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Capillary electrophoresis-mass spectrometry using an in-line sol–gel concentrator for the determination of methionine enkephalin in cerebrospinal fluid
Talanta 78 (2009) 638–642
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Talanta journal homepage: www.elsevier.com/locate/talanta
Capillary electrophoresis-mass spectrometry using an in-line sol–gel concentrator for the determination of methionine enkephalin in cerebrospinal fluid R. Ramautar a,∗ , C.K. Ratnayake b , G.W. Somsen a , G.J. de Jong a a b
Department of Biomedical Analysis, Utrecht University, P.O. Box 80082, 3508 TB Utrecht, The Netherlands Beckman Coulter, Inc., P.O. Box 3100, Fullerton, CA 92834-3100, USA
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Article history: Received 20 August 2008 Received in revised form 4 December 2008 Accepted 11 December 2008 Available online 24 December 2008 Keywords: Capillary electrophoresis-mass spectrometry Sol–gel concentrator Preconcentration Cerebrospinal fluid Methionine enkephalin
a b s t r a c t In this study, a CE-MS method using a monolithic sol–gel concentrator for in-line solid-phase extraction (SPE) is evaluated for the analysis of methionine enkephalin in biological samples. Operational SPE parameters such as sample pH, loading volume, elution volume and composition have been studied. After optimization of the in-line preconcentration methodology, a 40-fold preconcentration was demonstrated for a methionine enkephalin test solution using a loading volume of 3200 nL. The method was linear in the range from 62.5 to 1000 ng/mL (R2 > 0.99). R.S.D. values for migration times and peak areas were 1.2% and 8.4%, respectively. Finally, the analysis of cerebrospinal fluid samples spiked with methionine enkephalin and deproteinized with perchloric acid (1:1, v/v) showed a detection limit (S/N = 3) of approximately 1 ng/mL (ca. 5 nM). The recoveries of methionine enkephalin for three concentration levels (100, 10 and 1 ng/mL) were in the range of 74–91%, demonstrating the promising potential of the methodology for the analysis of biological samples. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Capillary electrophoresis (CE) is an efficient separation technique for a wide variety of analytes. However, for the analysis of biological samples CE suffers from an inherent low concentration sensitivity due to the limited sample volume that can be loaded onto the capillary. Several strategies can be used to improve the concentration sensitivity in CE [1,2]. In general, analyte preconcentration via electrophoresis-based or chromatography-based methods is used to improve the concentration sensitivity in CE [3,4]. Chromatography-based methods can offer two to four orders of preconcentration [5–8], and therefore are attractive to improve the concentration-sensitivity in CE. Typically, chromatographic preconcentration is carried out either in an off-line, on-line, or in-line mode [9]. In the on-line mode, the precolumn is not part of the CE system and the coupling of the precolumn is performed via an interface [10]. In the in-line mode, the preconcentration column is an integrated part of the CE system. When using these chromatographic techniques, on-line or in-line methods are regarded as advantageous compared to the off-line approach as a result of their shorter total analysis times, minimum of sample handling and possibility of automation. The main advantage of the
∗ Corresponding author. Fax: +31 30 253 5180. E-mail address: [email protected] (R. Ramautar). 0039-9140/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2008.12.025
in-line approach is that the entire preconcentrated amount of analyte can be transferred to the separation section of the CE capillary [9]. However, a disadvantage is that sample matrix components also pass through the separation capillary and may interfere with the CE analysis or they can adsorb onto the capillary wall, which can result in poor separations. Nevertheless, the in-line approach has the highest level of integration [9]. Various in-line SPE-CE approaches have been described such as an open-tubular capillary coated with a sorbent [11], a small (1–2 mm) packed section containing microsphere beads and retained by frits at the inlet of the capillary [12,13], and an immobilized particle-loaded membrane [14]. In the open-tubular configuration, the capacity is relatively low to load sufficient sample [15]. While packed SPE modules offer a relatively larger capacity, the introduction of a small packed section into the capillary can increase the back pressure, and rinsing the capillary using conventional CE instrumentation with low pressures can be problematic [15]. Moreover, the preparation of packed SPE modules in narrow bore capillaries can be difficult, especially with respect to the frit formation [15]. An alternative approach is the use of in situ formed monolithic materials [16]. By using UV-initiated polymerization, the monolithic SPE column can be made directly in the capillary. No frits are required as monolithic columns consist of a continuous piece of a highly porous microstructure. Monolithic columns can be used at high flow rates, thus minimizing sample loading time and backpres-
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sure. There are two types of monolithic columns, i.e. silica-based and polymer-based monoliths. Silica monoliths are prepared using sol–gel technology whereas polymer-based monoliths are made by in situ polymerization of monomers and cross-linkers [17]. Photopolymerized sol–gel monoliths have been used as a SPE sorbent and stationary phase in capillary electrochromatography for the preconcentration and separation of peptides [17–19]. However, sol–gel monoliths as preconcentration sorbents are still not used widely for in-line SPE-CE. So far, a few papers have described the applicability of monolithic columns for in-line SPE-CE to preconcentrate compounds. For instance, an in-line SPE-CE method using a monolithic methacrylate polymer was used for the preconcentration of Spropranolol from aqueous solutions. A limit of detection (LOD) in the low nanomolar range was achieved for S-propranolol with this method [20]. In another study, a polymeric monolithic SPE column was prepared in situ within a fused silica capillary from butyl methacrylate-co-ethylene dimethacrylate [15]. Using a 1 cm SPE column placed at the inlet of the capillary, the compounds sertraline, fluoxetine and fluvoxamine were extracted from aqueous solutions by applying a pressure rinse. Enrichment factors of over 500 were achieved for the compounds of interest. A sulphopropyl methacrylate monolith was used to preconcentrate amino acids from standard solutions using inline SPE-CE [21]. Monolithic columns have also been used for the preconcentration of inorganic anions using in-line SPE-CE [22]. A poly(butyl methacrylate-co-ethylene dimethacrylate-co-2acrylamido-2-methyl-1-propanesulfonic acid) monolithic column with cation-exchange sites was used for that purpose. An in-line SPE-CE time-of-flight mass spectrometry (TOF-MS) method using a silica-based monolith was developed for the determination of escitalopram in human urine [23]. The limit of detection (LOD) for escitalopram was 10 pg/mL obtained with a loading volume of approximately 2.25 L. The intraday precision of the escitalopram peak area was less than 6.3%. In the present paper the development and optimization of an in-line SPE-CE-MS method based on a silica-based monolithic sol–gel concentrator for the determination of methionine enkephalin in cerebrospinal fluid (CSF) is described. The main goal of this study was the evaluation of the feasibility of silicabased monolithic sol–gel capillaries for the analysis of CSF samples. The paper describes the systematic optimization of experimental parameters of the sol–gel concentrator, such as loading time, elution volume and composition of the elution solvent. Subsequently, repeatability of migration time and peak area, linearity and LODs have been determined. Finally, the potential of the in-line SPE-CE-ESI-MS method for CSF analysis is assessed. 2. Experimental 2.1. Chemicals Ammonium acetate, sodium hydroxide, perchloric acid and acetonitrile (all analytical grade or higher) were purchased from Merck (Darmstadt, Germany) and Biosolve BV (Valkenswaard, The Netherlands), respectively. Acetic acid (analytical grade) and acetate salts of Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu) and [Met5 ]-enkephalin (Tyr-Gly-Gly-Phe-Met) were obtained from Sigma–Aldrich (St. Louis, MO, USA). Human CSF was generously donated by the Utrecht Medical Centre (Utrecht, The Netherlands). Deionized water from a Milli-Q system (Millipore, Bedford, MA, USA) was used for all solutions. The background electrolyte (BGE) in all CE experiments was 5 mM ammonium acetate adjusted to pH 3.6 with acetic acid. The peptides were dissolved in BGE, unless otherwise stated.
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2.2. Preparation of sol–gel capillaries Sol–gel capillaries were obtained from Beckman Coulter (Fullerton, CA, USA). The monolithic sol–gel sorbent was prepared by mixing 500 L ethanol and 600 L tetraethoxysilane. Subsequently, 800 L of 1 M nitric acid was added to initiate hydrolysis and condensation. Then reversed phase silica particles (Beckman Coulter, Fullerton, CA, USA) were added to make a slurry. Bare fused silica capillaries were filled with this slurry to obtain an about 3–5 cm long plug which was heated to 120 ◦ C for 48 h. Finally, the capillary was cut to obtain a monolithic sol–gel plug of 5 mm at the inlet of the capillary.
2.3. Instrumentation and procedures CE-UV experiments were performed using a Beckman Coulter (Fullerton, CA, USA) PA 800 CE instrument equipped with a diode array detector (DAD). Capillaries with internal diameter (ID) of 50 m were from Composite Metal Services (The Chase, Hallow, UK). The sol–gel capillaries were flushed with acetonitrile (15 min) and 80% acetonitrile in BGE (10 min) prior to use. Sol–gel capillaries had a total length of 60 cm and effective length of 50 cm. Samples were injected for 5 min at 25 p.s.i. (injection volume of ca. 400 nL), unless otherwise stated, and the separation voltage was 30 kV. The capillary was thermostated at 20 ◦ C and detection was carried out at 200 nm with a data acquisition rate of 16 Hz. CE-ESIMS experiments were conducted using a PrinCE CE system from Prince Technologies B.V. (Emmen, The Netherlands) using 50 m ID sol–gel capillaries with a length of 90 cm. During sample injection, the nebulizer gas flow and the electrospray voltage of the CE-MS interface were turned off. CE was coupled to an Agilent Technologies 1100 Series LC/MSD SL IT mass spectrometer (Waldbronn, Germany) via a coaxial sheath-flow electrospray interface (Agilent). The CE capillary outlet was positioned at 0.2–0.5 mm from the tip of the interface. A sheath liquid of methanol–water–acetic acid (50:50:0.1, v/v/v) was supplied by a syringe pump at a flow rate of 10 L/min. The nebulizer-gas pressure was 10 p.s.i., and the flow and temperature of the drying gas were 4 L/min and 300 ◦ C, respectively. The electrospray voltage was 4.5 kV. In the Agilent CEMS set-up, the spray needle is grounded. MS detection was carried out in the positive ion mode and the scan range was 200–1200 m/z. All sol–gel capillaries for in-line SPE-CE-MS were first conditioned by consecutive flushes of acetonitrile (10 min), 80% acetonitrile in BGE (10 min) and BGE (10 min) at 25 p.s.i. Dilute samples of methionine enkephalin in BGE were hydrodynamically injected at 25 p.s.i. for 40 min (unless otherwise stated). A washing rinse with the BGE at 25 p.s.i. for 20 min was applied before the elution of methionine enkephalin. Elution was performed by injecting a solution of 80:20 (v/v) acetonitrile–BGE (apparent pH 3.6) at 10 p.s.i. for 0.5 min (ca. 50 nL). Separation was carried out applying a voltage of 30 kV (normal polarity). Between runs, the capillary was rinsed for 5 min with a solution of 80:20 (v/v) acetonitrile–BGE and BGE, in order to avoid carry-over between consecutive analyses.
2.4. Sample preparation The off-line pretreatment of blank and spiked CSF samples consisted of a protein precipitation step. This was carried out with 0.1 M perchloric acid in a ratio of 1:1 (v/v) (100 L CSF and 100 L perchloric acid). The supernatant was centrifuged (5 min at 9447 × g). Subsequently, the samples were hydrodynamically introduced at 25 p.s.i. for 40 min.
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2.5. Evaluation of analytical parameters The analytical parameters were calculated from data obtained by measuring the peak area and migration time from the extracted ion electropherogram of methionine enkephalin. The m/z of the molecular ion for leucine enkephalin was 556.0 and for methionine enkephalin 574.4. Repeatability studies (n = 5) were performed with methionine enkephalin dissolved in CSF at a concentration of 100 ng/mL. LOD (S/N = 3) for methionine enkephalin was obtained analyzing methionine enkephalin solutions of 10 ng/mL and 1 ng/mL. The linearity range was determined by injecting five concentrations in the range of 62.5–1000 ng/mL in BGE. Recoveries of methionine enkephalin spiked into CSF (at three concentration levels: 100, 10 and 1 ng/mL) were calculated using the calibration curve constructed for methionine enkephalin standards (62.5–1000 ng/mL range). 3. Results and discussion 3.1. SPE-CE optimization A preliminary study was performed to find a suitable BGE for enkephalin analysis. For BGE optimization, two closely related peptides, i.e. leucine enkephalin and methionine enkephalin, were used. Ammonium acetate at four different pH values (2.5, 3.1, 3.6 and 4.0) was investigated as it is a volatile BGE. With a BGE of 5 mM ammonium acetate (pH 3.6), a baseline separation for the two peptides was obtained (Fig. 1). Additionally, this low-ionic-strength BGE is well compatible with ESI-MS. Therefore, for the in-line SPE-CE-MS experiments, 5 mM ammonium acetate (pH 3.6) was chosen as BGE. The optimum composition of the sheath liquid was determined by pressure-induced infusion of a solution of methionine enkephalin in BGE (1 g/mL) using mixtures of methanol–water and acetonitrile–water containing 0.1% acetic acid. The optimum sheath liquid composition was methanol–water–acetic acid (50:50:0.1, v/v/v). The sheath liquid flow rate was optimized in the range from 2.5 to 12.5 L/min and the highest signal intensity for methionine enkephalin was obtained at a flow rate of 10 L/min.
Fig. 1. Separation of leucine enkephalin from methionine enkephalin. (A) Extracted ion electropherogram of 100 g/mL methionine enkephalin. (B) Extracted ion electropherogram of 100 g/mL leucine enkephalin. BGE, 5 mM ammonium acetate (pH 3.6); capillary: 90 cm × 50 m i.d.; separation voltage, +30 kV; injection: 50 nL.
For optimization of the in-line SPE-CE-MS procedure, several parameters have to be considered in order to achieve optimum performance, i.e. the pH of the sample solution, the loading volume, elution volume and composition of the elution solvent. These parameters have been evaluated with methionine enkephalin as test compound. First, the pH of the injected sample solution was determined as this parameter will affect the retention of the analyte on the monolithic sol–gel concentrator. The pH of the injected sample solution was studied between pH 2.5 and 6.8 using 5 mM ammonium acetate. A loading volume of ca. 400 nL (loading time 5 min) and an elution volume of ca. 50 nL (80% acetonitrile in BGE) were used. The sample solution was introduced hydrodynamically into the in-line SPE-CE-MS system. In this way, the volume of sample introduced can be calculated, taking into account the flow-rate through the sol–gel capillary which can be determined via the mass loss of a buffer-filled vial at a certain pressure and time interval. Using pH values above 4.5 for sample loading resulted in high back pressures and also current drops upon voltage application. Similar peak areas for methionine enkephalin were obtained at pH values below 4.0. Therefore, a sample pH of 3.6 was selected as acetate has a good buffer capacity at this pH. Next, the loading capacity of the sol–gel concentrator was evaluated using in-line SPE-CE-MS. The loading capacity of the monolithic sol–gel concentrator was determined by loading different volumes of a methionine enkephalin standard solution of 1000 ng/mL. In order to increase the speed of the analysis a loading pressure of 25 p.s.i. was chosen. Fig. 2A shows the effect of loading volume (i.e. loading time) on the peak area for methionine enkephalin. A linear relation of peak area versus loading volume could be observed up to a loading volume of ca. 3200 nL (40 min). The deviation from linearity above a loading volume of ca. 3200 nL indicated that the capacity of the sol–gel concentrator was exceeded after 40 min for methionine enkephalin. Therefore, for the preconcentration of methionine enkephalin, a loading volume of ca. 3200 nL was selected. The influence of the composition of the elution solvent on the desorption of methionine enkephalin (1000 ng/mL) was studied by in-line SPE-CE-UV using a loading volume of 1000 nL and an elution volume of ca. 50 nL. The percentage of acetonitrile in the BGE was varied from 20 to 80%. The amount of methionine enkephalin desorption increased with a higher concentration of acetonitrile. A percentage of 80% acetonitrile in the BGE was required for complete desorption of methionine enkephalin. Subsequently, optimization of the elution volume (acetonitrile/BGE, 8/2, v/v) was determined by loading 1000 nL of 1000 ng/mL methionine enkephalin onto the monolithic sol–gel concentrator while increasing the elution volume (ca. 13, 25, 50 and 100 nL). Fig. 2B shows that the peak area of methionine enkephalin increased with the elution volume until ca. 50 nL. Above this volume the peak area remained constant. Therefore, an elution volume of ca. 50 nL was used for desorption. On-line preconcentration using the sol–gel concentrator was evaluated with methionine enkephalin as test compound. When a 5000 ng/mL methionine enkephalin sample was loaded for 1 min (80 nL) a peak with an intensity of ca. 4.2 × 106 counts was observed (Fig. 3A). When a 100 ng/mL methionine enkephalin sample was loaded for 40 min (3200 nL) a peak with an intensity of ca. 3.4 × 106 counts was observed (Fig. 3B). The ratio of the peak intensities (1.23) corresponds well to the ratio of the amount of analyte loaded on the sol–gel concentrator (i.e., 0.40 ng/0.32 ng = 1.25). As a result, the 100 ng/mL methionine enkephalin sample was preconcentrated by a factor of 40. Using methionine enkephalin concentrations in the range of 62.5–1000 ng/mL, peak area (y) versus concentration (x) regression line was calculated (n = 5). Linearity was observed over the range between 62.5–1000 ng/mL (R2 > 0.995) and the equation is y = 12,471x + 56,127. Using an injection volume of ca. 3200 nL, the
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Fig. 3. On-line preconcentration of methionine enkephalin using the sol–gel concentrator. (A) Extracted ion electropherogram of an injection of 80 nL of 5000 ng/mL methionine enkephalin. (B) Extracted ion electropherogram of an injection of 3200 nL of 100 ng/mL methionine enkephalin. BGE, 5 mM ammonium acetate (pH 3.6); capillary: 90 cm × 50 m i.d.; separation voltage, +30 kV (5 p.s.i. forward); elution: 50 nL acetonitrile–BGE (8:2, v/v).
Fig. 2. (A) Effect of sample loading volume on peak area of methionine enkephalin (1000 ng/mL). A 5 mm monolithic sol–gel concentrator is employed. Capillary: 90 cm × 50 m i.d.; loading flow rate: 80 nL/min; elution: 50 nL acetonitrile–BGE (8:2, v/v). (B) Effect of elution volume on peak area of methionine enkephalin (1000 ng/mL) determined by CE-UV. A 5 mm monolithic sol–gel preconcentrator is employed. Capillary: 60 cm × 50 m i.d.; BGE, 5 mM ammonium acetate (pH 3.6); separation voltage, +30 kV (5 p.s.i. forward); injection: 25 p.s.i. for 5 min (ca. 400 nL); detection, UV at 214 nm.
injection. Therefore, for deproteinization spiked CSF samples were diluted with 0.5 M acetic acid (pH 2.5) in a 1:1 ratio and then centrifuged. The supernatant was subsequently loaded onto the sol–gel concentrator. Fig. 4 shows a total ion electropherogram (TIE) of a spiked CSF sample and an extracted ion electropherogram of methionine enkephalin. The signal intensity of methionine enkephalin in CSF was approximately 30% lower compared to the signal intensity obtained for methionine enkephalin in BGE, how-
limit of detection (S/N = 3) was approximately 1 ng/mL, which is comparable with the LOD value for methionine enkephalin obtained by in-line SPE-CE-MS method using microcartridges containing a C18 stationary phase [6]. A comparable LOD value for methionine enkephalin was also obtained by an on-line SPE-CEMS method [7]. In this case, a sample injection of 100 L resulted in a LOD value of ca. 1 ng/mL for methionine enkephalin. For five replicate analyses of a standard solution of methionine enkephalin (100 ng/mL), the within-day R.S.D. for migration time was less than 1.2%. The within-day R.S.D. for peak area was less than 8.4%. 3.2. Applicability for CSF analysis To demonstrate the feasibility of the SPE-CE-MS system for the analysis of biological samples, CSF spiked with methionine enkephalin was analyzed. Initially, the pH of the spiked CSF samples (10 ng/mL) were adjusted to pH 3.0 with acetic acid and then centrifuged (5 min at 13,200 rpm). When this sample was loaded onto the sol–gel concentrator, methionine enkephalin was not detected after elution and CE analysis. Probably the proteins in CSF compromised the sol–gel concentrator. Results using standard solutions of methionine enkephalin could not be repeated after this CSF
Fig. 4. Analysis of CSF sample spiked with 10 ng/mL methionine enkephalin using 0.5 M acetic acid for deproteinization. (A) Total ion electropherogram of the spiked CSF sample. (B) Extracted ion electropherogram of methionine enkephalin. BGE, 5 mM ammonium acetate (pH 3.6); capillary: 90 cm × 50 m i.d.; separation voltage, +30 kV (5 p.s.i. forward); injection: 3200 nL; elution: 50 nL acetonitrile–BGE (8:2, v/v).
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system shows good potential for the analysis of biological samples. 4. Conclusions
Fig. 5. Analysis of CSF sample spiked with 1 ng/mL methionine enkephalin using 0.1 M perchloric acid for deproteinization. (A) Total ion electropherogram of the spiked CSF sample. (B) Extracted ion electropherogram of methionine enkephalin. BGE, 5 mM ammonium acetate (pH 3.6). Capillary: 90 cm × 50 m i.d.; separation voltage, +30 kV (5 p.s.i. forward); injection: 3200 nL; elution: 50 nL acetonitrile–BGE (8:2, v/v).
ever, the peak areas were similar indicating that no analyte is lost. The high signal intensity between 28 and 30 min in the TIE indicates that also other compounds co-migrated with methionine enkephalin. Also the migration time of methionine enkephalin in CSF has been increased with approximately 5 min, which was probably due to adsorption of matrix components to the capillary wall. Moreover, the migration time of methionine enkephalin increased with ca. 10 min after a second injection of the spiked CSF sample on the same sol–gel capillary. Therefore, 0.1 M perchloric acid was evaluated for deproteinization of CSF. Using 0.1 M perchloric acid for deproteinization, the signal intensities and peak areas for different concentration levels of methionine enkephalin in CSF was similar to the signal intensities and peak areas obtained in BGE. The migration time of methionine enkephalin did not increase; indeed it even slightly decreased. Fig. 5 shows a TIE of a spiked CSF sample pretreated with perchloric acid and an extracted ion electropherogram of methionine enkephalin (1 ng/mL). For methionine enkephalin, the LOD (S/N = 3) was approximately 1 ng/mL (ca. 5 nM) using extracted ion electropherograms. To further evaluate the performance of the in-line SPE-CE-MS system for the analysis of CSF samples using perchloric acid for sample pretreatment, untreated CSF was spiked at three concentration levels (100, 10 and 1 ng/mL) with methionine enkephalin. The recoveries were 91, 84 and 74%, respectively, which are acceptable values. Therefore, the in-line SPE-CE-MS
A CE-MS method using an in-line sol–gel concentrator has been evaluated for the preconcentration and analysis of methionine enkephalin. After optimizing operational parameters for in-line SPE-CE-MS, preconcentration was demonstrated for methionine enkephalin solution using a loading volume of 3200 nL. A limit of detection (S/N = 3) below 1 ng/mL (<5 nM) was obtained using in-line preconcentration. Linearity was good over two orders of magnitude and R.S.D.s for migration times and peak areas were 1.2% and 8.4%, respectively. The suitability of the method for the analysis of biological samples was tested with CSF samples spiked with methionine enkephalin. To allow the analysis of methionine enkephalin in CSF, pretreatment of spiked CSF samples with perchloric acid was necessary in order to precipitate the proteins. For the determination of endogenous enkephalins in CSF, which are present in the pg/mL to the low ng/mL range [24], sensitivity improvement is still required. In the near future, the system will also be used for the analysis of exogenous compounds, such as pharmaceuticals in urine and CSF. Increased sensitivity and selectivity may be achieved by the use of specialized monolithic sol–gel concentrators, e.g. with immobilized antibodies and metal-affinity materials. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24]
P. Puig, F. Borrull, M. Calull, C. Aguilar, Trends Anal. Chem. 26 (2007) 664. B.M. Simonet, A. Ríos, M. Valcárcel, Trends Anal. Chem. 22 (2003) 605. S.L. Simpson Jr., J.P. Quirino, S. Terabe, J. Chromatogr. A 1184 (2008) 504. F.W.A. Tempels, W.J.M. Underberg, G.W. Somsen, G.J. de Jong, Electrophoresis 29 (2008) 108. S. Sentellas, L. Puignou, M.T. Galceran, J. Sep. Sci. 25 (2002) 975. E. Hernández, F. Benavente, V. Sanz-Nebot, J. Barbosa, Electrophoresis 28 (2007) 3957. F.W.A. Tempels, W.J.M. Underberg, G.W. Somsen, G.J. de Jong, Electrophoresis 28 (2007) 1319. L.H. Zhang, C.J. Zhang, X. Chen, Y.Q. Feng, X.Z. Wu, Electrophoresis 27 (2006) 3224. J.R. Veraart, H. Lingeman, U.A.Th. Brinkman, J. Chromatogr. A 856 (1999) 483. F.W.A. Tempels, W.J.M. Underberg, G.W. Somsen, G.J. de Jong, Anal. Chem. 76 (2004) 4432. M.C. Breadmore, A.S. Palmer, M. Curran, M. Macka, N. Avdalovic, P.R. Haddad, Anal. Chem. 74 (2002) 2112. N.A. Guzman, S.S. Park, D. Schaufelberger, L. Hernandez, X. Paez, P. Rada, A.J. Tomlinson, S. Naylor, J. Chromatogr. B 697 (1997) 37. F. Benavente, M.C. Vescina, E. Hernández, V. Sanz-Nebot, J. Barbosa, N.A. Guzman, J. Chromatogr. A 1140 (2007) 205. Q. Yang, A.J. Tomlinson, S. Naylor, Anal. Chem. 71 (1999) 183A. D. Schaller, E.F. Hilder, P.R. Haddad, Anal. Chim. Acta 556 (2006) 104. E.F. Hilder, F. Svec, J.M.J. Fréchet, J. Chromatogr. A 1044 (2004) 3. J.P. Quirino, M.T. Dulay, B.D. Bennett, R.N. Zare, Anal. Chem. 73 (2001) 3921. J.P. Quirino, M.T. Dulay, B.D. Bennett, R.N. Zare, Anal. Chem. 73 (2001) 5539. J.P. Quirino, M.T. Dulay, R.N. Zare, Anal. Chem. 73 (2001) 5557. N.E. Baryla, N.P. Toltl, Analyst 128 (2003) 1009. M.C. Breadmore, Anal. Chim. Acta 556 (2006) 121. J.P. Hutchinson, P. Zakaria, A.R. Bowie, M. Macka, N. Avdavolic, P.R. Haddad, Anal. Chem. 77 (2005) 407. N. Johannesson, J. Bergquist, J. Pharm. Biomed. Anal. 43 (2007) 1045. M. Langemark, F.W. Bach, R. Ekman, J. Olesen, Pain 63 (1995) 103.
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Capillary electrophoresis-mass spectrometry using an in-line sol–gel concentrator for the determination of methionine enkephalin in cerebrospinal fluid R. Ramautar a,∗ , C.K. Ratnayake b , G.W. Somsen a , G.J. de Jong a a b
Department of Biomedical Analysis, Utrecht University, P.O. Box 80082, 3508 TB Utrecht, The Netherlands Beckman Coulter, Inc., P.O. Box 3100, Fullerton, CA 92834-3100, USA
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i n f o
Article history: Received 20 August 2008 Received in revised form 4 December 2008 Accepted 11 December 2008 Available online 24 December 2008 Keywords: Capillary electrophoresis-mass spectrometry Sol–gel concentrator Preconcentration Cerebrospinal fluid Methionine enkephalin
a b s t r a c t In this study, a CE-MS method using a monolithic sol–gel concentrator for in-line solid-phase extraction (SPE) is evaluated for the analysis of methionine enkephalin in biological samples. Operational SPE parameters such as sample pH, loading volume, elution volume and composition have been studied. After optimization of the in-line preconcentration methodology, a 40-fold preconcentration was demonstrated for a methionine enkephalin test solution using a loading volume of 3200 nL. The method was linear in the range from 62.5 to 1000 ng/mL (R2 > 0.99). R.S.D. values for migration times and peak areas were 1.2% and 8.4%, respectively. Finally, the analysis of cerebrospinal fluid samples spiked with methionine enkephalin and deproteinized with perchloric acid (1:1, v/v) showed a detection limit (S/N = 3) of approximately 1 ng/mL (ca. 5 nM). The recoveries of methionine enkephalin for three concentration levels (100, 10 and 1 ng/mL) were in the range of 74–91%, demonstrating the promising potential of the methodology for the analysis of biological samples. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Capillary electrophoresis (CE) is an efficient separation technique for a wide variety of analytes. However, for the analysis of biological samples CE suffers from an inherent low concentration sensitivity due to the limited sample volume that can be loaded onto the capillary. Several strategies can be used to improve the concentration sensitivity in CE [1,2]. In general, analyte preconcentration via electrophoresis-based or chromatography-based methods is used to improve the concentration sensitivity in CE [3,4]. Chromatography-based methods can offer two to four orders of preconcentration [5–8], and therefore are attractive to improve the concentration-sensitivity in CE. Typically, chromatographic preconcentration is carried out either in an off-line, on-line, or in-line mode [9]. In the on-line mode, the precolumn is not part of the CE system and the coupling of the precolumn is performed via an interface [10]. In the in-line mode, the preconcentration column is an integrated part of the CE system. When using these chromatographic techniques, on-line or in-line methods are regarded as advantageous compared to the off-line approach as a result of their shorter total analysis times, minimum of sample handling and possibility of automation. The main advantage of the
∗ Corresponding author. Fax: +31 30 253 5180. E-mail address: [email protected] (R. Ramautar). 0039-9140/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2008.12.025
in-line approach is that the entire preconcentrated amount of analyte can be transferred to the separation section of the CE capillary [9]. However, a disadvantage is that sample matrix components also pass through the separation capillary and may interfere with the CE analysis or they can adsorb onto the capillary wall, which can result in poor separations. Nevertheless, the in-line approach has the highest level of integration [9]. Various in-line SPE-CE approaches have been described such as an open-tubular capillary coated with a sorbent [11], a small (1–2 mm) packed section containing microsphere beads and retained by frits at the inlet of the capillary [12,13], and an immobilized particle-loaded membrane [14]. In the open-tubular configuration, the capacity is relatively low to load sufficient sample [15]. While packed SPE modules offer a relatively larger capacity, the introduction of a small packed section into the capillary can increase the back pressure, and rinsing the capillary using conventional CE instrumentation with low pressures can be problematic [15]. Moreover, the preparation of packed SPE modules in narrow bore capillaries can be difficult, especially with respect to the frit formation [15]. An alternative approach is the use of in situ formed monolithic materials [16]. By using UV-initiated polymerization, the monolithic SPE column can be made directly in the capillary. No frits are required as monolithic columns consist of a continuous piece of a highly porous microstructure. Monolithic columns can be used at high flow rates, thus minimizing sample loading time and backpres-
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sure. There are two types of monolithic columns, i.e. silica-based and polymer-based monoliths. Silica monoliths are prepared using sol–gel technology whereas polymer-based monoliths are made by in situ polymerization of monomers and cross-linkers [17]. Photopolymerized sol–gel monoliths have been used as a SPE sorbent and stationary phase in capillary electrochromatography for the preconcentration and separation of peptides [17–19]. However, sol–gel monoliths as preconcentration sorbents are still not used widely for in-line SPE-CE. So far, a few papers have described the applicability of monolithic columns for in-line SPE-CE to preconcentrate compounds. For instance, an in-line SPE-CE method using a monolithic methacrylate polymer was used for the preconcentration of Spropranolol from aqueous solutions. A limit of detection (LOD) in the low nanomolar range was achieved for S-propranolol with this method [20]. In another study, a polymeric monolithic SPE column was prepared in situ within a fused silica capillary from butyl methacrylate-co-ethylene dimethacrylate [15]. Using a 1 cm SPE column placed at the inlet of the capillary, the compounds sertraline, fluoxetine and fluvoxamine were extracted from aqueous solutions by applying a pressure rinse. Enrichment factors of over 500 were achieved for the compounds of interest. A sulphopropyl methacrylate monolith was used to preconcentrate amino acids from standard solutions using inline SPE-CE [21]. Monolithic columns have also been used for the preconcentration of inorganic anions using in-line SPE-CE [22]. A poly(butyl methacrylate-co-ethylene dimethacrylate-co-2acrylamido-2-methyl-1-propanesulfonic acid) monolithic column with cation-exchange sites was used for that purpose. An in-line SPE-CE time-of-flight mass spectrometry (TOF-MS) method using a silica-based monolith was developed for the determination of escitalopram in human urine [23]. The limit of detection (LOD) for escitalopram was 10 pg/mL obtained with a loading volume of approximately 2.25 L. The intraday precision of the escitalopram peak area was less than 6.3%. In the present paper the development and optimization of an in-line SPE-CE-MS method based on a silica-based monolithic sol–gel concentrator for the determination of methionine enkephalin in cerebrospinal fluid (CSF) is described. The main goal of this study was the evaluation of the feasibility of silicabased monolithic sol–gel capillaries for the analysis of CSF samples. The paper describes the systematic optimization of experimental parameters of the sol–gel concentrator, such as loading time, elution volume and composition of the elution solvent. Subsequently, repeatability of migration time and peak area, linearity and LODs have been determined. Finally, the potential of the in-line SPE-CE-ESI-MS method for CSF analysis is assessed. 2. Experimental 2.1. Chemicals Ammonium acetate, sodium hydroxide, perchloric acid and acetonitrile (all analytical grade or higher) were purchased from Merck (Darmstadt, Germany) and Biosolve BV (Valkenswaard, The Netherlands), respectively. Acetic acid (analytical grade) and acetate salts of Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu) and [Met5 ]-enkephalin (Tyr-Gly-Gly-Phe-Met) were obtained from Sigma–Aldrich (St. Louis, MO, USA). Human CSF was generously donated by the Utrecht Medical Centre (Utrecht, The Netherlands). Deionized water from a Milli-Q system (Millipore, Bedford, MA, USA) was used for all solutions. The background electrolyte (BGE) in all CE experiments was 5 mM ammonium acetate adjusted to pH 3.6 with acetic acid. The peptides were dissolved in BGE, unless otherwise stated.
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2.2. Preparation of sol–gel capillaries Sol–gel capillaries were obtained from Beckman Coulter (Fullerton, CA, USA). The monolithic sol–gel sorbent was prepared by mixing 500 L ethanol and 600 L tetraethoxysilane. Subsequently, 800 L of 1 M nitric acid was added to initiate hydrolysis and condensation. Then reversed phase silica particles (Beckman Coulter, Fullerton, CA, USA) were added to make a slurry. Bare fused silica capillaries were filled with this slurry to obtain an about 3–5 cm long plug which was heated to 120 ◦ C for 48 h. Finally, the capillary was cut to obtain a monolithic sol–gel plug of 5 mm at the inlet of the capillary.
2.3. Instrumentation and procedures CE-UV experiments were performed using a Beckman Coulter (Fullerton, CA, USA) PA 800 CE instrument equipped with a diode array detector (DAD). Capillaries with internal diameter (ID) of 50 m were from Composite Metal Services (The Chase, Hallow, UK). The sol–gel capillaries were flushed with acetonitrile (15 min) and 80% acetonitrile in BGE (10 min) prior to use. Sol–gel capillaries had a total length of 60 cm and effective length of 50 cm. Samples were injected for 5 min at 25 p.s.i. (injection volume of ca. 400 nL), unless otherwise stated, and the separation voltage was 30 kV. The capillary was thermostated at 20 ◦ C and detection was carried out at 200 nm with a data acquisition rate of 16 Hz. CE-ESIMS experiments were conducted using a PrinCE CE system from Prince Technologies B.V. (Emmen, The Netherlands) using 50 m ID sol–gel capillaries with a length of 90 cm. During sample injection, the nebulizer gas flow and the electrospray voltage of the CE-MS interface were turned off. CE was coupled to an Agilent Technologies 1100 Series LC/MSD SL IT mass spectrometer (Waldbronn, Germany) via a coaxial sheath-flow electrospray interface (Agilent). The CE capillary outlet was positioned at 0.2–0.5 mm from the tip of the interface. A sheath liquid of methanol–water–acetic acid (50:50:0.1, v/v/v) was supplied by a syringe pump at a flow rate of 10 L/min. The nebulizer-gas pressure was 10 p.s.i., and the flow and temperature of the drying gas were 4 L/min and 300 ◦ C, respectively. The electrospray voltage was 4.5 kV. In the Agilent CEMS set-up, the spray needle is grounded. MS detection was carried out in the positive ion mode and the scan range was 200–1200 m/z. All sol–gel capillaries for in-line SPE-CE-MS were first conditioned by consecutive flushes of acetonitrile (10 min), 80% acetonitrile in BGE (10 min) and BGE (10 min) at 25 p.s.i. Dilute samples of methionine enkephalin in BGE were hydrodynamically injected at 25 p.s.i. for 40 min (unless otherwise stated). A washing rinse with the BGE at 25 p.s.i. for 20 min was applied before the elution of methionine enkephalin. Elution was performed by injecting a solution of 80:20 (v/v) acetonitrile–BGE (apparent pH 3.6) at 10 p.s.i. for 0.5 min (ca. 50 nL). Separation was carried out applying a voltage of 30 kV (normal polarity). Between runs, the capillary was rinsed for 5 min with a solution of 80:20 (v/v) acetonitrile–BGE and BGE, in order to avoid carry-over between consecutive analyses.
2.4. Sample preparation The off-line pretreatment of blank and spiked CSF samples consisted of a protein precipitation step. This was carried out with 0.1 M perchloric acid in a ratio of 1:1 (v/v) (100 L CSF and 100 L perchloric acid). The supernatant was centrifuged (5 min at 9447 × g). Subsequently, the samples were hydrodynamically introduced at 25 p.s.i. for 40 min.
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2.5. Evaluation of analytical parameters The analytical parameters were calculated from data obtained by measuring the peak area and migration time from the extracted ion electropherogram of methionine enkephalin. The m/z of the molecular ion for leucine enkephalin was 556.0 and for methionine enkephalin 574.4. Repeatability studies (n = 5) were performed with methionine enkephalin dissolved in CSF at a concentration of 100 ng/mL. LOD (S/N = 3) for methionine enkephalin was obtained analyzing methionine enkephalin solutions of 10 ng/mL and 1 ng/mL. The linearity range was determined by injecting five concentrations in the range of 62.5–1000 ng/mL in BGE. Recoveries of methionine enkephalin spiked into CSF (at three concentration levels: 100, 10 and 1 ng/mL) were calculated using the calibration curve constructed for methionine enkephalin standards (62.5–1000 ng/mL range). 3. Results and discussion 3.1. SPE-CE optimization A preliminary study was performed to find a suitable BGE for enkephalin analysis. For BGE optimization, two closely related peptides, i.e. leucine enkephalin and methionine enkephalin, were used. Ammonium acetate at four different pH values (2.5, 3.1, 3.6 and 4.0) was investigated as it is a volatile BGE. With a BGE of 5 mM ammonium acetate (pH 3.6), a baseline separation for the two peptides was obtained (Fig. 1). Additionally, this low-ionic-strength BGE is well compatible with ESI-MS. Therefore, for the in-line SPE-CE-MS experiments, 5 mM ammonium acetate (pH 3.6) was chosen as BGE. The optimum composition of the sheath liquid was determined by pressure-induced infusion of a solution of methionine enkephalin in BGE (1 g/mL) using mixtures of methanol–water and acetonitrile–water containing 0.1% acetic acid. The optimum sheath liquid composition was methanol–water–acetic acid (50:50:0.1, v/v/v). The sheath liquid flow rate was optimized in the range from 2.5 to 12.5 L/min and the highest signal intensity for methionine enkephalin was obtained at a flow rate of 10 L/min.
Fig. 1. Separation of leucine enkephalin from methionine enkephalin. (A) Extracted ion electropherogram of 100 g/mL methionine enkephalin. (B) Extracted ion electropherogram of 100 g/mL leucine enkephalin. BGE, 5 mM ammonium acetate (pH 3.6); capillary: 90 cm × 50 m i.d.; separation voltage, +30 kV; injection: 50 nL.
For optimization of the in-line SPE-CE-MS procedure, several parameters have to be considered in order to achieve optimum performance, i.e. the pH of the sample solution, the loading volume, elution volume and composition of the elution solvent. These parameters have been evaluated with methionine enkephalin as test compound. First, the pH of the injected sample solution was determined as this parameter will affect the retention of the analyte on the monolithic sol–gel concentrator. The pH of the injected sample solution was studied between pH 2.5 and 6.8 using 5 mM ammonium acetate. A loading volume of ca. 400 nL (loading time 5 min) and an elution volume of ca. 50 nL (80% acetonitrile in BGE) were used. The sample solution was introduced hydrodynamically into the in-line SPE-CE-MS system. In this way, the volume of sample introduced can be calculated, taking into account the flow-rate through the sol–gel capillary which can be determined via the mass loss of a buffer-filled vial at a certain pressure and time interval. Using pH values above 4.5 for sample loading resulted in high back pressures and also current drops upon voltage application. Similar peak areas for methionine enkephalin were obtained at pH values below 4.0. Therefore, a sample pH of 3.6 was selected as acetate has a good buffer capacity at this pH. Next, the loading capacity of the sol–gel concentrator was evaluated using in-line SPE-CE-MS. The loading capacity of the monolithic sol–gel concentrator was determined by loading different volumes of a methionine enkephalin standard solution of 1000 ng/mL. In order to increase the speed of the analysis a loading pressure of 25 p.s.i. was chosen. Fig. 2A shows the effect of loading volume (i.e. loading time) on the peak area for methionine enkephalin. A linear relation of peak area versus loading volume could be observed up to a loading volume of ca. 3200 nL (40 min). The deviation from linearity above a loading volume of ca. 3200 nL indicated that the capacity of the sol–gel concentrator was exceeded after 40 min for methionine enkephalin. Therefore, for the preconcentration of methionine enkephalin, a loading volume of ca. 3200 nL was selected. The influence of the composition of the elution solvent on the desorption of methionine enkephalin (1000 ng/mL) was studied by in-line SPE-CE-UV using a loading volume of 1000 nL and an elution volume of ca. 50 nL. The percentage of acetonitrile in the BGE was varied from 20 to 80%. The amount of methionine enkephalin desorption increased with a higher concentration of acetonitrile. A percentage of 80% acetonitrile in the BGE was required for complete desorption of methionine enkephalin. Subsequently, optimization of the elution volume (acetonitrile/BGE, 8/2, v/v) was determined by loading 1000 nL of 1000 ng/mL methionine enkephalin onto the monolithic sol–gel concentrator while increasing the elution volume (ca. 13, 25, 50 and 100 nL). Fig. 2B shows that the peak area of methionine enkephalin increased with the elution volume until ca. 50 nL. Above this volume the peak area remained constant. Therefore, an elution volume of ca. 50 nL was used for desorption. On-line preconcentration using the sol–gel concentrator was evaluated with methionine enkephalin as test compound. When a 5000 ng/mL methionine enkephalin sample was loaded for 1 min (80 nL) a peak with an intensity of ca. 4.2 × 106 counts was observed (Fig. 3A). When a 100 ng/mL methionine enkephalin sample was loaded for 40 min (3200 nL) a peak with an intensity of ca. 3.4 × 106 counts was observed (Fig. 3B). The ratio of the peak intensities (1.23) corresponds well to the ratio of the amount of analyte loaded on the sol–gel concentrator (i.e., 0.40 ng/0.32 ng = 1.25). As a result, the 100 ng/mL methionine enkephalin sample was preconcentrated by a factor of 40. Using methionine enkephalin concentrations in the range of 62.5–1000 ng/mL, peak area (y) versus concentration (x) regression line was calculated (n = 5). Linearity was observed over the range between 62.5–1000 ng/mL (R2 > 0.995) and the equation is y = 12,471x + 56,127. Using an injection volume of ca. 3200 nL, the
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Fig. 3. On-line preconcentration of methionine enkephalin using the sol–gel concentrator. (A) Extracted ion electropherogram of an injection of 80 nL of 5000 ng/mL methionine enkephalin. (B) Extracted ion electropherogram of an injection of 3200 nL of 100 ng/mL methionine enkephalin. BGE, 5 mM ammonium acetate (pH 3.6); capillary: 90 cm × 50 m i.d.; separation voltage, +30 kV (5 p.s.i. forward); elution: 50 nL acetonitrile–BGE (8:2, v/v).
Fig. 2. (A) Effect of sample loading volume on peak area of methionine enkephalin (1000 ng/mL). A 5 mm monolithic sol–gel concentrator is employed. Capillary: 90 cm × 50 m i.d.; loading flow rate: 80 nL/min; elution: 50 nL acetonitrile–BGE (8:2, v/v). (B) Effect of elution volume on peak area of methionine enkephalin (1000 ng/mL) determined by CE-UV. A 5 mm monolithic sol–gel preconcentrator is employed. Capillary: 60 cm × 50 m i.d.; BGE, 5 mM ammonium acetate (pH 3.6); separation voltage, +30 kV (5 p.s.i. forward); injection: 25 p.s.i. for 5 min (ca. 400 nL); detection, UV at 214 nm.
injection. Therefore, for deproteinization spiked CSF samples were diluted with 0.5 M acetic acid (pH 2.5) in a 1:1 ratio and then centrifuged. The supernatant was subsequently loaded onto the sol–gel concentrator. Fig. 4 shows a total ion electropherogram (TIE) of a spiked CSF sample and an extracted ion electropherogram of methionine enkephalin. The signal intensity of methionine enkephalin in CSF was approximately 30% lower compared to the signal intensity obtained for methionine enkephalin in BGE, how-
limit of detection (S/N = 3) was approximately 1 ng/mL, which is comparable with the LOD value for methionine enkephalin obtained by in-line SPE-CE-MS method using microcartridges containing a C18 stationary phase [6]. A comparable LOD value for methionine enkephalin was also obtained by an on-line SPE-CEMS method [7]. In this case, a sample injection of 100 L resulted in a LOD value of ca. 1 ng/mL for methionine enkephalin. For five replicate analyses of a standard solution of methionine enkephalin (100 ng/mL), the within-day R.S.D. for migration time was less than 1.2%. The within-day R.S.D. for peak area was less than 8.4%. 3.2. Applicability for CSF analysis To demonstrate the feasibility of the SPE-CE-MS system for the analysis of biological samples, CSF spiked with methionine enkephalin was analyzed. Initially, the pH of the spiked CSF samples (10 ng/mL) were adjusted to pH 3.0 with acetic acid and then centrifuged (5 min at 13,200 rpm). When this sample was loaded onto the sol–gel concentrator, methionine enkephalin was not detected after elution and CE analysis. Probably the proteins in CSF compromised the sol–gel concentrator. Results using standard solutions of methionine enkephalin could not be repeated after this CSF
Fig. 4. Analysis of CSF sample spiked with 10 ng/mL methionine enkephalin using 0.5 M acetic acid for deproteinization. (A) Total ion electropherogram of the spiked CSF sample. (B) Extracted ion electropherogram of methionine enkephalin. BGE, 5 mM ammonium acetate (pH 3.6); capillary: 90 cm × 50 m i.d.; separation voltage, +30 kV (5 p.s.i. forward); injection: 3200 nL; elution: 50 nL acetonitrile–BGE (8:2, v/v).
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system shows good potential for the analysis of biological samples. 4. Conclusions
Fig. 5. Analysis of CSF sample spiked with 1 ng/mL methionine enkephalin using 0.1 M perchloric acid for deproteinization. (A) Total ion electropherogram of the spiked CSF sample. (B) Extracted ion electropherogram of methionine enkephalin. BGE, 5 mM ammonium acetate (pH 3.6). Capillary: 90 cm × 50 m i.d.; separation voltage, +30 kV (5 p.s.i. forward); injection: 3200 nL; elution: 50 nL acetonitrile–BGE (8:2, v/v).
ever, the peak areas were similar indicating that no analyte is lost. The high signal intensity between 28 and 30 min in the TIE indicates that also other compounds co-migrated with methionine enkephalin. Also the migration time of methionine enkephalin in CSF has been increased with approximately 5 min, which was probably due to adsorption of matrix components to the capillary wall. Moreover, the migration time of methionine enkephalin increased with ca. 10 min after a second injection of the spiked CSF sample on the same sol–gel capillary. Therefore, 0.1 M perchloric acid was evaluated for deproteinization of CSF. Using 0.1 M perchloric acid for deproteinization, the signal intensities and peak areas for different concentration levels of methionine enkephalin in CSF was similar to the signal intensities and peak areas obtained in BGE. The migration time of methionine enkephalin did not increase; indeed it even slightly decreased. Fig. 5 shows a TIE of a spiked CSF sample pretreated with perchloric acid and an extracted ion electropherogram of methionine enkephalin (1 ng/mL). For methionine enkephalin, the LOD (S/N = 3) was approximately 1 ng/mL (ca. 5 nM) using extracted ion electropherograms. To further evaluate the performance of the in-line SPE-CE-MS system for the analysis of CSF samples using perchloric acid for sample pretreatment, untreated CSF was spiked at three concentration levels (100, 10 and 1 ng/mL) with methionine enkephalin. The recoveries were 91, 84 and 74%, respectively, which are acceptable values. Therefore, the in-line SPE-CE-MS
A CE-MS method using an in-line sol–gel concentrator has been evaluated for the preconcentration and analysis of methionine enkephalin. After optimizing operational parameters for in-line SPE-CE-MS, preconcentration was demonstrated for methionine enkephalin solution using a loading volume of 3200 nL. A limit of detection (S/N = 3) below 1 ng/mL (<5 nM) was obtained using in-line preconcentration. Linearity was good over two orders of magnitude and R.S.D.s for migration times and peak areas were 1.2% and 8.4%, respectively. The suitability of the method for the analysis of biological samples was tested with CSF samples spiked with methionine enkephalin. To allow the analysis of methionine enkephalin in CSF, pretreatment of spiked CSF samples with perchloric acid was necessary in order to precipitate the proteins. For the determination of endogenous enkephalins in CSF, which are present in the pg/mL to the low ng/mL range [24], sensitivity improvement is still required. In the near future, the system will also be used for the analysis of exogenous compounds, such as pharmaceuticals in urine and CSF. Increased sensitivity and selectivity may be achieved by the use of specialized monolithic sol–gel concentrators, e.g. with immobilized antibodies and metal-affinity materials. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24]
P. Puig, F. Borrull, M. Calull, C. Aguilar, Trends Anal. Chem. 26 (2007) 664. B.M. Simonet, A. Ríos, M. Valcárcel, Trends Anal. Chem. 22 (2003) 605. S.L. Simpson Jr., J.P. Quirino, S. Terabe, J. Chromatogr. A 1184 (2008) 504. F.W.A. Tempels, W.J.M. Underberg, G.W. Somsen, G.J. de Jong, Electrophoresis 29 (2008) 108. S. Sentellas, L. Puignou, M.T. Galceran, J. Sep. Sci. 25 (2002) 975. E. Hernández, F. Benavente, V. Sanz-Nebot, J. Barbosa, Electrophoresis 28 (2007) 3957. F.W.A. Tempels, W.J.M. Underberg, G.W. Somsen, G.J. de Jong, Electrophoresis 28 (2007) 1319. L.H. Zhang, C.J. Zhang, X. Chen, Y.Q. Feng, X.Z. Wu, Electrophoresis 27 (2006) 3224. J.R. Veraart, H. Lingeman, U.A.Th. Brinkman, J. Chromatogr. A 856 (1999) 483. F.W.A. Tempels, W.J.M. Underberg, G.W. Somsen, G.J. de Jong, Anal. Chem. 76 (2004) 4432. M.C. Breadmore, A.S. Palmer, M. Curran, M. Macka, N. Avdalovic, P.R. Haddad, Anal. Chem. 74 (2002) 2112. N.A. Guzman, S.S. Park, D. Schaufelberger, L. Hernandez, X. Paez, P. Rada, A.J. Tomlinson, S. Naylor, J. Chromatogr. B 697 (1997) 37. F. Benavente, M.C. Vescina, E. Hernández, V. Sanz-Nebot, J. Barbosa, N.A. Guzman, J. Chromatogr. A 1140 (2007) 205. Q. Yang, A.J. Tomlinson, S. Naylor, Anal. Chem. 71 (1999) 183A. D. Schaller, E.F. Hilder, P.R. Haddad, Anal. Chim. Acta 556 (2006) 104. E.F. Hilder, F. Svec, J.M.J. Fréchet, J. Chromatogr. A 1044 (2004) 3. J.P. Quirino, M.T. Dulay, B.D. Bennett, R.N. Zare, Anal. Chem. 73 (2001) 3921. J.P. Quirino, M.T. Dulay, B.D. Bennett, R.N. Zare, Anal. Chem. 73 (2001) 5539. J.P. Quirino, M.T. Dulay, R.N. Zare, Anal. Chem. 73 (2001) 5557. N.E. Baryla, N.P. Toltl, Analyst 128 (2003) 1009. M.C. Breadmore, Anal. Chim. Acta 556 (2006) 121. J.P. Hutchinson, P. Zakaria, A.R. Bowie, M. Macka, N. Avdavolic, P.R. Haddad, Anal. Chem. 77 (2005) 407. N. Johannesson, J. Bergquist, J. Pharm. Biomed. Anal. 43 (2007) 1045. M. Langemark, F.W. Bach, R. Ekman, J. Olesen, Pain 63 (1995) 103.