Abstract
Dopamine is a catecholamine neurotransmitter that degrades rapidly in aqueous solutions; hence, its analysis following brain microdialysis is challenging. The aim of the current study was to develop and validate a new microdialysis coupled LC-MS/MS system with improved accuracy, precision, simplicity and turnaround time for dopamine, serotonin, methamphetamine, amphetamine, 4-hydroxymethamphetamine and 4-hydroxyamphetamine analysis in the brain. Dopamine degradation was studied with different stabilizing agents under different storage conditions. The modified microdialysis system was tested in vitro, and was optimized for best probe recovery, assessed by %gain. LC-MS/MS assay was developed and validated for the targeted compounds. Stabilizing agents (ascorbic acid, EDTA and acetic acid) as well as internal and cold standards were added on-line to the dialysate flow. Assay linearity range was 0.01–100 ng/mL, precision and accuracy passed criteria, and LOQ and LLOQ were 0.2 and 1.0 pg, respectively. The new microdialysis coupled LC-MS/MS system was used in Wistar rats striatum after 4 mg/kg subcutaneous methamphetamine. Methamphetamine rapidly distributed to rat striatum reaching an average ~200 ng/mL maximum, ~82.5 min post-dose. Amphetamine, followed by 4-hydroxymethamphetamine, was the most abundant metabolite. Dopamine was released following methamphetamine injection, while serotonin was not altered. In conclusion, we proposed and tested an innovative and simplified solution to improve stability, accuracy and turnover time to monitor unstable molecules, such as dopamine, by microdialysis.
Keywords: Brain microdialysis, mass spectrometry, dopamine, serotonin, methamphetamine, amphetamine
1. Introduction
Monitoring the levels of neurotransmitters, notably, dopamine and serotonin, is of great importance in studying and diagnosing several neurological problems, such as drug dependence, Parkinson’s disease and schizophrenia [1–5]. Microdialysis is the state-of-the-art technique to assess the real-time brain levels of neurotransmitters, as well as other compounds such as drugs and metabolites. In this technique, a semi-permeable membrane, in a probe, is surgically inserted into the brain region of interest [6, 7]. A buffer perfused through the probe achieves pseudoequilibrium with the extracellular unbound solutes dissolved in the extracellular fluid of this brain region [6, 7]. The collected dialysates from different windows of time are then analyzed to determine the concentration of the targeted solute(s), which represents the recovered fraction of their levels in the brain region [6, 7]. Because dopamine is subjected to complete degradation within hours in dialysates [8, 9], its measurement by microdialysis is a challenging objective [10–12]. The dopamine degradation initially involves dopamine autoxidation and cyclization, reported to be catalyzed by redox-active metal ions and alkalinity, respectively [11, 12].
Several approaches have been adopted to mitigate the dopamine instability problem. The most common approach is to perform the analysis immediately upon collection of each sample to minimize the time between sample collection and analysis [13, 14]. However, this approach has a number of problems including that the dialysate collection from animals has to be performed in close physical/temporal proximity to the analytical instrumentation, which is often not feasible. Another method is to improve dopamine stability by the addition of antioxidants, chelating agents and/or acids to the collection bins [6, 15, 16], which can introduce variability in the sample volumes, compromising the quantitative results of dopamine assay [6]. Alternatively, low concentrations of ascorbic acid, as an endogenous reducing agent that improves dopamine stability, has been added to the microdialysis media before its passage through the brain probe [17, 18]. This avoids the addition of ascorbic acid to the collection bins afterwards, reducing volume variability and improving assay accuracy. However, it has been demonstrated that ascorbic acid (0.4 mM), delivered by reverse microdialysis, can induce the extracellular dopamine concentration in rat striatum [19].
In the current study, we addressed the challenges of measuring the physiological levels of dopamine by brain microdialysis, as a part of the determination of the pharmacokinetic profile of methamphetamine and its metabolites and the associated release of neurotransmitters in rat brains. Our aims were: 1) to study the stability of dopamine in media commonly used in microdialysis, Ringer’s solution and artificial cerebrospinal fluid (aCSF) with, and without, commonly used dopamine stabilizing agents, ascorbic acid, ethylenediaminetetraacetic acid (EDTA) and acetic acid, 2) to improve dopamine stability through a novel design of the microdialysis system, and 3) to develop and validate an LC-MS/MS method to accurately, precisely and rapidly measure dopamine, serotonin, methamphetamine and its metabolites simultaneously. Finally, 4) to use this new microdialysis design system coupled with the new LC-MS/MS method to assess, following methamphetamine treatment in vivo, the rat striatal dopamine and serotonin release and the pharmacokinetics of methamphetamine and its metabolites.
2. Materials and Methods
2.1. Materials
Dopamine, serotonin, methamphetamine, amphetamine, 4-hydroxymethamphetamine, 4-hydroxyamphetamine, ascorbic acid, EDTA disodium salt and acetic acid were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). High-performance-liquid-chromatography-grade acetonitrile, methanol and formic acid were purchased from EM Scientific (Gibbstawn, NJ). Other chemicals were purchased from Fisher Scientific Co. (Toronto, ON, Canada). Microdialysis system tubing, connectors, swivels, arms and cages were purchased from Bioanalytical System, Inc. (BASi) (West Lafayette, IN, USA).
2.2. Animals
Four adult male Wistar rats (Charles River Laboratories, Saint-Constant, Canada) were individually housed and food restricted to maintain a weight range of 350–450 g. Rats were maintained on a 12-hour light/dark cycle with experimentation occurring during light cycle. All procedures were approved by the Animal Care Committee at the University of Toronto.
2.3. Animal Surgery
Rats were anesthetized with isoflurane and injected with the analgesic meloxicam (2 mg/kg, subcutaneous). Thereafter, microdialysis guide-cannula (MD-2257, Bioanalytical System, Inc. (BASi), West Lafayette, IN, USA) were implanted into right striatum using a stereotaxic frame at the following coordinates relative to the bregma: anteroposterior: +1.2 mm; lateral: −3.0 mm; dorsoventral: −3.6 mm) [20]. Four screws (BASi) were implanted into the skull to serve as an anchor, and DuraLay inlay pattern resin (Reliance Dental Mfg. Co., Worth, IL, USA) was used as an adhesive to build a head cap around the microdialysis guide-cannula. Meloxicam was given daily (2 mg/kg, subcutaneous) up to 72-hour post-surgery. The experiments were initiated after at least one week of recovery.
2.4. Chromatographic Conditions and Mass Spectrometry Settings
The neurotransmitters (dopamine and serotonin), as well as, methamphetamine and its metabolites (amphetamine, 4-hydroxymethamphetamine and 4-hydroxyamphetamine) and the internal standards (dopamine-D4, serotonin-D4, methamphetamine-D5, and amphetamine-D6) were analyzed simultaneously using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). The analysis was performed on Agilent 1260 LC system, coupled with Agilent 6430 Triple Quadrupole system (Agilent Technologies, Santa Clara, CA). A gradient separation was performed on a Phenomenex PFP column (Kinetex® 1.7 μm F5 100 Å, 50 × 2.1 mm) at 30°C. The mobile phase A consisted of water with 0.1% formic acid (v/v), whereas mobile phase B consisted of acetonitrile with 0.1% formic acid (v/v). Linear gradient elution was used at a flow rate of 250 μL/min, as follows: 0 to 20% B in 0.1 min, held isocratically at 20% B for 2.4 min, returning to the initial condition and re-equilibration for 7.5 min. The first 0.92 min of the run (contains solvent front) was directed to the waste to avoid the contamination of the mass spectrometer ion source to improve dopamine sensitivity and the general robustness of the analytical method. The mass spectrometer was operated with multiple-ion monitoring under positive-ion mode. The gas temperature, flow and pressure were set at 350°C, 13 l/min and 40 psi, respectively, and the capillary voltage was 3.5 kV. The nebulizer gas was acquired from an in-house high purity nitrogen source.
2.5. Stability Study
The stability of dopamine, serotonin, methamphetamine, amphetamine, 4-hydroxymethamphetamine and 4-hydroxyamphetamine were studied in two rounds of stability experiments. These experiments examined a variety of solvents at a range of temperatures.
In round one, 1 μg/mL of the six compounds were dissolved in different solvents: 1) Milli-Q water, 2) Ringer’s solution (7.25 mg/mL NaCl, 0.17 mg/mL CaCl2, 0.37 mg/mL KCl, pH 7.4), 3) aCSF (7.25 mg/mL NaCl, 0.37 mg/mL KCl, 0.06 mg/mL CaCl2, 0.3 mg/mL MgCl2, 2.18 mg/mL NaHCO3, 1.8 mg/mL D-glucose, 0.14 mg/mL Na2HPO4, pH 7.4), 4) aCSF without magnesium (7.25 mg/mL NaCl, 0.37 mg/mL KCl, 0.06 mg/mL CaCl2, 2.18 mg/mL NaHCO3, 1.8 mg/mL D-glucose, 0.14 mg/mL Na2HPO4, pH 7.4) or 5) control brain dialysate (Ringer’s solution passed through microdialysis probe inserted in control rat brain at a flow rate of 2 μL/min). These were stored at different temperatures: A) room temperature, B) 4°C, C) −20°C or D) −80°C for different storing durations of: i) 0 h, ii) 18 h, iii) 24 h or iv) 36 h.
In round two, 1 μg/mL of the six compounds dissolved in a mixture of: 1) aCSF and Ringer’s solution (1:1), 2) aCSF and Ringer’s solution (1:1) with ascorbic acid at 90 μg/mL, 3) aCSF and Ringer’s solution (1:1) with ascorbic acid at 900 μg/mL, 4) aCSF and Ringer’s solution (1:1) with EDTA-Na2 at 100 μg/mL, 5) aCSF and Ringer’s solution (1:1) with EDTA-Na2 at 1000 μg/mL, 6) aCSF and Ringer’s solution (1:1) with1% acetic acid or 7) aCSF and Ringer’s solution (1:1) with stabilizing mixture (90 μg/mL ascorbic acid, 100 μg/mL EDTA and 1% acetic acid). These were stored at HPLC autosampler ambient temperature for different storing durations of: i) 0 h, ii) 24 h, iii) 36 h, iv) 48 h, v) 72 h or vi) 168 h.
Samples were then analyzed by LC-MS/MS. A full-ion scan between m/z of 50–500 under positive-ion mode was applied using the same chromatographic conditions and mass spectrometry source settings as described for the analytical method above. Also, 0.1 μg/mL of the six compounds dissolved in aCSF and Ringer’s solution (1:1) with and without stabilizing mixture (90 μg/mL ascorbic acid, 100 μg/mL EDTA and 1% acetic acid) were stored at room temperature for different storing durations of: i) 0 h, ii) 24 h, iii) 48 h, iv) 72 h or v) 168 h. These samples were analyzed by MRM following the analytical method described above.
2.6. Method Validation and Sample Recovery
Calibration curves constructed on three separate days were analyzed to evaluate the linearity in the range of 0.01 to 100 ng/mL for dopamine, serotonin, methamphetamine, amphetamine, 4-hydroxymethamphetamine and 4-hydroxyamphetamine. The accuracy and precision were determined using quality control samples at four concentrations, 0.05, 0.5, 5 and 50 ng/mL for all compounds, which was in the range of expected concentrations in dialysates. Five quality control samples at each of the four concentrations were prepared on three different days to enable the assessment of intraday and interday accuracy and precision. Accuracy was determined by calculating the concentration of each quality control sample relative to the calibration curve, and bias was assessed by calculating percent error (%error = (Ccalculated−Cnominal)/Cnominal×100) for the quality control samples at each of the four concentrations. Precision was assessed by calculating the coefficient of variation (%CV = SD/Mean×100) of the calculated concentration for the quality control samples at each of the four concentrations.
The effect of the addition of the stabilizing mixture (90 μg/mL ascorbic acid, 100 μg/mL EDTA and 1% acetic acid) on the sensitivity of the analytical method (matrix effect) was also studied. Five quality control samples at each of the four different concentrations (0.05, 0.5, 5 and 50 ng/mL) of methamphetamine amphetamine, 4-hydroxymethamphetamine and 4-hydroxyamphetamine prepared in a mixture of aCSF and Ringer’s solution (1:1) with, and without, the stabilizing mixture were analyzed and the peak areas of each compound for each concentration were compared.
2.7. On-Line Sample Preparation Microdialysis
We modified a regular swivel-based microdialysis system to accommodate an auxiliary flow of a solution of the deuterated internal and cold standards and stabilizing mixture in addition to the original flow of the microdialysis medium passing through the probe (dialysate flow), and to combine the two flows immediately after the microdialysis probe (Fig. 1). Dialysate flow were modified on-line by the following, 1) adding the stabilizing mixture immediately to the dialysate to protect dopamine from degradation, 2) adding the internal standards to eliminate time needed for sample preparation for analysis, and 3) spiking the dialysate with low concentration of cold standards to increase assay sensitivity. Moreover, combining the dialysate flow with equal auxiliary flow enabled us to decrease perfusion flow rate without the need of special measures to handle ultrasmall sample volume. This was performed in an automated fashion and in a fixed proportion to the original perfusion rate to reduce variability in volume due to time and evaporation, and therefore, improving the quantitative results of the assay. The system consisted of a dual syringe pump (Pump 11 Pico Plus Elite; Harvard Apparatus, Holliston, MA, USA), a cage with a metal arm attached to a swivel (BASi, West Lafayette, IN, USA), an in-house binary static mixer and a refrigerated microfraction collector (CMA 470; Harvard Apparatus, Holliston, MA, USA).
Figure 1. A graphical representation of the on-line sample preparation microdialysis with static mixer.
Ringer’s (R), and a mixture (S) are simultaneously pumped. R-flow is directed through the microdialysis probe to be subsequently mixed with the S-flow, and the combined flow is collected by a fraction collector. S-mixture consists of 180 μg/mL of ascorbic acid, 200 of μg/mL EDTA, 2% of acetic acid, 2 ng/mL of methamphetamine-D5, amphetamine-D6, dopamine-D4 and serotonin-D4, and 0.2 ng/mL of dopamine, serotonin and 4-hydroxyamphetamine.
The in-house mixers were made from a T-shaped arrangement of three 21-gauge needles, connected together with a mixing chamber made of polytetrafluoroethylene, sealed and secured in clear epoxy resin molded into a triangle prism shape. These in-house mixers were left for two days to cure. Methylene blue dye was used to check mixer integrity and measure mixer dead volume. The mixing performance of the in-house mixers was measured by pumping two equal and steady flows at either 0.5, 1.0 or 2.0 μL/min with the first flow containing 1 ng/mL amphetamine, and the second flow containing 1 ng/mL of amphetamine-D6. The combined flow was collected over 90 min (six microdialysate samples x 15 min) and was analyzed by LC-MS/MS.
Two solutions were installed to the dual syringe pump, the first solution was the microdialysis medium (Ringer’s solution), whereas, the second solution contained 180 μg/mL ascorbic acid, 200 μg/mL EDTA and 2% acetic acid, 2 ng/mL of dopamine-D4, serotonin-D4, methamphetamine-D5, and amphetamine-D6, and 0.2 ng/mL of dopamine, serotonin and 4-hydroxyamphetamine (Fig. 1). The microdialysis medium was allowed to pass through the swivel to the microdialysis probe inserted into the animal brain at a fixed flow rate. The dialysate flow was combined with equal rates of auxiliary flow containing 1) the stabilizing mixture to prevent dopamine from degradation, 2) the cold standards to further improve the sensitivity of the analytical method, and 3) the internal standards to readily complete sample preparation for the analysis following the microdialysis run (Fig. 1). The combined flow was collected in 300-μL polypropylene LCMS vial inserts (Agilent Technologies, Santa Clara, CA) using the fraction collector (Fig. 1).
2.8. Recovery of the microdialysis probe
The microdialysis probe recoveries for dopamine, serotonin, methamphetamine, amphetamine, 4-hydroxymethamphetamine and 4-hydroxyamphetamine were tested in vitro. The microdialysis probes attached to the two-flow microdialysis system were incubated in well-stirred Ringer’s solution bath containing 10 or 100 ng/mL of the six compounds at 37°C. The two flows of Ringer’s solution and mixture of stabilizing agents and internal and cold standards were initiated at 1 μL/min each. The probe equilibration was allowed for 30 min. The combined flow of 2 μL/min was collected every 15 min for 180 min (12 microdialysate samples) and the samples were subsequently analyzed by LC-ESI-MS/MS. The probe recoveries were measured as the %gain of each of the six compounds in the dialysate (after passing through the probe) relative to the original concentration in the bath (%gain =Cout/CBath × 100).
2.9. Microdialysis
Brain levels of dopamine and serotonin as well as methamphetamine and its metabolites were assessed by brain microdialysis using the modified microdialysis coupled LC-MS/MS system. Microdialysis probes (2 mm, MD-2201, BASi) were introduced in the microdialysis guide-cannulae, and animals (N=3) were allowed to habituate for at least 30 minutes while perfusion medium (Ringer’s solution) was pumped at a flow rate of 1 μl/min. In 15 min time intervals, five baseline microdialysis samples (−75 min to 0 min) were collected prior to administration of 4 mg/kg subcutaneous methamphetamine, and 11 microdialysis samples were collected afterwards (0 min to 165 min). The concentration of the six compounds in each dialysate sample represents the average concentration for that time bin and is plotted at the mid-interval time point.
3. Results and Discussion
3.1. Dopamine stability at different conditions
It has been reported that dopamine is stable for several hours in dextrose in water and lactated Ringer’s solutions [21, 22]; however, this is not consistent with dopamine’s instability in aqueous solutions [8, 9, 11, 12]. We found that dopamine was stable in Milli-Q water at room temperature for at least 36 h (Fig. 2A). However, water cannot be used as the perfusate during in vivo central nervous system microdialysis, as it could deplete electrolytes from the brain through the semipermeable membrane. Similar to Milli-Q water, dopamine was stable in Ringer’s solution at room temperature for at least 36 h (Fig. 2B), while, in aCSF, dopamine degraded almost completely over the 36 h (Fig. 2C). The stability of dopamine in aCSF was slightly improved by lowering the storage temperature to −80°C (Fig. 2D, E & F). Redox-active metal dications, such as magnesium, have been reported to accelerate dopamine degradation [11, 12]. The absence of magnesium in aCSF improved dopamine stability over 36 h at room temperature and at 4°C (Supplementary Fig. 1), suggesting that the absence of redox-active dications in Ringer’s solution may be responsible for the greater dopamine stability. However, Ringer’s solution is expected to gain magnesium, as well as, other cations, from brain tissue as Ringer’s solution passes through the microdialysis probe. To test this, we examined dopamine stability in the rat brain dialysate following microdialysis with Ringer’s solution; the dialysate showed accelerated degradation of dopamine over 36 h (Fig. 2G), at a rate similar to aCSF alone (Fig. 2C). In all test conditions, serotonin was stable for at least 36 h (Fig. 2).
Figure 2. Dopamine is unstable in aCSF solution regardless of the storage temperature.
Dopamine at 1 μg/mL dissolved in aCSF showed a time-dependent decline in quantity at room temperature, 4°C, −20°C or −80°C, compared to dopamine at 1 μg/mL in water or in Ringer’s solutions over 36 h. Total-ion scan was performed by LC-ESI-MS to generate the extracted-ion chromatograms at the m/z of dopamine (154.1) and m/z of serotonin (177.1).
3.2. Improving dopamine stability
We next examined dopamine stability in aCSF and Ringer’s solution 1:1 mixture, which better mimics the composition of Ringer’s solution dialysate. Also, we examined dopamine stability at the ambient temperature of the LCMS autosampler without temperature control (~26°C) (e.g. a worst-case scenario). Dopamine was almost completely degraded within 36 h, while, serotonin and the other compounds were stable for at least 7 days (Fig. 3A and Supplementary Table 1). The addition of EDTA, a chelating agent, at concentrations reported to improve dopamine stability [9, 23], failed to improve dopamine stability after 24 h under the tested conditions (Fig. 3B & C). However, the addition of an antioxidant, ascorbic acid, slowed the rate of dopamine degradation in a concentration-dependent manner (Fig. 3 D&E). The lower concentration of ascorbic acid (90 μg/mL) provided protection from dopamine autoxidation for 24 h whereas, the higher concentration of ascorbic acid (900 μg/mL) provided partial protection over the 7 days (Fig. 3 D&E). Likewise, acidification using 1% acetic acid stabilized dopamine for 24 h (Fig. 3F). The mixture of the three dopamine stabilizing agents, EDTA, ascorbic acid and acetic acid, substantially slowed the rate of dopamine degradation, stabilizing dopamine for at least 7 days under the tested storage conditions (Fig. 3G and Supplementary Table 1). This stabilization was achieved using the lower concentrations of EDTA and ascorbic acid, 100 and 90 μg/mL, respectively (Fig. 3G and Supplementary Table 1). In agreement, Van Schoors et al studied oxalic acid, L-cysteine, ascorbic acid, EDTA and acetic acid alone and in combination as stabilizing agents for dopamine and norepinephrine, reporting that the best improvement in stability came from the mixture of ascorbic acid, EDTA and acetic acid for up to 6 months at −20°C or −80°C [8]. Thus, EDTA (100 μg/mL), ascorbic acid (90 μg/mL) and acetic acid (1%) stabilizing mixture was used to mitigate dopamine instability in the new microdialysis system described in the current study.
Figure 3. Ascorbic acid, EDTA and acetic acid were added to improve the stability of dopamine in a mixture of Ringer’s solution and aCSF at ambient temperature over 7 days.
Dopamine at 1 μg/mL dissolved in Ringer’s solution and aCSF (1:1) combined with 90 μg/mL of ascorbic acid, 100 of μg/mL EDTA, 1% of acetic acid did not show a decline in quantity at the HPLC autosampler ambient temperature. Ascorbic acid (90 or 900 μg/mL) or EDTA (100 or 1000 μg/mL) alone did not improve dopamine stability. Total-ion scan was performed by LC-ESI-MS to generate the extracted-ion chromatograms at the m/z of dopamine (154.1) and m/z of serotonin (177.1).
3.3. Developing and validating highly sensitive LCMS assay
In microdialysis the perfusate is to be isotonic to avoid disruption of brain tissue through the semipermeable membrane, and therefore, microdialysis perfusate and resulting samples have high salt content. However, this causes ion-suppression of polar compounds with low retainability by reverse phase HPLC, such as dopamine and serotonin, during LCMS analysis. To avoid an additional desalting and extraction step, a pentafluorophenyl LC column was used to prolong the dopamine retention time away from the solvent front (0.65 min for the column used). The solvent front was directed to the waste to avoid contaminating the ion source. The assay was adapted to simultaneously measure dopamine, serotonin, methamphetamine and its metabolites. To optimize the dwell time, for cleaner baseline and peaks of sufficient resolution (Table 1), the 6-min MRM was divided into three segments, one for monitoring dopamine and dopamine-D4, another for monitoring serotonin, serotonin-D4 and 4-hydroxyamphetamine, and the third for monitoring methamphetamine, methamphetamine-D5, amphetamine, amphetamine-D6 and 4-hydroxymethamphetamine (Fig. 4A). We used two transitions to monitor dopamine ions to improve method selectivity to dopamine, and one transition for serotonin and methamphetamine and its metabolites, as well as the internal standards (Fig. 4B and Table 1). The assay was linear from 0.01 to 100 ng/mL, with an average slope of 1.818 and r2 of >0.999, for dopamine and serotonin, and an average slope of 1.265 and r2>0.998 for methamphetamine. The addition of the stabilizing mixture (ascorbic acid, EDTA and acetic acid) did not give interfering peaks or supress the sensitivity toward our targeted compounds tested at 0.05, 0.5, 5 and 50 ng/mL (Supplementary Table 2). The criteria of intraday and interday accuracy and precision, of a %error and a %CV that ranged between ±15%, and ±20% at the lower limit of quantification, was met at all the tested concentrations (Table 2) [24]. The limit of quantification based on S/N >10 was 0.01 ng/mL, whereas, the lower limit of quantification based on intraday and interday accuracy and precision criteria was 0.05 ng/mL for all compounds, both at 20 μL injection volume (Table 2).
Table 1.
The mass spectrometry parameters used for dopamine, serotonin, methamphetamine, amphetamine, 4-hydroxymethamphetamine and 4-hydroxyamphetamine.
| Transition | Collision Energy (V) | Fragmentor Voltage (V) | Dwell Time (msec) | |
|---|---|---|---|---|
| → 91.1 | 25 | |||
| Dopamine-D4 | 158.1 → 141 | 8 | 60 | 150 |
| Serotonin | 177.1 → 160 | 8 | 65 | 150 |
| Serotonin-D4 | 181.1 → 164 | 8 | 65 | 150 |
| Methamphetamine | 150.1 → 91.1 | 20 | 70 | 100 |
| Methamphetamine-D5 | 155.1 → 92.1 | 20 | 70 | 100 |
| Amphetamine | 136.1 → 91.1 | 16 | 65 | 100 |
| Amphetamine-D6 | 142.1 → 93.1 | 16 | 65 | 100 |
| 4-Hydroxymethamphetamine | 166.1 → 107 | 24 | 70 | 100 |
| 4-Hydroxyamphetamine | 152.1 → 107 | 20 | 53 | 100 |
Figure 4. The mass spectra and representative MRM chromatograms of dopamine, serotonin, methamphetamine, amphetamine, 4-hydroxymethamphetamine and 4-hydroxyamphetamine.
The six compounds at 1000 ng/mL and 0.05 ng/mL were used to generate the mass spectra and the MRM chromatograms, respectively, by LC-ESI-MS/MS.
Table 2.
The validation of the LC-ESI-MS/MS assay for dopamine, serotonin, methamphetamine, amphetamine, 4-hydroxymethamphetamine and 4-hydroxyamphetamine in dialysates.
| QC (ng/ml) | Intraday | Interday | |||
|---|---|---|---|---|---|
| %Error | %CV | %Error | %CV | ||
| Dopamine | 50 | 13.9 | 3.0 | 11.5 | 3.2 |
| 5 | 6.6 | 4.1 | 0.3 | 4.3 | |
| 0.5 | −10.7 | 5.3 | 0.0 | 12.1 | |
| 0.05 | 17.2 | 12.3 | 0.0 | 16.6 | |
| Serotonin | 50 | 12.2 | 1.7 | 9.0 | 3.3 |
| 5 | 6.9 | 3.7 | 0.2 | 5.8 | |
| 0.5 | −11.4 | 1.9 | 0.0 | 9.5 | |
| 0.05 | 15.1 | 10.4 | 0.0 | 10.9 | |
| Methamphetamine | 50 | 7.4 | 2.7 | 5.1 | 4.7 |
| 5 | 11.0 | 3.9 | 0.6 | 7.6 | |
| 0.5 | −0.2 | 1.7 | 0.1 | 6.5 | |
| 0.05 | 12.1 | 12.3 | 0.0 | 7.9 | |
| Amphetamine | 50 | 11.2 | 3.3 | 7.7 | 4.2 |
| 5 | 10.1 | 4.2 | 0.6 | 5.1 | |
| 0.5 | −3.1 | 2.3 | 0.1 | 9.1 | |
| 0.05 | 1.7 | 8.7 | 0.0 | 6.7 | |
| 4-Hydroxymethamphetamine | 50 | 2.4 | 3.1 | 0.3 | 6.4 |
| 5 | 13.3 | 3.8 | 0.8 | 6.9 | |
| 0.5 | 2.3 | 2.1 | 0.1 | 5.3 | |
| 0.05 | 7.4 | 10.3 | 0.0 | 7.2 | |
| 4-Hydroxyamphetamine | 50 | 12.8 | 4.0 | 11.6 | 3.5 |
| 5 | −2.1 | 4.5 | 0.2 | 5.6 | |
| 0.5 | −13.5 | 3.8 | −0.1 | 7.7 | |
| 0.05 | −3.6 | 7.1 | 0.0 | 6.1 | |
3.4. Validating the in-house static microdialysis mixer
The novel aspect of the new microdialysis system is the static mixer (Fig. 1), merging an auxiliary flow with the original flow of the perfusate after the microdialysis probe (dialysate) (Fig. 1). We made two in-house mixers and confirmed that there were no leakages by pumping dye solution for 12 h. We also investigated whether the two in-flow arms had similar resistance in the mixer, in order to achieve a stable 1:1 mixing of the microdialysis perfusate and the auxiliary flow (Fig. 5A). The two mixers were able to maintain 1:1 mixing over 90 min at three different in-flow rates, 0.5, 1.0 and 2.0 μL/min (Fig. 5B & C). Lastly, the mixing needs to be confined to the mixing chamber in the mixer (Fig. 5A). There was no amphetamine-D6 (representing the auxiliary flow), in the amphetamine flow (representing the microdialysis perfusate) after 90 min of stable double flows at 0.5, 1.0 or 2.0 μL/min of amphetamine and amphetamine-D6.
Figure 5. The in-house mixers are effectively mixing the perfusate flow and auxiliary flow.
Amphetamine flow and deuterated amphetamine flow were used to represent the perfusate flow and auxiliary flow, respectively, tested at 0.5, 1.0 and 2.0 μL/min flow rates. The peak areas of amphetamine and deuterated amphetamine in dialysate were measured by LC-ESI-MS/MS.
3.5. Recovery of the microdialysis probe
During microdialysis the perfusate is constantly passing through the probe with very limited time for the exchange with the surroundings [6, 25], and thus, only a fraction of the extracellular unbound solute concentration is recovered in the dialysate [6, 25]. The recovery can be improved by using longer membrane probes (a larger surface area of the semipermeable membrane), and by using slower flow rates [6, 25]. The semipermeable membrane length used in brain microdialysis ranges between 1–4 mm; however, the size of the targeted brain structure often limits the maximum membrane length that can be precisely inserted within the structure [26]. Our target was the striatum, which receives several dopaminergic projections from the substantia nigra and raphe nucleus [27, 28]. The striatum has a volume of about 100 mm3 [26, 29], and therefore, we used microdialysis probe with 2 mm semipermeable membrane.
The fraction recovery is inversely proportional to perfusion flow rate, but slow flow leads to smaller collection volumes. When studying a time-dependent change in a solute either the collection time, has to be lengthened, losing important time-specific information, or this results in an ultrasmall collection volume, which could lower the absolute recovery of the solute [6]. In most cases, increasing perfusate flow rate to a certain limit leads to an increase in the total amount of the solute in the collected dialysate [6, 30]. The %recovery of a microdialysis probe at a certain perfusate flow rate can be determined by measuring %gain of the solute by the perfusate as the solute is exchanged with the surroundings, or by measuring %loss of the solute from the perfusate to the surroundings [6, 25]. We measured %recovery by gain, which is mimicking the real microdialysis situation compared with %recovery by loss; moreover, because dopamine and serotonin are endogenously present in the brain, which will interfere with the exchange between the perfusate and the surroundings, we preferred to measure %recovery in vitro.
The average %recovery of the 2 mm-membrane microdialysis probe at 1 μL/min was 36.8% (dopamine, serotonin, methamphetamine, amphetamine. 4-hydroxymethamphetamine and 4-hydroxyamphetamine, were 35.5, 36.5, 39.7, 40.4, 37.4 and 31.4% respectively) (Fig. 6). The in vitro %recovery was measured three to four individual times over three hours at two different solute concentrations to confirm consistency, i.e. there is no concentration-related or time related alteration in probe efficiency (Fig. 6). Increasing the flow rate to 2 μL/min, under the same other settings, led to a decrease in microdialysis probe %recovery to an average of 16.5% (methamphetamine, amphetamine, 4-hydroxymethamphetamine and 4-hydroxyamphetamine were 14.3, 13.1, 19.5 and 18.9%, respectively). In line with our results, the %recovery of dopamine was 12.0% at 2 μL/min of aCSF using 3.0 mm-membrane microdialysis probe [31], 18.8% at 1 μL/min of Ringer’s solution using 2.0 mm-membrane microdialysis probe [32], and 13.12% at 1.5 μL/min of modified aCSF using 2.0 mm-membrane microdialysis probe [33]. For serotonin, the %recovery was an average of 37.5% at 0.41 μL/min of aCSF using 2.0 mm-membrane microdialysis probe [34], ~15% at 2.0 μL/min of Ringer’s solution using 3.0 mm-membrane microdialysis probe [35], and 16.2% at 1.1 μL/min of Ringer’s solution using 3.5 mm-membrane microdialysis probe [36]. The %recovery is also dependent on the membrane material of the microdialysis probe, such as cuprophane, polyarylethersulfones, cellulose, and polyacrylonitrile. Polyacrylonitrile is the membrane material of the microdialysis probe used in the current study.
Figure 6. The microdialysis probe recoveries for dopamine, serotonin, methamphetamine, amphetamine, 4-hydroxymethamphetamine and 4-hydroxyamphetamine.
Probe recoveries were measured by incubating the probes in 50 ml of 10 or 100 ng/mL concentrations of the six compounds under well-stirred conditions in vitro at 37°C. The two-flow microdialysis system was used to perfuse1 μL/min of Ringer’s solution through the probe. The concentrations of the analytes in the collected dialysates were measured by LC-ESI-MS/MS. Recoveries were assessed as %gain in the dialysate over 180 min. Results are presented as mean and SD, based on three to four individual experiments.
3.6. The pharmacokinetics study and the release of dopamine and serotonin
Using the new microdialysis system, the intrastriatal pharmacokinetic profile of methamphetamine and its metabolites and the associated time-dependent release of dopamine and serotonin was determined (Fig. 7). Methamphetamine, amphetamine, 4-hydroxymethamphetamine and 4-hydroxyamphetamine were not detected above the limit of quantification in the baseline samples (Fig. 7A–D). For dopamine and serotonin, the average concentration in the baseline samples were 0.34 and 0.57 ng/mL, respectively, after the correction for microdialysis probe recovery, representing their basal extracellular unbound concentration in the striatum before exposure to methamphetamine (Fig. 7E & F). Consistent with this, the basal extracellular unbound dopamine concentration in striatum ranges between 0.25–0.75 ng/mL in rats using the no-net-flux method [37–41], whereas; for serotonin it was about 0.5 ng/mL in mice also using the no-net-flux method [42, 43]. No-net-flux method is a multistep microdialysis-based method used to measure the physiological levels of a solute, by perfusing different concentrations of this solute and determining the concentration that is changed by passing through the microdialysis probe (no-flux); this concentration is, therefore, equal to the extracellular unbound concentration of the targeted solute [6].
Figure 7. The brain time-concentration curves of methamphetamine, its metabolites, amphetamine, 4-hydroxymethamphetamine and 4-hydroxyamphetamine, and associated dopamine and serotonin.
Intrastriatal microdialysis was performed on rats receiving 4 mg/kg subcutaneous methamphetamine using the two-flow microdialysis system, with analytes in the collected dialysates measured by LC-ESI-MS/MS. The concentrations were corrected for microdialysis probe recovery. Results are presented as mean and SEM, based on experiments in three individual rats.
Methamphetamine was first detected in the 0–15 min microdialysis sample (1.6 ng/mL), and reached an average Cmax of 202.2 ng/mL at an average Tmax of 82.5 min (Fig. 7A and Table 3). Methamphetamine is metabolized by cytochrome P450 enzymes to two primary metabolites, amphetamine and 4-hydroxymethamphetamine, and one secondary metabolite, 4-hydroxyamphetamine (subsequent to amphetamine) [44]. The first methamphetamine metabolite detectable in rat brain after methamphetamine injection was 4-hydroxymethamphetamine, which was also detected in the 0–15 min microdialysis time bin (0.24 ng/mL); 4-hydroxymethamphetamine reached an average Cmax of 0.6 ng/mL at an average Tmax of 87.5 min (Fig. 7C and Table 3). Amphetamine was the most abundant methamphetamine metabolite, reaching an average Cmax of 39.1 ng/mL at an average Tmax of 107.5 min (Fig. 7B and Table 3). The primary metabolite of amphetamine, 4-hydroxyamphetamine, followed the amphetamine concentration-time curve, reaching an average Cmax of 0.2 ng/mL at an average Tmax of 107.5 min (Fig. 7D and Table 3). Dopamine was increased following the methamphetamine injection, reaching an average Cmax of 2.2 ng/mL at an average Tmax of 57.5 min (Fig. 7E and Table 3). Serotonin was not altered by the methamphetamine injection (Fig. 7F). The dopamine concentration-time curve appears to mirror the methamphetamine curve, which could suggest that the methamphetamine played the major role, compared with its metabolites, in stimulating the release of dopamine in rat striatum (Supplementary Figure 2). Striatal dopamine levels increased to almost 700% of the basal concentration. Methamphetamine at 2 and 4 mg/kg intraperitoneal, induced dopamine levels by 550% and 1200% in male rat striatum, respectively [13], and after 2 mg/kg intraperitoneal, induced dopamine by 200% and 1300% in female rat prefrontal cortex and nucleus accumbens, respectively [15]. However, brain levels of methamphetamine and its metabolites were not measured during the dopamine release in these studies [13, 15].
Table 3.
The Cmax and Tmax (mean value and SEM) for dopamine, methamphetamine and its metabolites, amphetamine, 4-hydroxymethamphetamine and 4-hydroxyamphetamine in rat striatum, after 4 mg/kg subcutaneous methamphetamine injection. Values were corrected for microdialysis probe recovery and microdialysis system dead volume.
| Analyte | Animal | aCmax (ng/mL) | Time bin (min) | Averageb Cmax (ng/mL) | Averagec Tmax (min) |
|---|---|---|---|---|---|
| Dopamine | 1 | 1.2 | 75–90 | 2.2±1.0 | 57.5±5.0 |
| 2 | 0.7 | 60–75 | |||
| 3 | 4.6 | 45–60 | |||
| Methamphetamine | 1 | 197.5 | 45–60 | 202.2±31.1 | 82.5±15.0 |
| 2 | 150.9 | 90–105 | |||
| 3 | 258.3 | 90–105 | |||
| Amphetamine | 1 | 37.3 | 105–120 | 39.1±2.7 | 107.5±13.2 |
| 2 | 35.5 | 75–90 | |||
| 3 | 44.5 | 120–135 | |||
| 4-Hydroxymethamphetamine | 1 | 0.8 | 60–75 | 0.6±0.1 | 87.5±21.8 |
| 2 | 0.3 | 75–90 | |||
| 3 | 0.7 | 120–135 | |||
| 4-Hydroxyamphetamine | 1 | 0.2 | 90–105 | 0.2±0.1 | 107.5±10.0 |
| 2 | 0.1 | 90–105 | |||
| 3 | 0.2 | 120–135 |
Footnotes
the average concentration in the time bin listed for maximal concentration
the average from the three animals during the time bin of maximal concentration
the average of the midpoint time for each the time bin where the concentration was maximal
3.7. The significance of the new system
In the novel microdialysis coupled LC-MS/MS system described here, our goal was to address the challenges associated with measuring dopamine in dialysates. These challenges were handled in previous papers by unusual sample-preparation techniques, by specialized equipment or by sophisticated modifications in the system. As mentioned, doing the analysis immediately after dialysate collection is a commonly used solution for dopamine instability. This often results in using the less accurate and precise external standard analytical technique, i.e. injecting without the addition of any internal standard [45–48]. In some studies, a fixed amount of the internal standards were added beforehand to the dialysate collection bins. For example, Gottås et al used dopamine-D3 as the internal standard adding a fixed volume to each collection pin, in order to improve the quantitative results of the assay [49]. However, in practice, maintaining a stable dialysate flow during the microdialysis run on a conscious animal can be problematic, because of the constant animal movements, causing flow interruptions and variability in the volume of the dialysate samples. Using a fixed amount of the internal standard with a variable amount of dialysate (i.e. not relative to sample volume) compromises accuracy and precision. This was likely less of a problem in the Gottås et al study, because of the sedative effect of heroin administered to animals in their study [49]. In the current study, we used a central nervous system stimulant, methamphetamine, at a relatively high dose of 4 mg/kg, subcutaneous, (e.g. usual range in rats is 0.1 to 10 mg/kg [50, 51]) to stimulate animal movement. Human methamphetamine users typically consume between 0.7 and 15 mg/kg (assuming a standard 70 kg weight) over the course of a day, with heavy users taking up to approximately 4 mg/kg in a single dose [52].
An external standard technique was also followed in studies utilizing the automated online sample injection systems or on-line sensors. The automated on-line sample injection systems is a sophisticated modification of the microdialysis system that allows the dialysate flow to be collected directly into the sample loop of the HPLC to automatically inject after a predefined time interval, allowing the immediate analysis after dialysate collection [14, 19, 53–55]. This approach needs specialized technical skills to prevent interruptions in perfusate flow during sample injections, such as the two-sample-loop modification [55]. Ngo et al used two 0.6 μL sample loops, switching between receiving and injecting the dialysate every one min, with a special flow switcher to achieve one-min resolution of monitoring dopamine levels in brain [55]. In addition, for on-line sample injection systems, or on-line sensors, the detection instrument needs to be directly attached to the animal on microdialysis which limits the selection of the detection method. Accordingly, electrochemical detection has been the detection method of choice for dopamine quantitation [56]. Electrochemical detection usually exploits dopamine ease of oxidation, i.e. dopamine catechol moiety is oxidized to orthoquinone losing a pair of electrons/molecule, to detect dopamine with good sensitivity - LOD can reach 0.003 ng/mL using specially modified gold electrode [56]. However, in comparison with MS/MS, electrochemical detectors are less selective and versatile. For example, polar electrochemical reactive compounds, notably serotonin, ascorbic acid and divalent metal ions, will cause interfering peaks near dopamine using the electrochemical detector, contrary to the superior selectivity of MS/MS [56, 57]. Because of the nature of electrochemical detection and being a destructive method of detection, the compounds that can simultaneously be measured with dopamine are very limited, for example drugs used to alter dopamine release, such as opioids and amphetamines. In the current study, the new microdialysis system coupled the new LC-MS/MS assay measured dopamine without any interference from its electrochemically interfering compounds, serotonin and ascorbic acid, and simultaneously measured several other compounds, methamphetamine and its metabolites. Therefore, we could directly assess the association between the change in brain concentration of methamphetamine and its metabolites with dopamine release.
4. Conclusion
In the current study, dopamine stability was shown to be dependent on the composition of its aqueous solution. We found that dopamine is relatively stable in Ringer’s solution, while in aCSF and brain dialysate, it is rapidly degraded (Fig. 2B, 2C & 2G). We found that the combination of EDTA, ascorbic acid and acetic acid remarkably improves dopamine stability (Fig. 3G). This stabilizing mixture, in addition to internal and cold standards, were added on-line to the dialysate flow by modifying a regular swivel-based microdialysis system (Fig. 1). This new microdialysis system was coupled with a LC-MS/MS assay that simultaneously measures the targeted analytes: dopamine, serotonin and methamphetamine and its metabolites, amphetamine, 4-hydroxymethamphetamine and 4-hydroxyamphetamine, with high sensitivity. A series of experiments were done to validate the on-line mixing step (Fig. 5), microdialysis probe performance (Fig. 6) and the LC-MS/MS assay (Table 2). The new microdialysis coupled LC-MS/MS system was then used to assess the brain levels of our targeted analytes in rat striatum following methamphetamine treatment (Fig. 7). In conclusion, we proposed and tested a new microdialysis system that improves stability, accuracy and turnover time to monitor dopamine, serotonin and methamphetamine and its metabolites by brain microdialysis.
Supplementary Material
Highlights.
A new microdialysis/LCMS system was developed and optimized for dopamine stability.
Stabilizers/standards were added on-line to improve LCMS quantitative results.
Rat striatal dopamine was increased by 7-fold post methamphetamine 4 mg/kg sc dose.
Serotonin, methamphetamine and its metabolites were concurrently quantified.
The new method was accurate and precise (<15%) with good probe recovery (~37%).
Acknowledgements
This research was undertaken, in part, thanks to funding a Canadian Institutes of Health Research (Foundation grant FDN-154294); an R01 grant from National Institutes of Health (DA043526-01A1), the Canada Research Chairs program (Dr. Tyndale, the Canada Research Chair in Pharmacogenomics), the Centre for Addiction and Mental Health and the CAMH Foundation. We also acknowledge the support of Dr. Bin Zhao for LC-MS/MS analyses.
Footnotes
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Declarations of competing interests
R.F.T. has consulted for Quinn Emanuel and Ethismos on unrelated topics. All other authors declare no competing interests.
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