Brain Disposition and Catalepsy After Intranasal Delivery of Loxapine: Role of Metabolism

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 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

3 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. Table S1 Optimized LC and MS conditions for positive and negative ionization modes for the analyses of various neurotransmitters in the current study.

Positive mode Negative mode LC conditions Isocratic mobile ACN–0.1% acetic acid in ACN–0.1% acetic acid in phase H2O (1:1) H2O (3:7) Flow rate (ml/min) 1 0.25 Column Waters Nova-Pak C18, Phenomenex Gemini C18, 150 × 3.9 mm, 150 × 2.0 mm, 4 µm particle size 5 µm particle size Guard filter Thermo Unifilter Agilent Eclipse XDB-C8, 0.5 µm 2.1 × 15 mm, 3.5 µm particle size Injection volume (µl) 20 20 Run time (min) 9 7 MS conditions Gas temperature (°C) 350 340 Gas flow (l/min) 8.5 8.5 Nebulizer (psi) 33 25 Capillary (V) 1900 1700

Table S2 Optimized LC–MS/MS parameters for each tested neurotransmitters and their metabolites.

Ionizatio Analyte Q1 Q3 CE Fragmentor Dwell Rt n mode m/z m/z (V) (V) time (ms) (min) Positive DA 154.1 91.0 21 65 80 2.0 3-MT 168.1 151.1 5 65 80 2.3 5-HT 177.1 160.1 5 65 80 2.3 Ephedrine (IS) 166.1 148.1 9 75 80 3.7 Negative DOPAC 167.0 123.0 5 50 80 2.3 HVA 181.1 137.1 5 60 80 2.8 5-HIAA 190.1 144.0 17 65 80 2.6 Ferulic acid (IS) 193.1 134.1 9 85 80 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.

5 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 intra- day 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

±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.

7 Table S3 Intra- and inter-day accuracy and precision of the

neurotransmitter assay.

Analyte Range Spiked Intra-day (n=3) Inter-day (n=3) Ionization (ng/g) (ng/g) Accuracy (%) RSD (%) Accuracy (%) RSD (%) mode DA 80–600 100 110 1 102 11 Positive 400 97 8 100 9 3-MT 40–800 60 98 7 103 8 Positive 600 107 4 101 9 5-HT 60–600 80 103 3 104 4 Positive 400 95 3 97 5 DOPAC 100–12000 200 100 7 101 8 Negative 8000 97 1 99 2 HVA 100–8000 200 104 5 89 10 Negative 4000 102 2 96 3 5-HIAA 200–4000 400 102 7 105 6 Negative 1200 97 7 92 7

Fig. S1 Representative extracted ion chromatograms of an occipital- temporal 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.

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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