Brain disposition and catalepsy after intranasal delivery of
loxapine: role of metabolism in PK/PD of intranasal CNS
drugs
Supplementary material
1
LC–MS/MS ASSAY DEVELOPMENT FOR QUANTIFICATION
OF NEUROTRANSMITTERS AND THEIR METABOLITES IN
RAT BRAIN TISSUE
(i)
Preparation of stock solutions, calibration standards and
quality control samples
Master stock solutions of DA and 5-HT and their metabolites were prepared
separately in methanol at concentration of 1 mg/ml as free base. The stock standard
mixture solution was prepared by mixing and diluting the six master stock solutions
with methanol to reach a concentration of 50 µg/ml for each analyte. These stock
solutions were stored at –80°C, which were reported to be stable for at least 6 to 12
months when kept refrigerated (1,2). The working standard mixture solutions were
freshly prepared by serial dilution of the stock standard mix solution with 0.3%
acetic acid in water before analysis. The master stock solutions of the internal
standards ephedrine hydrochloride and ferulic acid were prepared in methanol at 1
mg/ml. The working standard containing the two internal standards was prepared by
mixing and diluting the master stock solutions with 50% ACN in water to achieve
50 ng/ml ephedrine hydrochloride and 200 ng/ml ferulic acid.
Calibration standards and quality control (QC) samples were prepared by
spiking the appropriate amount of working standards to occipital-temporal cortex
obtained from untreated rats. To 20 mg (wet weight) brain tissue, 20 µl of internal
standard working standard and 200 µl of working standard mixture were spiked to
yield analyte concentrations from 40 ng/g to 12000 ng/g, with 50 ng/g ephedrine
hydrochloride and 200 ng/g ferulic acid. QC samples were prepared at two
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concentrations (low and high). All calibration standards and QC samples were
subjected to the sample extraction as described below.
(ii)
Sample extraction procedure
For the brain samples obtained from PK/PD study, 20 µl of IS working standard and
then 200 µl of 0.3% acetic acid in water were added as homogenization medium.
The sample was homogenized on ice by an ultrasonic probe (Microson XL-2000,
Misonix, USA) for 12 sec. Ethyl acetate (400 µl) was added to the homogenate,
followed by centrifugation at 20000 × g (4 °C) for 20 min. Three hundred microliter
of the organic phase was evaporated to dryness under nitrogen stream and the
residue was reconstituted with 100 µl of ACN–0.1% acetic acid in water (3:7, v/v).
After centrifugation at 20000 × g (4 °C) for 10 min, supernatant was obtained for
LC–MS/MS analysis in negative ionization mode. One hundred microliter of the
supernatant in the aqueous phase was mixed with 300 µl of ACN–0.1% acetic acid
in water (1:1, v/v). After centrifugation at 20000 × g (4 °C) for 10 min, supernatant
was obtained for LC–MS/MS analysis in positive ionization mode.
(iii)
LC–MS/MS conditions
The liquid chromatography–tandem mass spectrometry (LC–MS/MS) system
consisted of Agilent 1290 Infinity LC System, coupled with an Agilent 6430 triple
quadrupole mass spectrometer with an electrospray ionization source (Agilent, CA,
USA). The MS/MS system was operated under multiple reaction monitoring (MRM)
mode. Positive ionization mode was used for the simultaneous analysis of basic
analytes (DA, 3-MT, and 5-HT), while negative ionization mode was used for the
simultaneous analysis of acidic analytes (DOPAC, HVA, and 5-HIAA). The LC and
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MS conditions for positive and negative ionization modes (Table S1) and the LC–
MS/MS parameters for each analyte (Table S2) were optimized.
(iv)
Method validation
Linearity was considered satisfactory if the coefficient of determination (R2) of the
plot was higher than 0.99. The lower limit of quantification (LLOQ) was defined as
the lowest concentration of the calibration curve at which the accuracy (relative
error) was within ±20% of the nominal concentration and the precision (relative
standard deviation, RSD) was less than 20%, and with a signal-to-noise peak height
ratio greater than 5:1.
Intra-day accuracy and precision were determined within one day by
analyzing three replicates of the QC samples at two concentrations (low and high).
The inter-day accuracy and precision were determined on three separate days.
Concentrations of the analytes were determined from calibration curve prepared
above. Accuracy within ±15% of the nominal concentration and precision with RSD
less than ±15% were considered to be acceptable.
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Table S1
Optimized LC and MS conditions for positive and negative
ionization modes for the analyses of various neurotransmitters in
the current study.
LC conditions
Isocratic mobile
phase
Flow rate (ml/min)
Column
Guard filter
Injection volume (µl)
Run time (min)
MS conditions
Gas temperature (°C)
Gas flow (l/min)
Nebulizer (psi)
Capillary (V)
Table S2
Positive mode
Negative mode
ACN–0.1% acetic acid in
H2O (1:1)
1
Waters Nova-Pak C18,
150 × 3.9 mm,
4 µm particle size
Thermo Unifilter
0.5 µm
20
9
ACN–0.1% acetic acid in
H2O (3:7)
0.25
Phenomenex Gemini C18,
150 × 2.0 mm,
5 µm particle size
Agilent Eclipse XDB-C8,
2.1 × 15 mm,
3.5 µm particle size
20
7
350
8.5
33
1900
340
8.5
25
1700
Optimized LC–MS/MS parameters for each tested
neurotransmitters and their metabolites.
Ionization Analyte
mode
Positive
DA
3-MT
5-HT
Ephedrine (IS)
Negative DOPAC
HVA
5-HIAA
Ferulic acid (IS)
Q1
m/z
154.1
168.1
177.1
166.1
167.0
181.1
190.1
193.1
Q3
m/z
91.0
151.1
160.1
148.1
123.0
137.1
144.0
134.1
CE
(V)
21
5
5
9
5
5
17
9
Fragmentor
(V)
65
65
65
75
50
60
65
85
Dwell
time (ms)
80
80
80
80
80
80
80
80
Rt
(min)
2.0
2.3
2.3
3.7
2.3
2.8
2.6
3.7
Q1 m/z, mass-to-charge ratio of precursor ion; Q3 m/z, mass-to-charge ratio of
fragment ion; CE, collision energy; Rt, retention time; IS, internal standard.
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RESULTS
LC–MS/MS assay for quantification of neurotransmitters and their
metabolites in rat brain tissue
An analytical method based on liquid-liquid extraction and LC–MS/MS was
developed and validated for quantifying the neurotransmitters DA and 5-HT, and
their major metabolites in rat brain tissue. Limits of quantification (40–200 ng/g
tissue) as listed in Table S3 for each analyte were sufficient for analysis of multiple
neurotransmitters in rat brain regions including striatum and olfactory bulb. During
the assay development, other analytical columns (cyano, phenyl, C8 and other C18
columns), mobile phases (formic acid, ammonium acetate, methanol), and extraction
methods (liquid-liquid extraction with ACN; solid-phase extraction with
Oasis® HLB Cartridge) had also been evaluated, but only the current method
achieved optimal peak shapes and signal strength.
The LC–MS/MS method was validated by spiking neurotransmitter
standards to 20 mg of occipital-temporal cortex obtained from rats receiving no drug
treatment. Occipital-temporal cortex tissue was chosen because, when compared
with other cortical regions, it contains the lowest endogenous levels of DA and
relatively low levels of 5-HT (3). The cortical tissue lateral to the hypothalamus was
discarded since it contains several nuclei including the nucleus amygdaloideus
centralis and relatively high levels of DA and 5-HT (3). For all determinations of
spiked samples, the endogenous levels of all neurotransmitter analytes in blank
tissues were quantified and subtracted from the tested samples. The results for intraday and inter-day accuracy and precision of the assay are shown in Table S3. For all
the analytes, both the accuracy (within ±15% bias) and precision (RSD less than
6
±15%) met the criteria. Representative chromatograms of an occipital-temporal
cortex sample spiked with 600 ng/g of analytes and a brain sample of striatum from
a non-treated rat are shown in Fig. S1 and Fig. S2, respectively. The column
effluent was diverted to waste for the first 1.5 min of each run to reduce the amount
of hydrophilic impurities introduced to the MS.
For over thirty years HPLC coupled with electrochemical detection has been
the most common technique for the quantification of neurotransmitters. In the last
decade, there are a number of reports on the use of LC–MS/MS for quantification of
neurotransmitters. Compared with the electrochemical detection which relies on
consistent retention times for the HPLC peaks for analyte identification, LC–
MS/MS allows specific mass spectrometric detection using MRM transitions (1) and
is believed to provide more accurate identification and quantification of
neurotransmitters in complex biological matrices like brain tissues which contains
numerous molecules that could interfere with the quantification process. Thus, LC–
MS/MS was adopted for the current study to quantitatively monitor the levels of
neurotransmitters from different treatment groups.
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Table S3
Intra- and inter-day accuracy and precision of the
neurotransmitter assay.
Analyte
DA
Range
(ng/g)
80–600
3-MT
40–800
5-HT
60–600
DOPAC
100–12000
HVA
100–8000
5-HIAA
200–4000
Spiked
(ng/g)
100
400
60
600
80
400
200
8000
200
4000
400
1200
Intra-day (n=3)
Accuracy (%) RSD (%)
110
1
97
8
98
7
107
4
103
3
95
3
100
7
97
1
104
5
102
2
102
7
97
7
Inter-day (n=3)
Accuracy (%) RSD (%)
102
11
100
9
103
8
101
9
104
4
97
5
101
8
99
2
89
10
96
3
105
6
92
7
Ionization
mode
Positive
Positive
Positive
Negative
Negative
Negative
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Fig. S1
Representative extracted ion chromatograms of an occipitaltemporal cortex sample (20 mg) from a non-drug-treated rat
spiked with 600 ng/g of analytes and internal standards (50 ng/g
ephedrine hydrochloride and 200 ng/g ferulic acid) under (A)
positive and (B) negative ionization modes.
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Fig. S2
Representative extracted ion chromatograms of a brain sample of
striatum from a non-drug-treated rat spiked with internal
standards (50 ng/g ephedrine hydrochloride and 200 ng/g ferulic
acid) under (A) positive and (B) negative ionization modes.
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REFERENCES
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and metabolites using liquid chromatography/electrospray tandem mass
spectrometry and comparison with liquid chromatography/electrochemical detection.
Rapid Commun Mass Spectrom. 2007;21(23):3898-3904.
2. Van Haard PMM, Pavel S. Chromatography of urinary indole derivatives. J
Chromatogr. 1988 7/29;429:59-94.
3. Reader TA. Distribution of catecholamines and serotonin in the rat cerebral cortex:
Absolute levels and relative proportions. J Neural Transm. 1981;50(1):13-27.
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