0 44764 143.2 16035
500 3.22 533.34 106.7 246547 209.4 18096
Table 1: The linearity of Bromantane determination.

+ MRM (308.1 - 135.2) 2CAL1.d

x102 RT=3.214
Counts

Name=Bromantan
1.4 Calc. Conc. =1.0232
Height=87.2307
1.2 Area=661.2254
1
RT=2.342
0.8 Name=Bromantan
Calc. Conc. =0.0698
0.6 Height=20.5265
Area=190.5198
0.4
0.2

2.3 2.4 2.5 2.6 2.7 2.8 2.9 3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4 4.1 4.2
Acquisition Time (min)

Figure 4: MRM mass chromatograms of LLOQ (1 ng/mL) of Bromantane.

Calculated C (ng/ml) Found C (ng/ml) Intra-day precision R.S.D. (%) Inter-day precision R.S.D. (%) Accuracy RE (%)
4 4.26 7.5 8.6 6.5
45 44.26 9.4 8.9 -1.6
135 138 5.1 7.3 1.9

Table 2: Accuracy and precision of the assay for determination of Bromantane in plasma (n=5).

J Sports Med Doping Stud Volume 3 • Issue 1 • 1000120

ISSN: 2161-0673 JSMDS, an open access journal
Citation: Miroshnichenko II, Sergeeva SA, Platova AI, Krasnykh LM (2013) A Rapid and Sensitive LC–MS/MS Assay for the Quantitation of Bromantane
in Human Plasma. J Sports Med Doping Stud 3: 120. doi:10.4172/2161-0673.1000120

Page 5 of 6

xx xx
x1021 + TIC MAM 97.d
1
0.98
0.96
0.94
0.92
0.9 Br
0.88
0.86
0.84
0.82
0.8
0.78
0.76
0.74
0.72
0.7
0.68
0.66
0.64
0.62
0.6
0.58
0.56
0.54
0.52
0.5
0.48
0.46
0.44
0.42
0.4
0.38
IS
0.36
0.34
0.32
0.3
0.28
0.26
0.24
0.22
0.2
0.18
0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6
Counts [X] vs. Acquisition Time [min]

Figure 5: Typical mass chromatograms of plasma spiked with Bromantane (45 ng/mL) and the IS (1 µg/mL), validation of Bromantane Quantitation.

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Rev Anal Chem 2: 485–507.
Acknowledgments
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The authors are thankful to prof. N.N. Zolotov from the Institute of Pharmacology The mechanisms of the neurotropic action of bromantan. Eksper Klin Farmak
Russian Academy of Medical Sciences for providing sample of Bromantane for the 62: 11-14.
research work.
8. Gui K, Wu M, Liu X, Zhang Y, Wang S (1999) Study on the metabolites of
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J Sports Med Doping Stud Volume 3 • Issue 1 • 1000120

ISSN: 2161-0673 JSMDS, an open access journal
Citation: Miroshnichenko II, Sergeeva SA, Platova AI, Krasnykh LM (2013) A Rapid and Sensitive LC–MS/MS Assay for the Quantitation of Bromantane
in Human Plasma. J Sports Med Doping Stud 3: 120. doi:10.4172/2161-0673.1000120

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