i!J ELSEVIER Journal of Neuroscience Methods 53 (1994) 19-22

JOURNALOF NEUROSCIENCE

METHODS

A simplified method for the measurement of caffeine in plasma and brain: evidence for a cortical-subcortical caffeine concentration

differential in brain

Robert J. Carey *, Gail DePalma VA Medical Center and SUNY Health Science Center, 800 Irving Avenue, Syracuse, NY 13210, USA

Received 30 July 1993; revised 16 December 1993; accepted 22 December 1993

Abstract

We describe a much simplified high-performance liquid chromatography (HPLC) method for the measurement of caffeine in plasma and brain. A particularly attractive feature of this method is that a simple methanol/water (60 : 40) mobile phase can be used both for plasma and brain samples. In addition, the method is compatible with solid-phase extraction for plasma samples and conventional brain tissue preparation for biogenic amine analysis with HPLC. Using this method to measure the concentrations of caffeine in plasma and brain of rats which received 10 or 50 mg/kg caffeine injections, we found substantial concentration differences between cortical and subcortical brain tissue. Specifically, at the 10 mg/kg dose, a nearly 2-fold difference between cortex and striatum caffeine concentrations was observed. A shortcoming of many neurobehavioral studies of caffeine effects is the absence of caffeine concentration measurements. The simplicity of the present method for the measurement of caffeine in plasma and brain tissue makes it a practical and feasible procedure to incorporate into neurobehav- ioral studies designed to elucidate the CNS actions of caffeine.

Key words: Caffeine; High-performance liquid chromatography; Plasma cortex striatum

1. Introduction

The neuroanatomical and neuroreceptor mecha- nisms which mediate the CNS activation induced by caffeine have been the subject of numerous investiga- tions. Although caffeine is widely used in neurobehav- ioral studies (Hughes and Gerig, 1976; Holtzman, 1983; Dews, 1984; Meliska and Loke, 1984; Herz and Beninger, 1987; Mumford et al., 1988), it is still rela- tively uncommon that plasma and brain levels are measured. A wide variety of analytical techniques are available for the measurement of caffeine in tissue (e.g., radioimmunoassay (RIA)) (Matsumoto et al., 1988), gas chromatography (GC) (Naline et al., 1987), h igh-performance liquid chromatography (HPLC) (Parra et al. 1991). While HPLC appears to be the most practical method for the determination of car-

* Corresponding author. Tel. (315) 476-7461 (ext. 2447 or 2319); Fax: (315) 476-5348.

0165-0270/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0165-0270(94)00026-D

feine in tissue the existing procedures have sufficient complexity to prevent their ready incorporation into neurobehavioral experimentation (Kaplan et al., 1989). Furthermore, significant differences between plasma and brain levels have been reported (Stahle et al., 1991) indicating the importance of making both m e a - surements. The present method substantially simplifies the measurement by HPLC of caffeine concentrations in plasma and brain since a convenient me thano l /wa te r mobile phase can be used for both determinations. Employing this method, the present study indicates that significant differences can occur in caffeine con- centrations not only between plasma and brain but also between cortical and subcortical tissue.

2. Methods

2.1. An imals

Twenty-four male Sprague-Dawley rats weighing ap- proximately 500 g were used. The animals were main-

20 R.J. Carey, G. DePalma /Journal of Neuroscience Methods 53 (1994) 19-22

tained in individual wire cages at 22°C +__ 2°C using a (12-h) dark/light cycle. Food and water were provided ad libitum.

2. 2. Drugs

umn temperature was maintained at 28°C with a flow rate of 1 ml/min. The samples were detected with a Bioanalytical Systems (West Lafayette IN) variable wave length UV detector. The setting was 254 nm.

Anhydrous caffeine (Sigma, St. Louis, MO) was used. The caffeine solutions were prepared by dilution in sterile water. All caffeine injections were (i.p.).

2.3. Procedure

Three groups of rats were used. One group (n = 8) received saline, 1 group (n = 8) 10 mg/kg caffeine and the third group (n = 8) was given 50 mg/kg caffeine. Forty minutes after injection animals were placed in plastic restraint cones (Braintree Products, Braintree, MA) and killed by guillotine decapitation. Trunk blood was collected and, over ice, brain samples of frontal cortex and striatum were dissected. The neocortical tissue sample contains 1 mm x 2 mm medial prefrontal cortex sections from each hemisphere. The neostriatal samples contain bilateral samples of the caudate nu- cleus dorsal to the crossing of the anterior commissure (Carey, 1991).

2. 4. Sample preparation

2. 4.1. Plasma After the blood samples were centrifuged to sepa-

rate plasma from blood ceils, a solid-phase extraction procedure was followed to prepare the plasma sample for analysis. The extraction column was a C18 3-ml (500 mg) column. Under vacuum, the column was first conditioned with 2 x 3 ml of methanol followed by 2 x 3 ml of HPLC-grade water. Before the column could dry 1.5 to 2.0 ml of plasma (depending upon availability of sample) was passed through the column and this was immediately followed with 2 ml HPLG- grade water and then a 2 ml of HPLC-grade water/acetonitrile wash (90: 10). Next, the column was air-dried for 3 min. Finally, the sample was eluted with 2 x 5 ml methanol and then directly injected into the HPLC column.

2. 4. 2. Brain tissue The bilateral tissue samples were sonicated in 0.5 ml

of 0.1 M HCIO 4 for 30 s and then centrifuged. The supernatant was then filtered through 0.2 /xm 8 mm cellulose filters and injected directly into the HPLC column.

2.5. HPLC procedure

A 250 X 4.6 mm 5 ~m C18 column was used. A methanol/water (60:40) mobile phase was used. Col-

3. Results

Fig. 1 shows a chromatograph for a caffeine stan- dard (20 /xg/ml) and for a plasma sample of rats treated with either 10 or 50 mg/kg caffeine. As is evident in Fig. 1 there is a rapid elution of caffeine (3.6 m) and under the amplification conditions used the signals from all other chemical constituents of the plasma sample are attenuated such that only caffeine is evident. Sample concentrations were calculated using the measured peak heights which were converted to concentrations by means of a linear regression equa- tion derived from a standard curve shown in Fig. 2. Table 1 summarizes the results for the experimental treatments with regard to plasma and brain concentra- tion of caffeine for the 10 and 50 mg/kg treatment groups. The saline group is not represented since caf- feine was not detected in any plasma or brain samples. As can be seen in Table 1 the plasma concentrations revealed a nearly 5-fold difference in concentration between the dose levels. This result is congruent with the 5-fold difference in drug dosage. More importantly, however, the concentrations obtained from cortical and striatal brain samples revealed substantial differences. A statistical assessment of differences was performed using 1-way ANOVAs in conjunction with Duncan's multiple range test to determine differences between specific samples. For the cortex samples the concentra-

rD ~9

-

Column: 250ram x 4.6 mm minutes Packing: Biophase ODS, 5u Mobile Phase: 60% Methanol, 40% Water Flowrate: lmL/min Detector: 0.005 AUFS, UV 254 nm

Fig. 1. Representative chromatograms obtained from a caffeine standard (20 p~g/ml) and two plasma samples of rats injected with 10 and 50 mg/kg caffeine, respectively. The peak heights were recorded at a 0-100 mV sensitivity range. The calculations of caffeine plasma concentration need to be adjusted to plasma volumes. The injection deflection is made by a manually activated event marker.

R.J. Carey, G. DePalma /Journal of Neuroscience Methods 53 (1994) 19-22 21

200 -

y=5.3798x +.24324

150 -

.

5 10 15 20 25 30

conc. caffeine ug/rnl

Fig. 2. Standard curve obtained from caffeine peak heights generated by caffeine standard solutions.

tions of caffeine were comparable to plasma concentra- tions although for the 10 mg/kg group the cortex concentrations were significantly lower than the plasma concentration (p < 0.05). This difference between brain and plasma was further exaggerated in the striatal tissue samples for the 10 mg/kg group. The difference between the cortical and striatal samples was highly significant statistically (p < 0.01) and the concentration of the striatal tissue was only about 50% the level of the cortex. While a similar pattern was evident for the 50 mg/kg treatment group the concentration differen- tial between cortex and striatum was considerably less than for the 10 mg/kg group. As a validation of the tissue samples, dopamine concentrations were also measured for the two brain samples. As expected the striatal sample was rich in dopamine (9.2/~g/g vs. 0.1 /~g/g for the cortex). There were no statistical differ- ences in dopamine concentration among the three treatment groups.

Fig. 3 presents representative chromatographs for cortical and striatal brain samples in an animal treated with 50 mg/kg caffeine. The caffeine standard (1 /~g/ml in 0.1 M perchloric acid) is shown as a refer- ence. In saline rats there were no detectable peaks at this location in the chromatograms. Additionally, to directly evaluate whether caffeine detection was some- how differentially affected by cortical vs. striatal tissue

Table 1 Means and SEM of caffeine concentrations in plasma ( /zg/ml) and brain ( / zg /g wet tissue)

Treatment Plasma Cortex Striatum

Caffeine 10 m g / k g 5.895:0.17 * 4.585:0.54 ** 2.825:0.25 Caffeine 50 mg /kg 28.055:0.60 26.865:0.75 + 23.905:0.57

* P < .05, plasma vs. cortex. ** P < .01, striatum vs. cortex and striatum vs. plasma. + P < .05, striatum vs. cortex and striatum vs. plasma.

:e

J

v~

L)

Column: Packing: Mobile Phase: FIowrate: Detec to r :

o

250mm x 4.6 mm Biophase ODS, 5u 60% Methanol, 40% Water I mLJmin 0.005 A U F S , U V 254 n m

-o

minutes

Fig. 3. Representative chromatograms obtained from a caffeine standard (1 /~g/ml) in 0.1 M perchloric acid and two brain samples (cortex and striatum) of rats treated with 50 mg /kg caffeine. The peak heights were recorded at a 0-10 mV sensitivity range. The calculation of tissue concentration need to be adjusted to the tissue weight of the samples. The injection deflection is made by a manu- ally activated event marker.

samples caffeine standards were added to cortical and striatal samples in saline-treated rats. The standard curves for caffeine were identical for cortical and stri- atal samples thereby confirming that the neuroanatom- ical source of the tissue sample did not differentially affect the chromatographic measurement of caffeine.

4. Discussion

In the use of HPLC for the analysis of plasma, sample clean-up is the first critical step. In the present experiment a solid-phase extraction was used. For effi- ciency, a vacuum manifold as used in the present experiment is desirable but not necessary. It is possible to draw the solutions through the solid-phase column with a syringe and with an appropriate size tubing to connect the syringe to the column. One critical step in the present procedure is the solution used to wash the extraction column. We employed a variety of organic solvents and found that water followed by the acetoni- trile water solvent 10 : 90 provided the most satisfactory combination of sample clean-up and caffeine preserva- tion. That is, with this procedure no caffeine could be detected in the wash solution and caffeine recovery was greater than 99% but as the acetonitrile concentra- tion is increased above this level then caffeine begins to be eluted in the wash solution. Conversely, using lower concentrations of the organic solvent diminished the efficacy of the sample clean-up. Another consider-

22 R.J. Carey, G. DePalma /Journal of Neuroscience Methods 53 (1994) 19-22

ation is the volume of the plasma sample. While we used 0.5 -- 2.0 ml in the present experiment, we have found the method reliable for volumes of 0.25 ml. Thus, the present method would appear to be compati- ble with blood extractions in studies which indwelling catheters are used to collect blood samples.

As indicated in the results section the caffeine peak can be measured at an amplification level at which no other plasma constituents are present in the record. If a more sensitive setting is used then other peaks are evidenced including the methanol or perchloric acid peaks. Caffeine, however, is not co-eluted so that sub- stantially lower concentrations than were detected in the present study can be readily quantitated.

Although the primary topic of this report is the method, the observation of a substantial differential in the caffeine concentration in different brain structures is of importance to neurobehavioral assessments of caffeine effects. That is, attempts to relate CNS effects of caffeine to specific brain structures need to consider the possibility of differences in concentrations of caf- feine in different brain structures. A number of studies have been conducted (Lee et al., 1987; Kirch et al., 1990) in which caffeine induced alterations in brain neurotransmitters have been reported to have a non- homogenous distribution in brain tissue. Since the pre- sent findings also indicate that the caffeine concentra- tion in brain structures can differ substantially it then becomes necessary to perform concomitant measure- ments of caffeine and neurotransmitter concentrations in the same brain tissues. This combined determination is readily accomplished with the present method since the same tissue sample can be injected into either analytical system. The marked difference in caffeine concentration between cortex and striatum observed in the present study does not appear to be unique to caffeine, however, as we have recently found (Carey and Carrera, 1993) following L-3,4-dihyroxypheny- lalanine (L-dopa) treatment in rats that L-dopa is more highly concentrated in cortex than striatum. Thus, the present results, while not specific to caffeine, point to the importance of assaying drug concentrations in cor- tex as well as other brain areas in any attempt to relate drug effects to selected brain structures. Another im- portant topic in understanding the central effects of caffeine has been that of tolerance. Thus far, studies of the development of tolerance to caffeine have not considered the possibility of differential caffeine con- centrations in different brain structures. Since the con- centration relationships between brain areas may change with repeated caffeine treatment such a shift in the brain distribution of caffeine may be a relevant component to tolerance phenomenon.

Acknowledgement

This research was supported by NIDA Grant R01DA0536607.

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