Evaluation of reversed phase columns designed for polar compounds and porous graphitic carbon in “trapping” and separating neurotransmitters

Journal of Chromatography A, 1122 (2006) 97–104

Evaluation of reversed phase columns designed for polar compounds and porous graphitic carbon in “trapping” and separating neurotransmitters Didier Thi´ebaut a,∗ , J´erˆome Vial a , Monika Michel a,1 , Marie-Claire Hennion a , Tyge Greibrokk b a

b

Laboratoire Environnement et Chimie Analytique (CNRS, UMR 7121), ESPCI, 10 rue Vauquelin, 75005 Paris, France Analytical Chemistry Research Group, Department of Chemistry, University of Oslo, P.O. Box 1033, Blindern, N-0315 Oslo, Norway Received 26 October 2005; received in revised form 6 April 2006; accepted 18 April 2006 Available online 24 May 2006

Abstract Quantification of neurotransmitters as biologically active analytes in neurological samples is of high interest for studying their effect on multiple targets. This work is part of a strategy involving two-dimensional liquid chromatography (2D LC) system with mass spectrometry (MS) detection. The concept of the on-line LC system is the coupling of reversed phase liquid chromatography (RPLC, the second separation dimension) to ion-exchange chromatography (IEC, the first dimension). Our objective in this study is to find the appropriate second dimension column, ensuring that samples of neurotransmitters are refocused and separated on it. Silica-based columns designed specifically to retain polar compounds were tested in LC conditions and compared with results obtained with a porous graphitic carbon (PGC, Hypercarb) column. These polar embedded, polar endcapped, and high-density alkyl chain columns successfully separated analytes in question using mobile phase systems with high percentage of water, or even pure water. Only Hypercarb column provided efficient retention of the most polar neurotransmitters and could be used for trapping and preconcentrating the compounds without rapid breakthrough. © 2006 Elsevier B.V. All rights reserved. Keywords: Neurotransmitters; Stationary phases; LC; Porous graphitic carbon

1. Introduction The nervous system is the human body’s control and communication network. In some ways, neurons act like computers. That is, they receive messages, process those messages, and send out the results as new messages to other cells. In the case of neurons, the message consists of chemicals, named neurotransmitters, that interact with the outer surface of the cell membrane [1]. Biologists are on the verge of understanding the compounds that take part in the regulation of the response to stress, psychomotor activity, emotional processes, learning, sleep, and memory. There are many types of chemicals that act as neurotransmitter substances, such as amino acids, amines, and peptides. Some representatives of these categories, studied in this work, are shown in Table 1. The amino acid and amine neurotransmitters are all small organic molecules containing ionizable functional

∗ 1

Corresponding author. Tel.: +33 1 40 79 46 48; fax: +33 1 40 79 47 76. E-mail address: [email protected] (D. Thi´ebaut). On leave from Plant Protection Institute, Toru´n, Poland.

0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2006.04.074

groups such as carboxyl, amino, or phenol, so they exhibit acidic, basic, or both properties. Among them, noradrenaline and adrenaline, are polar compounds (log P = −1.24 for noradrenaline and log P = −1.37 for adrenaline). Due to high receptor specificity, highly potent and large effects are produced by only very small amount [2]. Accurate and specific measurements of these molecules in brain tissue, urine, plasma, serum, and cerebrospinal fluid samples have been used to develop effective treatment strategies for neuropsychiatric and neurodegenerative diseases and for the clinical diagnosis of pathologies, such as more known Alzheimer’s disease, Down’s syndrome, multiple sclerosis, Parkinson’s disease, and schizophrenia [3,4]. Owing to the low amount of neurotransmitters, the potential interferences with a large number of endogenous and exogenous compounds, the time-consuming analysis, and the tendency of the catechol group to be oxidized, measurement of these analytes presents numerous difficulties. A commonly accepted method of neurotransmitters analysis [5–8] in biological samples is reversed phase liquid chromatography (RP) in conjunction with ion-pairing reagents, required for retention of the more polar compounds, coupled with electrochemical

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Table 1 Structures, abbreviations (Abb), pKa , log P, and molecular weight (MW) values for the neurotransmitters used in this study

(ECD) [9–11], fluorescent [12], or UV [13–15] detection. A variety of mobile phases has been developed which provide good resolution for complex mixtures. These mobile phases usually contain one or more of the following components: methanol (MeOH) or acetonitrile (MeCN) as the organic modifier, and trifluoroacetic acid (TFA) or heptafluorobutyric acid (HFBA) as the ion-pair reagent [16,17]. Interest in testing the capability of MS in screening and determining the neurotransmitters has also increased recently. Especially, LC–MS with electrospray ionization (ESI) has demonstrated its potential in pharmaceutical and biomedical fields [18–22]. To our knowledge, the simultaneous determination of all the neurotransmitters of interest and their metabolites in complex biological samples remains to be achieved. This type of samples requires analytical methods of high resolving power in order to provide reliable analysis of the sample components. Thus, this work is part of a strategy involving 2D LC system with MS detection for the analysis of biological samples. This concept is based on the coupling of RP chromatography (the second separation dimension) to ion-exchange (IE) chromatography (the first dimension), this combination being in agreement

with the orthogonal approach set up by Giddings and co-workers [23–25] for multidimensional separations. Preconcentration and trapping of the compounds eluting from the first column is possible in the second dimension due to the fact that elution solvents in IEC are almost pure aqueous solvents, i.e. weak solvents for RPLC [26,27]. Thus, the choice of the RP stationary phase for the trapping column is of utmost importance in 2D LC because it must be compatible with highly aqueous conditions used in the first dimension to avoid collapse of RP chains in the aqueous environment [28–32] and poor wetting of the stationary phase which forces the mobile phase out of the pores [33]. The aim of this work is the investigation of second dimension RP column properties to ensure that neurotransmitters eluted from the first dimension are refocused and separated on it and keeping in mind that a multidimensional system would give higher selectivity in MS. Eight stationary phases including polar embedded, hydrophilic polar end capped (enhanced polar selectivity), high-density bonded alkyl, and “Aqua type” phases were selected and compared to PGC which meets a hydrophobic yet highly polarizable surface [34,35].

Interchim Agilent

Waters

Phenomenex

YMC

0–14 100 120 0.7 250 5 100 Hypercarb

NA: not available; ODS: octadecylsilane.

250 Kromasil KR 100-5C18

4.6

2–9.5 19 330 0.9 100 5

150 150 Uptisphere 5HDO (Aqua type) Zorbax SB-Aq (Aqua type)

4.6

NA 2–8 NA NA NA 180 NA NA NA 80 5 5

150 Atlantis dC18 (Aqua type)

4.6 4.6

2–7 12 320 NA 100 5

150 Synergi 4u Hydro-RP 80 (Aqua type)

4.6

1.5–7.5 19 475 1.05 80 4

150 YMC-Pack ODS Aq (Aqua type)

4.6

3.5

120

NA

300

14

2–6.5

Alkyl chain with polar embedded group (carbamate) Polar endcapped ODS Polar endcapped ODS Polar endcapped ODS ODS StableBond technology High-density endcapped ODS Porous graphitic carbon 2–8 17 340 NA 150 SymmetryShield RP18

Column ID (mm) Column length (mm) Column

Table 2 Characteristics of columns used in this study

The chromatographic apparatus consisted of a HP 1050 quaternary pump (Palo Alto, CA, USA), a Waters 715 Ultra WISP autosampler, and a Waters 2487 UV detector (Waters, SaintQuentin en Yvelines, France) set at 215 nm. Data were handled using CLASS-VP 4.2 Chromatography Laboratory Automated Software System (Shimadzu Scientific Instruments, Columbia, MD, USA). For identity confirmation, MS detection coupled with micro LC (1 mm column ID) was used. It was performed on a Thermo Finnigan LCQ ion trap (Thermo Finnigan, San Jose, CA, USA). The API source was operated in the positive ESI mode. A spray voltage of 4 kV and a capillary temperature of 150 ◦ C were employed. The nitrogen sheath and auxiliary gas flow rates were set at 30 and 0.4 arbitrary LCQ units, respectively. Separations were achieved on RP columns specifically designed to perform in highly aqueous solutions (Table 2). All columns were placed in an Alltech water jacket connected to a Bioblock 18205 water bath set at 25 ◦ C (±0.1 ◦ C, Fisher Bioblock Scientific, Illkirch, France). Mobile phases were different mixtures of MeOH or MeCN with aqueous solutions of TFA or HFBA. Prior to use, mobile phases were filtered through Millipore GV type filters (47 mm diameter, 0.22 ␮m pore size; Millipore, Molsheim, France), degassed by sonication (Transsonic T-460, Elma GmbH, Sin-

Particle size (␮m)

2.3. Apparatus and chromatographic conditions

3

Pore size ˚ (A)

Pore volume (mL/g)

Surface area (m2 /g)

Compounds of interest were not stable in solution. They were sensitive to light, oxygen, temperature, and extremely high or low pH. Consequently, special care had to be taken during preparation of solutions, analysis, and storage [36–39]. Stable stock solutions of neurotransmitters were prepared by dissolving the individual compounds in 0.05 M HCl at a concentration between 1000 ␮g/mL and 2000 ␮g/mL. They were kept frozen in darkness at −20 ◦ C and aliquots were diluted with ultrapure water to appropriate concentration (10–100 ␮g/mL) and used within one day. Ammonium acetate buffer was prepared by weighing 3.85 g of acetate ammonium salt; the solution was adjusted to pH 9.2 using concentrated sodium hydroxide.

100

2.2. Standard solutions

3.5

Carbon content (%)

pH range

Nature of bonding

All the neurotransmitters of interest, listed in Table 1, were purchased from Sigma (St. Quentin Fallavier, France). Dissociation constants (pKa ) and partition coefficients (log P) were obtained from Beilstein and internet Database. HPLC-grade MeCN and MeOH were obtained from Carlo Erba (Val de Reuil, France). TFA, HFBA were purchased from Sigma. Concentrated hydrochloric acid (HCl, 37%, w/w) and ammonium acetate (AA) were from Merck (Darmstadt, Germany). Ultrapure water (H2 O, 18.2 M
) was deionized with a MilliQ system from Millipore (Milford, MA, USA). Unless otherwise stated, all reagents were of analytical quality.

4.6

Supplier

2.1. Chemicals and reagents

Waters

2. Experimental

Thermo Electron Corp.

99 Eka Chemicals

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gen, Germany). At the end of each chromatographic session, the columns were washed for 15 min with deionized water and stored with the solvent advised by the manufacturer. The flow rate was 0.5 mL/min and the samples were injected by means of a 10 ␮L loop. 3. Results and discussion The chromatographic behavior of the selected neurotransmitters on PGC and on seven ODS stationary phase mainly designed for polar compounds was evaluated, discussed, and compared. 3.1. Chromatographic behavior of neurotransmitters on PGC The ionic nature of compounds of interest made them suitable candidates for analysis by ion-pairing chromatography. In preliminary experiments using PGC, isocratic elution with mobile phases prepared from different ratios of MeOH and MeCN in water with TFA, was only achieved for small neurotransmitters including dipeptides while the neuroactive peptides remained on the column and could not be studied. This observation is consistent with the strong interactions observed on PGC, either by hydrophobic effects [40] or by dipolar and ionic interactions [34]. Fig. 1 shows the plots of k as a function of percent of MeOH in mobile phase with TFA or HFBA, for compounds that could be eluted. As expected, the more hydrophobic the ion-pairing reagent is (HFBA), the greater the retention of the analytes. Increasing TFA concentration from 10 mM to 100 mM increased the retention of the neurotransmitters; further increase of the concentration of TFA did not affect the retention. Acceptable separations were observed for MeOH content in the range 65–75% for all the compounds except for adrenaline and noradrenaline which were unretained. Below 65% of MeOH retention became far too high except for adrenaline and noradrenaline. Fig. 2(A and B) shows the effect on the k values of the neurotransmitters of MeOH and MeCN concentration as organic modifier in mobile phases containing 0.1% TFA. According to what is described in the literature [29–32,41–43] the observed elution strengths of MeOH and MeCN were different: The magnitude of the difference was rather high as retention factors were between 0.6–15.6 and 0.3–1.0 for 40% MeOH and MeCN, respectively. A slight variation in the percentage of organic solvent induced a dramatic variation of the retention. As an example, varying the percentage of MeCN from 20% to 10% can change k value from 0 to more than 20 for tyrosine. A similar behavior has recently been described by Cherkaoui et al. [44] for the separation of acarbose. The coeluting NOR and ADR began to separate below 10% of MeCN. An alternative to ion pairing is to work at high pH to take profit of the differential ionizations of the solutes. When the pH of the mobile phase was changed from 2.5 to 9, as shown in Fig. 2(B and C), using 50 mM AA buffer instead of TFA in MeCN/H2 O, slight retention for NOR and ADR was observed at 20% of MeCN. At 10% of MeCN, NOR and ADR were completely resolved giving better results compared to pH 2.5 for these two compounds. Differential and reduced ioniza-

Fig. 1. Retention factor k vs. percentage of MeOH with (A) TFA and (B) HFBA as ion-pair reagents in mobile phase and PGC as stationary phase.

tion at high pH is an alternative to ion pairing; the main drawback of this approach is that stability of the compounds became questionable in basic conditions. 3.2. Chromatographic behavior of neurotransmitters on RP columns The following RP columns selected for neurotransmitters analysis could be separated into four distinct groups: (1) polar embedded: Symmetry Shield RP18; (2) polar endcapped: YMC ODS Aq, Synergi Hydro RP, Atlantis dC18; (3) high-density bonding: Kromasil C18 and (4) others: Uptisphere 5HDO, Zorbax SB-Aq. The fourth group corresponds to columns for which little or no information is available: Uptisphere 5HDO is only known to have C18 bonding; for Zorbax SB-Aq it is only known that it profits from the StableBond technology based on steric hindrance at the bottom of the alkyl chain. In the analysis of ionizable substances, the pH of the eluent will affect the degree of ionization of both the analytes and of residual silanols present on the surface of the stationary phase. Therefore, the pH will influence the retention through ionic interactions between the analytes and the stationary phase. For separation purpose, the composition of the mobile phase

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Fig. 3. Retention factors of eight neurotransmitters on the evaluated columns: (A) MeCN/H2 O + 0.1% TFA (5:95, v/v, %) and (B) H2 O + 0.1% TFA as mobile phase.

Fig. 2. Retention factor k vs. percentage of (A) MeOH, pH 2.5; (B) MeCN, pH 2.5 and (C) MeCN, pH 9 as organic modifier in mobile phase and PGC as stationary phase.

was selected in such a way that all the neurotransmitters were resolved in the shortest analysis time. It has been observed that retention of neurotransmitters was possible only with a highly aqueous mobile phase with 0.1% of TFA (26.5 mM) at pH 2.5. The percentages of water in mobile phases with MeCN

as organic modifier were 90%, 95%, and 100%. For the sake of simplicity, isocratic elution with constant temperature was used. Fig. 3 shows the retention of the eight neurotransmitters on the considered stationary phases using 95% and 100% of water in the mobile phase (data obtained using 90% are not presented owing to insufficient retention). Results on the Hypercarb (PGC) column are also given for comparison. Data corresponding to retention factor higher than 20 are not presented. The polar embedded phase Symmetry Shield RP18 produced a good separation of analytes in mobile phase with 95% of water. It could be linked to the presence of carbamate polar functionalities within the alkyl graft. Nevertheless, overall retention was rather low with retention factors between 0.3 and 4.5. The same behavior was observed for YMC, Zorbax, and “Aqua type” columns, specially designed for high aqueous environment. A significant increase of retention was observed when switching to pure water mobile phases: it was more pronounced for phases belonging to groups, 3 and 4 (Uptisphere only). The Zorbax column presented a peculiar behavior for all compounds compared to the other stationary phases; no information was provided by the column manufacturer; the experimental results seem to indicate a rather different bonding chemistry than the other stationary phases investigated; as they involve polar endcapping or embedded polar group, one may suppose that no polar functionality is involved in Zorbax SB-Aq retention mechanism and another approach has been used by the manufacturer to provide improved retention. Synergi Hydro-RP, Atlantis dC18, and Uptisphere 5HDO showed very similar behavior in all the mobile phases tested. They required TFA to improve the peak shape, and good

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separations were achieved using both MeOH and MeCN in the mobile phase. This effect, as suggested by Walter et al. [33], is related to the enhanced wettability of the stationary phase owing to the decrease of surface tension by the acidic additive. This behavior could indicate that the Uptisphere 5HDO presents very similar treatment strategy towards polar compounds, probably a polar endcapping. Very surprisingly, Kromasil C18, the “conventional” ODS phase with high-density alkyl chains, appeared to be a potential alternative for the separation owing to the retention of polar compounds. For mobile phase containing 95% and 100% of water, k values were between 0.7–9.8 and 0.9–18.3, respectively. In the case of aqueous conditions, no elution of serotonin, gly-tyr, and lys-phe was observed after 60 min of analysis. These results should be used with caution because unlike the other columns, the Kromasil has not been designed for purely aqueous conditions; it should be checked that neither collapse nor dewetting of the pores [33] phenomena could degrade the observed performances in long-term use. The separation of NOR and ADR was only possible in pure aqueous conditions with Synergy hydro RP, Atlantis, and Upti-

sphere columns. For all seven columns tested, retention factors did not exceed a value of 2.5 for ADR and of 1.5 for NOR whatever the mobile phase composition. Thus, retention is too low to expect efficient trapping of NOR and ADR on such phases. Fig. 4 shows representative chromatograms using polarembedded SymmetryShield RP18, polar-endcapped Atlantis dC18, and high-density ODS Kromasil C18 columns. Mobile phases are described in the caption of Fig. 4. 3.3. Comparison of ODS and PGC columns Retention on PGC was significantly higher (k  2) in all tested conditions. Fig. 5 shows representative chromatograms for PGC column (Hypercarb) obtained with 5% and 10% of MeCN in the mobile phase. No elution of analytes was observed on PGC using only aqueous mobile phase with TFA for 60 min. Thus, only PGC could allow efficient trapping of the most polar neurotransmitters, NOR and ADR, without rapid breakthrough of the compounds (using 5% MeCN in the solution to be percolated for trapping of catecholamines, the breakthrough volume

Fig. 4. Representative chromatograms obtained on three C18 columns of different grafting chemistry: (A) MeCN/H2 O + 0.1% TFA (5:95, v/v, %) and (B) H2 O + 0.1% TFA as mobile phase.

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Table 3 Practical hints for trapping and separating neurotransmitters using tested columns Columns

Process/conditions Trapping

SymmetryShield RP18 YMC-Pack ODS Aq Synergi 4u Hydro-RP 80 Atlantis dC18 Uptisphere 5HDO Zorbax SB-Aq Kromasil KR 100-5C18 Hypercarb

Separating

MeCN/H2 O + 0.1% TFA (10:90, v/v, %)

MeCN/H2 O + 0.1% TFA (5:95, v/v, %)

MeCN/H2 O + 0.1% TFA (0:100, v/v, %)

MeCN/H2 O + 0.1% TFA (10:90, v/v, %)

MeCN/H2 O + 0.1% TFA (5:95, v/v, %)

MeCN/H2 O + 0.1% TFA (0:100, v/v, %)

+ + + + + + + ++

+ + + + + + ++ ++

+ + ++ ++ ++ + ++ ++

− − − − − − + −

− − + + + − ++ −

+++ + + + + − +/++ −

++: excellent; +: good; −: poor.

of NOR should be more than 7.5 mL; using 100% water, the breakthrough volume would exceed 30 mL). The elution order of eight neurotransmitters on ODS stationary phases tested and PGC were only the same for the first three eluting compounds, NOR, ADR, and DOP. For other compounds, elution order was DOPA, TYR, SER, GT, LP, and TYR, GT, DOPA, SER for ODS and PGC, respectively. As the elution order was the same on all the tested ODS phases, the difference in the neurotransmitters elution order between ODS and PGC suggested that the molecular interactions determining solute retention are quite different for the two materials for these compounds as expected from the literature. This difference is believed to be associated with the sensitivity of PGC towards the number of contact points or total contact area of the solutes with the surface of PGC and/or electronic

interactions [34]. On the other hand, PGC has the disadvantage of being very retentive for higher molecular mass solutes that may not elute from the column. Neuropeptides, for example, are strongly retained even in strong elution conditions (tetrahydrofuran). High retention was also observed for lys-phe eluted as a broad peak with strong tailing at 34.5 min using MeOH/H2 O (90:10, v/v) with 0.1% TFA as the mobile phase operated at 1.5 mL/min flow rate (chromatogram is not presented). 3.3.1. Practical considerations As summary, Table 3 presents practical hints concerning stationary phases used in highly aqueous media for “trapping” and/or “separating” purpose in the second dimension of a 2D LC system. It could be used to (1) identify equivalent phases; (2) select columns of widely differing characteristics; and (3) as assistance in the rational selection of suitable stationary phases. 4. Conclusions Owing to its high retention towards polar compounds, PGC is the best choice for the trapping of very polar neurotransmitters at highly aqueous conditions for eventual implementation in a multidimensional system. However, PGC is not the best choice for separating a complex mixture of neurotransmitters owing to the strong retention of most of the compounds: for separating a wide range of transmitters, special C18 columns should be preferred using a elution gradient. In further studies, HILIC column (bare silica used with hydroorganic eluent) as trapping support of neurotransmitters should be investigated. Micro columns should be used for the final implementation in a multidimensional system coupled to MS [14]. Acknowledgements

Fig. 5. PGC Representative chromatograms of ADR, NOR, and DOP using: (A) MeCN/H2 O + 0.1% TFA (10:90, v/v, %) and (B) MeCN/H2 O + 0.1% TFA (5:95, v/v, %) as mobile phase.

This research was supported by the COM-CHROM project no. HPRN-CT-2001-00180 Training young researchers in miniaturized comprehensive liquid chromatography in the Commission of European Communities 5th framework program.

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