Journal of Pharmaceutical and Biomedical Analysis 107 (2015) 217–222

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Analysis of amino acid neurotransmitters from rat and mouse spinal cords by liquid chromatography with fluorescence detection Nurullah S¸anlı a,∗ , Sarah E. Tague b , Craig Lunte c a b c

Faculty of Engineering, Us¸ak University, Us¸ak, 64100, Turkey Kansas Intellectual and Developmental Disabilities Research Center, University of Kansas Medical Center, KS, 66160, USA Ralph N. Adams Institute for Bioanalytical Chemistry, Department of Chemistry, University of Kansas, Lawrence, KS, 66045, USA

a r t i c l e

i n f o

Article history: Received 3 October 2014 Received in revised form 10 December 2014 Accepted 14 December 2014 Available online 19 December 2014 Keywords: Amino acid neurotransmitters Rat spinal cord Fluorescence detection 3-(4-carboxybenzoyl)-2quinolinecarboxaldehyde (CBQCA)

a b s t r a c t A RP-LC-FL detection method has been developed to identify and quantitate four amino acid neurotransmitters including glutamic acid, glycine, taurine and ␥-aminobutyric acid in rat and mouse spinal cord tissue. 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde (CBQCA) was employed for the derivatization of these neurotransmitters prior to RP-LC-FL analysis. Different parameters which influenced separation and derivatization were optimized. Under optimum conditions, linearity was achieved within the concentration ranges of 0.50–50.00 ␮M for all analytes with correlation coefficients from 0.9912 to 0.9997. The LODs ranged from 0.03 ␮M to 0.06 ␮M. The proposed method has been successfully applied to the determination of amino acid neurotransmitters in biological samples such as rat and mouse spinal cord with satisfactory recoveries. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Amino acids play a major role in energy metabolism, neurotransmission, and lipid transport and their quantitative analysis is increasingly important in disease diagnostics, and in elucidating nutritional influences on physiology [1]. Over the past several years, amino acid neurotransmitters have been the focus of much attention in biomedical research, medical diagnostics, clinical chemistry, and the pharmaceutical industry, because they play essential roles in control and regulation of various functions in the central and peripheral nervous system [2,3]. The most studied amino acid neurotransmitters are glutamic acid (Glu), ␥-aminobutyric acid (GABA), glycine (Gly) and taurine (Tau) (Fig. 1). As the major excitatory neurotransmitters in the mammalian central nervous system (CNS), Glu is present in more than half of all CNS synapses, which underscores their important involvement in learning, memory, sleep, movement, and feeding [4]. GABA, Gly, and Tau are inhibitory neurotransmitters in the CNS [5]. In fact, as many as 10–40% of nerve terminals in the hippocampus and cerebral cortex may use GABA as a neurotransmitter to transmit “closure” signals [6]. Gly,

∗ Corresponding author. Tel.: +90 276 2212121; fax: +90 276 2212121. E-mail addresses: [email protected], [email protected] (N. S¸anlı). http://dx.doi.org/10.1016/j.jpba.2014.12.024 0731-7085/© 2014 Elsevier B.V. All rights reserved.

plays key roles in postsynaptic inhibition, sensorimotor function, and abnormal startle responses. The inhibitory amino acid Tau is an osmoregulator and neuromodulator, and also exerts neuroprotective actions in neural tissue [7,8]. In addition, evidences showed that the changes in amino acid neurotransmitter levels in biological samples was correlated with a number of neurological diseases such as Alzheimer’s [9], Parkinson’s [10], stroke [11], epilepsy [12], and schizophrenia [13]. Hence, determination of these neurotransmitter levels in biological samples may provide a means of diagnosis and possible treatment of neuropsychiatric diseases. Moreover, the components of these biological samples are quite complex (from amino acids, peptides, to protein). It is therefore necessary to develop an improved, new, rapid, selective, accurate, precise, sensitive, fully validated technique for the determination of neurotransmitters in rat spinal cord tissues. There are multiple separation methods reported for these amino acid neurotransmitters including separation approaches such as gas chromatography (GC), high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) coupled with different detection techniques [14–16]. HPLC is commonly used for quantification, because these systems are widely commercially available and can be very robust. In recent years, several papers have reported the use of HPLC coupled with fluorescence detection (FLD) [17] or mass spectrometric (MS) detection [18] to resolve mixtures.

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Fig. 1. Chemical structures of studied compounds.

Derivatization (pre- or post-separation) is often necessary due to the lack of a chromophore for UV–vis and fluorescence detection [19,20]. The main advantage of manual derivatization is the relatively free choice of the reaction conditions. Most of the research on amino acids in biological samples has been performed with six different labeling agents: NDA (naphthalene dicarboxaldehyde) [21,22], OPA [23], FQ (3-(2-furoyl)quinoline-2-carboxaldehyde) [24], CBQCA (3(4-carboxybenzoyl)-2-quinolinecarboxaldehyde) [25], fluorescein derivatives as FITC (fluorescein-5-isothiocyanate) or CFSE (carboxyfluorescein succinimidyl ester) [26]. The CBQCA reagent is virtually nonfluorescent in aqueous solution; however, in the presence of cyanide, it reacts with primary

amines at room temperature such as those found in spinal cord to form highly fluorescent derivatives. The reaction scheme for derivatizing primary amines with CBQCA is given in Fig. 2. The high sensitivity, freedom from background and long-wavelength excitability makes this a potential reagent for research and biological sample applications. Compared to fluorescein based reagents, less interference in chromatograms can be expected because of the decreased number of side products and minimized effects of the excess reagent. This work describes, a rapid, selective, accurate, precise, sensitive, fully validated RP-LC-FL method to quantify the Glu, GABA, Tau, Gly in rat and mouse spinal cord tissues as well as its application to real sample analysis.

N. S¸anlı et al. / Journal of Pharmaceutical and Biomedical Analysis 107 (2015) 217–222

COOH O COOH N

CHO

+

R-NH2

CBQCA

CN-

N-R N

CN

Fluorophor

Fig. 2. Schematic illustration of the fluorogenic amine derivatization reagent CBQCA.

2. Materials and methods 2.1. Chemicals and reagents All chemicals and solvents were of analytical-reagent grade and used without further purification. CBQCA was purchased from Invitrogen (Molecular Probes, Eugene, OR, USA). Potassium cyanide (KCN) and ammonium acetate were purchased from Fluka (Buchs, Switzerland). Glu, GABA, Tau, Gly, 2-aminoadipic acid (2-AAP) (Fig. 1), sodium tetraborate, boric acid, and perchloric acid were purchased from Sigma (St Louis, MO, USA). Methanol (MeOH, HPLC grade) and tetrahydrofuran (THF) and dimethylsulfoxide (DMSO) were purchased from Fisher Scientific (Pittsburgh, PA, USA). Ultrapure water was obtained with a Milli-Q system (Millipore, Bedford, MA, USA). A 10 mM solution of CBQCA was prepared by dissolving 5 mg of CBQCA in 1.64 mL DMSO. Stock solutions of Glu, GABA, Tau, Gly were prepared by dissolving approximately 40 mM of studied compounds in 0.1 M perchloric acid. The stock solution of 10 mM 2-AAP was also prepared by dissolving it in 0.1 M perchloric acid and used as an internal standard. All reagents were protected from light. Prepared solutions were stored at 4 ◦ C for several days or at −20 ◦ C for long-term storage. CBQCA was kept at 4 ◦ C or below, the solution was allowed to warm to room temperature and mixed well before opening. Reaction buffer was prepared by adding 500 mM boric acid to 125 mM sodium tetraborate until pH 8.7. 2.2. HPLC equipment and conditions The LC analysis was carried out on a Shimadzu HPLC system with system controller (SLC- 10AVP, Tokyo, Japan) with a 10 ␮L loop (Rheodyne 7725-I, CA, USA) and a fluorescence detector (FLDShimadzu spectrofluorometric detector RF-10AXL, Tokyo, Japan), coupled to an LC-20 AD pump. The Synergi Hydro-RP analytical ˚ 150 mm × 2 mm × 4 ␮m) and guard column (Phencolumn (80 A, omenex, Torrance, CA, USA) were used as stationary phases at ambient temperature. An integrator (Shimadzu EZStart, V7.4) was used to analyze the chromatographic data. To separate the amino acid neurotransmitters and avoid interference from other concomitant amino acids at the same time, gradient elution was used. Before analysis, the column was preequilibrated for 30 min with the mobile phase. The mobile phase consisted of A: water-THF with 50 mM ammonium acetate buffer (95:5, v/v) and B: pure MeOH. The mobile phases were filtered through Fisher Scientific 0.22 ␮m nylon membrane filters and degassed with argon gas prior to use. Chromatographic analyses were performed at ambient temperature. A 10-␮L aliquot of the sample solution was injected into the chromatograph and the flow rate was 0.350 mL min−1 . The fluorescent detector was set at an excitation wavelength of 465 nm and an emission wavelength of 550 nm. After injection, the mobile phase was changed as follows: linear gradient change of mobile phase B to 5–30%

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for 0.0–8.0 min, then 70:30 (A:B) for 8.0–15.90 min, 40:60 (A:B) for 15.90–16.0 min, linear gradient change of mobile phase B to 60–5% for 16.0–17.5 min, then 95:5 (A:B) for 17.5–20.0 min for reequilibration of the column. 2.3. Animals Female Sprague–Dawley rats weighing 200–250 g were used in this study. The animals were housed in plastic cages, maintained on a 12:12 h light–dark cycle and fed ad libitum. They were decapitated, their spinal cords were removed and the tissues were separated and processed as described in Section 2.4. Care and use of animals in this study were done according to the National Institutes of Health Guide for Care and Use of Laboratory Animals [27]. Additionally, spinal cords were removed from 13-week-old male A/J mice, which were housed in forced-air ventilated cages on a 14:10 light–dark cycle and fed ad libitum. The mice were decapitated, the spinal columns were removed, and the spinal cords were forcefully ejected from the spinal column by injection of ice cold isotonic saline solution into the caudal end of the vertebral canal using a 3 mL syringe and a tight-fitting needle (18–23 gauge). The spinal cords were immediately frozen in a foil sleeve on dry ice and stored at −80 ◦ C. 2.4. Sample preparation procedures The spinal cords were rapidly removed, rinsed in saline (0.9% w/v sodium chloride), dried by blotting on tissue paper, weighed and immersed in liquid nitrogen, then placed in 1.5 mL microcentrifuge tubes and stored at −80 ◦ C until use. Approximately 0.1 g of sample was dissected and homogenized in 500 ␮L of 0.1 M perchloric acid–MeOH (50:50, v/v); vortexed for 3 min and placed in an ultrasonic bath for 5 min, and then centrifuged at 12,400 rpm for 15 min. Aliquots of the supernatants were stored at −20 ◦ C until derivatization with CBQCA for neurotransmitter analysis. 2.5. Derivatization procedure For simultaneous assay of the neurotransmitter amino acids Glu, Gly, Tau and GABA, RP-LC-FL detection was employed following derivatization with CBQCA using a modification of the product protocol [28]. The derivatization was performed by mixing 10 ␮L sample or standards solutions with 10 ␮M internal standard, 20 ␮L of 40 mM CBQCA in DMSO, 10 ␮L of 20 mM KCN and 260 ␮L reaction buffer (pH 8.7). The resulting solution was vortexed and analyzed after 1 h incubation in the dark at room temperature. 2.6. Analytic identification and quantification The neurotransmitter amino acids were identified by their characteristic retention times as determined by standard injections. Sample peak areas were measured through the integrator system and compared with the calibration curve standard in order to quantify the amino acids concentrations. 3. Results and discussion 3.1. Method development and validation The provided LC method presents a simple procedure to simultaneously determine the concentrations of Glu, Gly, Tau and GABA in comparison to the internal standard (IS), 2-AAP, in spinal cord tissues by fluorescence detection. The analytical process has difficulties due to the lack of chromophore group and the high polarity of the amino acids. Extremely polar analytes are not always retained

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and separated well on conventional C18 columns. The Synergi Hydro-RP column was selected and used successfully as a stationary phase for the simultaneous determination of the studied compounds. This column is a C18 bonded phase endcapped with a polar group to provide extreme retention of both hydrophobic as well as polar compounds under highly aqueous conditions. The high (475 m2 /g) 4 ␮m silica surface area combined with dense bonded phase coverage allows for substantial interaction between the sample analyte and the bonded phase. The obtained results are very retentive for C18 phase and well suited to separate polar and nonpolar analytes such as amino acid neurotransmitters. Most of the common methods used in the literature have more or less limitations. UV detection is not sensitive and selective for monoamines and helpless for amino acids. Electrochemical detection tends to lack reproducibility mainly because of hysteretic degradation of the electrode [29]. CE-LIFD is limited for analyzing monoamine neurotransmitters in biological fluids containing complex mixtures due to the similar electrophoretic behavior of these compounds [30]. LC–MS methods are more complex and expensive compare to the others. HPLC method with fluorescence detection with pre-column derivatization procedures is preferred because most groups of amino acids have weak luminescence. Also, fluorescence detection improves reproducibility, further sensitivity and achieves shorter analysis time. The pre-column derivatization also offers the advantage of increasing the hydrophobicity of the analytes sufficiently to retain on the reversed-phase stationary phase. Gradient elution was used to separate the compounds and avoid interference from other concomitant amino acids at the same time. To develop an efficient and reproducible method, different mobile phase compositions and ratios were employed. The order of elution of the four amino acid neurotransmitters derivatives and IS did not change substantially when the concentration of MeOH in the mobile phase was varied from 25, 35 to 70% (v/v). If the MeOH content was below 30% (v/v), the separation was found to be excessively long. Therefore, mostly 60% (v/v) mobile phase A and 30% (v/v) mobile phase B were used for this experiment as described in Section 2.2. In LC methods, precision and accuracy can often be enhanced by the use of an appropriate internal standard, which also serves to correct for fluctuations in the detector response. One of the main reasons for using an IS is for samples requiring significant pretreatment or preparation. Ideally, an IS should share similar physico-chemical properties with the analytes. Generally, sample preparation steps including reaction, filtration, precipitation, and extraction may cause unexpected results because of sample losses. When added prior to sample preparation, a properly chosen internal standard can be used to compensate for these sample losses. The IS is a different compound from the analytes, but one that is well resolved in the separation. 2-AAP was chosen as the IS because it showed a shorter retention time with better peak shape and better resolution. Also, the chemical structure of 2-AAP is similar to Glu (structures differ in only –CH2 – group).

Fig. 3. Representative chromatogram of CBQCA derivatives of amino acid neurotransmitters (a) standard mixture of studied compounds 1) Glu, 2) 2-AAP, 3) Gly, 4) Tau, 5) GABA; (b) blank sample; (c) rat spinal cord sample. Derivatization and RP-LC conditions are described in the Section 2.

Fig. 3 shows a typical chromatogram obtained for analysis of amino acid neurotransmitters derivatives of standard solutions of studied compounds (a), blank sample (b) and a rat spinal cord sample (c), respectively. As shown in Fig. 3, the substances were base line separated, forming well-shaped, symmetrical, single peaks, that were well separated from the peaks produced from the derivatization process. No interfering peaks were obtained in the chromatogram due to the derivatization process or sample matrix. Results obtained from the proposed method for the analysis of amino acid neurotransmitters derivatives in spinal cord samples indicate that the proposed technique can be used for simultaneous quantitation and routine analysis of these mixtures in spinal cord samples. The calibration curves and equations for compounds in standard solutions and biological samples were calculated by plotting the peak area ratio of compound to IS vs. concentration of compound in the range of 0.50–30.00 ␮M for Gly, Tau and GABA and 3.33–50.00 ␮M for Glu, respectively (Table 1). These results showed highly reproducible calibration curves with correlation coefficients of >0.99. The low standard error (SE) values of the slope and the intercept show the precision of the proposed method. The LOD and LOQ were calculated from the following equations and using the standard deviation (s) of response and the slope (m) of the corresponding calibration curve [31,32]. LOD =

3.3s ; m

LOQ =

10s m

(1)

Table 1 Statistical evaluation of the calibration data of Gly, Tau, GABA and Glu by RP-LC. Compounds

Glycine

Taurine

GABA

Glutamic acid

Linearity range/␮M (n = 6) Slope Intercept Correlation coefficient SE of slope SE of intercept Limit of detection (LOD) Limit of quantification (LOQ) Retention time (min) (n = 16) RSD% of retention time

0.50–30.00 0.891 0.405 0.996 0.039 0.601 0.031 0.095 9.52 1.38

0.50–30.00 1.092 −0.163 0.999 0.024 0.363 0.040 0.123 11.53 1.96

0.50–30.00 1.683 0.025 0.999 0.019 0.296 0.045 0.135 13.84 2.92

3.33–50.00 (n = 5) 0.065 0.181 0.998 0.002 0.064 0.059 0.179 2.97 1.82

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Table 2 Recovery of studied compounds from rat spinal cord samples.

Glutamic acid Glycine Taurine GABA

Sample concentration of amino acid (␮M)

Concentration of amino acid added (␮M)

Concentration of amino acid found (␮M)

%Recovery

15.1 20.9 6.2 4.6

13.3 13.3 13.3 13.3

29.0 34.2 19.2 17.5

104.5 100.0 97.7 97.0

Table 3 Glu, Gly, Tau and GABA levels in spinal cord tissue of (a) female Sprague–Dawley rat and (b) mouse. Concentration ␮M Glutamic acid Glycine Taurine GABA *

19.57 ± 0.48* ,(a) 21.93 ± 0.59(b) 16.14 ± 0.34 19.25 ± 0.17 6.05 ± 0.17 13.72 ± 0.10 5.37 ± 0.14 5.28 ± 0.10

% RSD

␮g

␮g/g tissue

2.4692.670

71.9980.65

574.061186.57

2.0770.866

30.2836.14

241.50531.64

2.8080.755

18.9242.91

150.86631.34

2.5741.863

13.8513.62

110.41200.31

Results are expressed as mean ± standard deviation (n = 5).

of the recovery analysis for all techniques are shown in Table 2. Table 2 shows that the recovery obtained from these amino acids was between 97.29% and 104.23%. 3.2. Spinal cord tissue levels of Glu, Gly, Tau and GABA Following the validation of the method, the levels of Glu, Gly, Tau and GABA in the female Sprague–Dawley rat and male A/J mouse spinal cord tissue were determined. Chromatograms of spinal cord samples of (a) rat and (b) mouse spiked with standards were shown in Fig. 4. The concentrations of neurotransmitters are given as ␮g/g of tissue. The results are expressed as the average of five replicates. The results are given in Table 3. 4. Conclusions

Fig. 4. Chromatogram of spinal cord sample of (A) rat and (B) mouse spiked with 13.33 ␮M of standards of CBQCA derivatives and 10 ␮M of 2-AAP (IS).

Recovery studies were determined from spinal cord samples for accuracy and precision of the proposed method. The recovery of the procedure was carried out by spiking the already analyzed samples at the moment of homogenization with the known concentrations of standard solutions of Glu, Gly, Tau and GABA. The results

A rapid, selective, accurate, precise, sensitive and validated determination of Glu, Gly, Tau and GABA in rat spinal cord tissue using CBQCA as the derivatization reagent was achieved in present study. Using the described chromatographic conditions, Glu, Gly, Tau, GABA and 2-AAP (IS) were well separated. The proposed method gives good resolution between selected compounds and IS within an analysis time of 20 min. The sample preparation step is easy and gives clean chromatogram without interferences. High percentage recovery shows that the method is free from the interferences of the endogenous substances in spinal cord tissues. The CBQCA reagent has proven useful for quantitating amines in solution, including the accessible amines in proteins. The CBQCA quantitation assay also functions well in the presence of substances, such as lipids, which interfere with many other protein determination methods. The method developed would serve as a versatile analytical tool suitable for the simultaneous analysis of these amino acid neurotransmitters with low LOD and LOQ values and would be of interest for clinical laboratories and investigators. Acknowledgment This work was supported by the Higher Education Council of Turkey (YOK).

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