Capillary Column Switching Restricted-Access Media-Liquid

Jul 12, 2007 - Capillary online restricted-access media-liquid chromatography-electrospray ionization-tandem mass spectrometry (RAM-LC-ESI-MS/MS) for ...
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Anal. Chem. 2007, 79, 6359-6367

Capillary Column Switching Restricted-Access Media-Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry System for Simultaneous and Direct Analysis of Drugs in Biofluids Alvaro J. Santos-Neto,†,‡ Karin E. Markides,† Per J. R. Sjo 1 berg,† Jonas Bergquist,† and ,‡ Fernando M. Lancas*

Department of Physical and Analytical Chemistry, Analytical Chemistry, Uppsala University, P.O. Box 599, 75124 Uppsala, Sweden, and Laboratory of Chromatography, Institute of Chemistry at Sao Carlos, University of Sao Paulo, P.O. Box 780, 13566-590 Sao Carlos, Brazil

Capillary online restricted-access media-liquid chromatography-electrospray ionization-tandem mass spectrometry (RAM-LC-ESI-MS/MS) for direct analysis of drugs and metabolites spiked in biological fluids was developed. Using a column switching setup it was possible to perform effective sample preparation and analysis of raw biological fluids (plasma and urine) without matrix effects in the electrospray mass spectrometric detection step. The peak focusing efficiency of the extraction column was more effective in backflush compared to foreflush mode. The system was able to concentrate diminished samples of polar drugs and their metabolites reaching quantifiable results as low as 1 ng/mL utilizing a sample volume of only 333 nL of biofluids. New column hardware was developed to circumvent clogging problems experienced with plasma injections. The glass fiber filter frit, which is commonly used, was replaced with a short piece of 20 µm i.d. fused silica capillary. The extraction columns were able to handle up to 60 injections and showed a high loading capacity, making the saturation of the MS detector the limiting factor on the linear dynamic range. The simultaneous separation and detection of 10 drugs and metabolites was obtained in 8 min of analysis, including the online sample preparation and enrichment step. Liquid chromatography on a capillary scale (cLC) has known advantages including higher mass sensitivity when utilizing concentration sensitive detectors, and a decrease in sample, stationary phase, and solvent consumption, with respect to the conventional process.1 However, for good transition from conventional HPLC to cLC, a down-scale factor proportional to the square of the column radius (f ) r2conv/r2micro) should be followed.2 In this way, the claim that cLC exhibits better sensitivity using * To whom correspondence should be addressed. Phone: +55 16 33739983. Fax: +55 16 33739984. E-mail: [email protected]. † Uppsala University. ‡ University of Sao Paulo. (1) Ishii, D.; Takeuchi, T. Trends Anal. Chem. 1990, 9, 152-157. (2) Szumski, M.; Buszewski, B. Crit. Rev. Anal. Chem. 2002, 32, 1-46. 10.1021/ac070671g CCC: $37.00 Published on Web 07/12/2007

© 2007 American Chemical Society

concentration-sensitive detectors is only true if proportionally higher amounts of sample can be injected.3 Regarding the downscale factor, which requires a reduction in the injected volume, a higher mass loading can only be obtained using on-column focusing. This approach, which can be accomplished using either single-column or column switching modes, has been reviewed by Vissers and colleagues.4 Demands in the pharmaceutical and life sciences are driving the development of minimal sample preparation and faster and more sensitive analytical tools able to handle and consume lower amounts of sample. Drug analysis in biological fluids using LC coupled to mass spectrometry (MS) is sensitive to the sample preparation, and incomplete elimination of the matrix effect can be detrimental.5-10 Thus the development of efficient sample preparation techniques has been considered an important step.11,12 Different sample preparation techniques have been evaluated regarding matrix effects.13-15 For the majority of drugs, electrospray ionization (ESI) is still the most sensitive ionization technique, although it has been reported as the most susceptible to suppression effects, however the sample was prepared.13 In (3) Vissers, J. P. C. J. Chromatogr. A 1999, 856, 117-143. (4) Vissers, J. P. C.; de Ru, A. H.; Ursem, M.; Chervet, J.-P. J. Chromatogr. A 1996, 746, 1-7. (5) Dams, R.; Huestis, M. A.; Lambert, W. E.; Murphy, C. M. J. Am. Soc. Mass Spectrom. 2003, 14, 1290-1294. (6) Muller, C.; Schafer, P.; Stortzel, M.; Vogt, S.; Weinmann, W. J. Chromatogr. B 2002, 773, 47-52. (7) King, R.; Bonfiglio, R.; Fernandez-Metzler, C.; Miller-Stein, C.; Olah, T. J. Am. Soc. Mass Spectrom. 2000, 11, 942-950. (8) Buhrman, D. L.; Price, P. I.; Rudewicz, P. J. J. Am. Soc. Mass Spectrom. 1996, 7, 1099-1105. (9) Matuszewski, B. K.; Constanzer, M. L.; Chavez-Eng, C. M. Anal. Chem. 1998, 70, 882-889. (10) Tiller, P. R.; Romanyshyn, L. A. Rapid Commun. Mass Spectrom. 2002, 16, 92-98. (11) Smith, R. M. J. Chromatogr. A 2003, 1000, 3-27. (12) Moldoveanu, S. C. J. Chromatogr. Sci. 2004, 42, 1-14. (13) Marchi, I.; Rudaz, S.; Selman, M.; Veuthey, J.-L. J. Chromatogr. B 2007, 845, 244-252. (14) Souverain, S.; Rudaz, S.; Veuthey, J.-L. J. Pharm. Biomed. Anal. 2004, 35, 913-920. (15) Souverain, S.; Rudaz, S.; Veuthey, J.-L. J. Chromatogr. A 2004, 1058, 6166.

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1985, Hagestam and Pinkerton presented the internal surface reversed-phase (ISRP) concept for drug analysis in biofluids.16 The restricted-access media (RAM) expression was introduced by Desilets et al. in 1991 as a general term for chromatographic supports allowing the direct injection of biological fluids and limiting the interaction within the pores to small molecules only.17 The use of RAM and classifications for the several supports have been reviewed,18-21 and very recently RAM has gained attention with some more reexaminations.22-24 It can be used in singlecolumn or column switching mode, with the latter showing several advantages (i.e., larger sample injection in a higher flow rate and reduced interference).25 Electrospray ionization operating at low flow rates (a few microliters per minute) usually behaves as a concentration-sensitive detector.26 Therefore, combining the advantages of cLC in RAM column switching with ESI-MS seems to be an attractive alternative to the current methods of drug analysis in complex samples. Swart and co-workers27 used a column switching cLC-MS/MS system for the concentration of ultrafiltered samples. Cai and Henion28 coupled microbore immunoaffinity LC with cLC-MS/ MS for urine samples. In a review of Saito and Jinno29 some applications of online solid-phase extraction (SPE) and microcolumn LC were presented, but no plasma sample was treated in a fully capillary system. In a nice approach, Liu et al.30 analyzed plasma samples in a cLC system; however, they needed to perform offline sample preparation by protein precipitation and pipet tip SPE. Fast analysis of biosamples was obtained by Hsieh et al.31 using a conventional scale monolithic LC-MS/MS system. Christiaens and co-workers32 developed a semi-microbore RAM-LC-MS/ MS system for human plasma analysis. Lanckmans et al.33 demonstrated practical advantages of using cLC and nanoLC for small molecule quantification; however, they had matrix effect problems with biosamples and needed to use microdialysis in sample preparation. Finally, Suenami and co-workers34 used a capillary SPE-LC-MS/MS system, but they did not include the use of RAM. Additionally, as the analytical columns had 520 µm (16) Hagestam, I. H.; Pinkerton, T. C. Anal. Chem. 1985, 57, 1757-1763. (17) Desilets, C. P.; Rounds, M. A.; Regnier, F. E. J. Chromatogr. 1991, 544, 25-39. (18) Pinkerton, T. C. J. Chromatogr. 1991, 544, 13-23. (19) Anderson, D. J. Anal. Chem. 1993, 65, R434-R443. (20) Boos, K. S.; Rudolphi, A. LC-GC 1997, 15, 602-611. (21) Souverain, S.; Rudaz, S.; Veuthey, J.-L. J. Chromatogr. B 2004, 801, 141156. (22) Cassiano, N. M.; Lima, V. V.; Oliveira, R. V.; de Pietro, A. C.; Cass, Q. B. Anal. Bioanal. Chem. 2006, 384, 1462-1469. (23) Mullett, W. M. J. Biochem. Biophys. Methods 2007, 70, 263-273. (24) Sadilek, P.; Satinsky, D.; Solich, P. Trends Anal. Chem. 2007, 26, 375384. (25) Boos, K. S.; Rudolphi, A. LC-GC 1997, 15, 814-823. (26) Bruins, A. P. J. Chromatogr. A 1998, 794, 345-357. (27) Swart, R.; Koivisto, P.; Markides, K. E. J. Chromatogr. A 1998, 828, 209218. (28) Cai, J.; Henion, J. J. Chromatogr. B 1997, 691, 357-370. (29) Saito, Y.; Jinno, K. Anal. Bioanal. Chem. 2002, 373, 325-331. (30) Liu, S.; Griffiths, W. J.; Sjovall, J. Anal. Chem. 2003, 75, 791-797. (31) Hsieh, Y.; Wang, G.; Wang, Y.; Chackalamannil, S.; Korfmacher, W. A. Anal. Chem. 2003, 75, 1812-1818. (32) Christiaens, B.; Fillet, A.; Chiap, P.; Rbeida, O.; Ceccato, A.; Streel, B.; De Graeve, J.; Crommen, J.; Hubert, P. J. Chromatogr., A 2004, 1056, 105110. (33) Lanckmans, K.; Van Eeckhaut, A.; Sarre, S.; Smolders, I.; Michotte, Y. J. Chromatogr. A 2006, 1131, 166-175. (34) Suenami, K.; Lim, L. W.; Takeuchi, T.; Sasajima, Y.; Sato, K.; Takekoshi, Y.; Kanno, S. J. Chromatogr. B 2007, 846, 176-183.

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i.d., that analysis consumed 10 µL of sample which should be injected and washed manually. This work presents, for the first time, the feasibility of RAMLC-ESI-MS/MS in a fully capillary approach for the direct and simultaneous analysis of biological fluids containing pharmaceutical drugs and metabolites. Peak focusing and sample cleanup were developed and evaluated in a simple column switching setup. Efforts were made to develop a reusable and efficient extraction column able to tolerate the injection of a biological matrix. Matrix effects were assessed on the column extraction and on the ESI as well. Therefore, the performance of the RAM-LC-ESI-MS/MS was tested in the separation and adequate detection of 10 different compounds. EXPERIMENTAL SECTION Chemicals and Materials. The packing materials evaluated as RAM were 25 µm LiChrospher ADS-C18, pore size 60 Å (Merck, Darmstadt, Germany), 5 µm Semi-Permeable Surface (SPS) C18, pore size 100 Å (Regis Technologies, Morton Grove, IL), and the in-house in situ prepared 10 µm bovine serum albumin-C18 (BSA-C18), pore size 120 Å. The packing material used for preparation of the capillary analytical columns was YMC ODS-AQ, pore size 120 Å (YMC Europe, Schermbeck/Weselerwald, Germany), with a particle size of 5 µm. All fused silica capillaries were purchased from Polymicro Technologies (Phoenix, AZ). Methanol (MeOH) and acetonitrile (ACN) of LiChrosolv grade, as well as ammonium acetate, acetic acid, ammonia solution (25%), and 1,2-dichloroethane of analytical grade, were purchased from Merck. All solutions were prepared using water purified with a Milli-Q Plus system (Millipore, Bedford, MA). Analytical standards of desipramine, nortriptyline, imipramine, amitriptyline, clomipramine, fluoxetine, and fluoxetine-d5 (ADs group) were bought from Sigma-Aldrich (St. Louis, MO) as hydrochloride salts. Analytical standards of albendazole, mebendazole, albendazole sulfoxide, and albendazole sulfone (AHs group) were kindly provided by Prof. Dr. Demerval de Carvalho (UNAERP, Ribeira˜o Preto, Brazil). Standard and Plasma/Urine Sample Preparation. Human plasma was obtained from healthy controls via the blood bank at the University Hospital (Uppsala, Sweden) and stored at -80 °C before use. Human urine was collected from a healthy male donor and stored at 4 °C for 24 h before use. Stock solutions of each antidepressant (AD) compound were prepared in ACN at 1 mg/mL. Intermediate solutions at 10 µg/ mL were obtained by dilution with ACN. The same was repeated for stock and intermediate solutions of each antihelmintic (AH), but at 100 and 1 µg/mL, respectively. Mixtures at 1250 and 100 ng/mL in ACN were obtained for each group (ADs and AHs) by mixing adequate aliquots of stock or intermediate solutions. Those solutions were kept at -20 °C. Working solutions of the analytical standards were obtained by drying adequate aliquots of the mixtures in a SpeedVac system (ISS110, Savant Instruments, Holbrook, NY) at a medium drying rate. The working standards were then resuspended in the adequate matrix (plasma, urine, or water) by agitation in a vortex. That step was followed by a 1:2 dilution with water if not otherwise stated. Afterward they were centrifuged at 14926g for 10 min in a Biofuge 13 centrifuge (Heraeus Sepatech, Osterode, Germany). When not at work, those

solutions were kept at 4 °C for a short term (max. 3 days). Fresh working solutions were prepared in the beginning of each new study. For the loading test, aqueous standard solutions were prepared, as stated above, in order to obtain growing loaded masses in the range from 83 pg to 4 ng for each analyte. The loaded volume in this experiment was 1 µL. Preparation of Capillary Columns. Extraction columns were prepared using fused silica capillaries with 200 and 320 µm i.d, as described below. The analytical columns were prepared with 200 and 250 µm i.d. tubings. Frits with approximately 0.2 mm length were placed inside capillaries by punching their tips against a glass fiber filter (Whatman GF/A, W & R Balston, UK) laid on a hard and smooth surface. Capillaries with 50 µm i.d. were used to insert and retain the glass fiber frits about 3 mm from the column tip. Capillaries with 184 µm o.d. were coupled together with 200 µm i.d. capillaries, while capillaries with 200 µm o.d. were coupled to capillaries with 250 and 320 µm i.d. One drop of epoxy resin (Araldite Hobby 10 min, Brascola, Sa˜o Paulo, Brazil) was used to keep the junction between the column capillary tubing and the connecting tubing. Several pieces of column hardware could be prepared in a reasonable time and be left overnight for a complete cure. One Jasco pump (model PU-980, Tokyo, Japan) delivered MeOH as packing solvent. Empty column hardware (ca. 20 cm) was connected to the packing reservoir, which then was filled with the slurry. Slurries were made of 8 mg of packing material sonicated for 1 min in 200 µL of 1,2-dichloroethane. ADS particles, exclusively, were suspended in MeOH because of their better wetability in that solvent. Using a flow rate of 0.2 mL/min, pressure was increased until 320 bar and kept constant for 1 h. The packing set (column and reservoir) was sonicated during the whole time inside an Elma Transsonic Digital S bath (Singen, Germany) operated at 100% of potency. In the case of RAM columns, they were packed for 30 min under 250 bar. After being packed, columns were cut to the correct length. In the case of analytical columns, just about 0.2 mm of the column tip was gently unpacked using the heated reflector nozzle of a heat gun (Ungar/ Weller model 6966B, Apex, NC) and then filled with a piece of glass fiber filter as described above. RAM particles were not easily unpacked just by heating. The packed bed was first gently dried at the tip with the heat gun and then unpacked by tapping with a twice-folded sheet of paper. The RAM columns had 3 mm of their tip unpacked in order to get a correct length of extraction bed. Afterward another capillary connector (50 µm i.d.) was inserted and glued, retaining a piece of frit. The second version of RAMADS columns employed fused silica capillaries of 0.5 mm (153 µm o.d. × 20 µm i.d.), instead of glass fiber, as the column “frit”. The column tips were emptied as described above. Then, precut capillaries (153 µm o.d.) were carefully introduced until the cutting scratch caused by the ceramic cutter was reached; afterward the short segments were released inside the columns by bending 90° and pulling apart the capillaries. One fused silica capillary with 184 µm o.d. and 50 µm i.d. was placed tightly holding the inner slighter capillary and glued with epoxy resin. A scheme of the column hardware setups is detailed in Figure 1. For the preparation of the RAM-BSA-C18, at first, one capillary column (320 µm i.d. × 15 mm) was packed using Kromasil C-18, pore size 120 Å with 10 µm particle size. The hardware employed

Figure 1. Instrumental setup of the capillary column switching LCESI-MS/MS system. The backflush mode is depicted in the switching valve, and the two possibilities of RAM column end-fitting hardware developed in this work (glass filter fritted and “fritless”) are illustrated in the inset.

was constructed as described above, but the column was packed under supercritical conditions and in situ modified as described elsewhere.35 Exclusion Profile Measurement. One Jasco pump PU-980 delivering water under flow rates of 20 and 40 µL/min was connected to a six-port manual injection valve (C6W, Valco Instruments, Houston, TX) equipped with an external capillary loop of 1 µL. Extraction columns were connected between this valve and a Jasco UV-975 detector (280 nm) equipped with a capillary cell (model UZ-JA97-CAP) from LC Packings (Amsterdam, The Netherlands). To evaluate the total amount of injected protein, the extraction column was replaced by a piece of fused silica capillary (300 mm × 50 µm i.d.). Flow Injection and Single-Column Test (FIA). The flow injection tests were performed by connecting a 25 cm fused silica capillary of 50 µm i.d. between the internal loop manual injection valve (C14W, Valco) and the ESI source. The valve was set with either 60 or 500 nL internal loop, and the signal was acquired in selected ion monitoring (SIM) mode by the MS system. In the single-column mode one analytical column was directly connected to the injection valve and with its 50 µm i.d. outlet capillary connected to the ESI. Chromatography. A 20 mM ammonium acetate/acetic acid buffer, pH 5.4, was calculated using PHoEBus program (Analis, Orleans, France) and confirmed using a pH meter. The prepared buffer was mixed with ACN obtaining 70% of organic solvent in the separation mobile phase. For the cleanup step, water and 20 mM ammonium acetate/ammonia electrolyte, pH 7.4, were tested (35) Santos Neto, A. J.; Rodrigues, J. C.; Fernandes, C.; Titato, G. M.; Alves, C.; Lancas, F. M. J. Chromatogr. A 2006, 1105, 71-76.

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with the addition of up to 10% of ACN. Mobile phases were degassed daily for 10 min in an ultrasonic bath. A scheme of the instrumental setup is depicted in Figure 1. The first chromatographic setup was composed of two Jasco pumps PU-980, one delivering the cleanup mobile phase at 20 µL/min and another delivering the separation mobile phase at 5 µL/min. The cleanup pump was connected to the six-port manual injection valve equipped with one external capillary loop (1 µL, if not otherwise stated). That valve was connected to an electronically actuated six-port switching valve (C6WE, Valco) holding the RAM column. The separation pump was also connected to the switching valve supplying mobile phase to the analytical column. The analytical column was directly connected to the valve (no capillary connection), and its outlet, a 15 cm capillary tubing (50 µm i.d.), was plugged to the ESI. The second setup was obtained using one Agilent 1100 series HPLC system (Germany) that basically consisted of a G1377A micro-autosampler and a G2226A nanopump. The nanopump replaced the Jasco separation pump of the previous setup, while the micro-autosampler replaced the manual injection valve. The rest of the system was kept unchanged. With these setups, in both foreflush and backflush modes, the valve was switched at 3.5 and switched back at 6.5 min. Mass Spectrometry. API I single quadrupole and API III plus triple quadrupole mass spectrometers, both from PE-Sciex (Concord, ON, Canada), were used in different stages of the work. Initially the API I was used for the first studies with the RAM and analytical columns, as well as development of setup and reduction of dead volume. Later the API III plus was used for the final tests, setup of the final approach, and coupling with the Agilent HPLC system. Both equipments were operated in positive mode using the same pneumatically assisted electrospray interface at off-axis positioning. To make the integration with the LC system easier, one zero dead volume Teflon sleeve (180 µm i.d.) was used to butt connect the column outlet with the 50 µm i.d. square-cut. bare-fused silica capillary ESI tip emitter. The ion spray and orifice potentials were 3500 and 50 V, respectively. The nebulization gas pressure was 40 psi for both instruments. During MRM and daughter scan experiments, the collision gas thickness was 2.10 × 1015 atoms/cm2 and collision energy 20 eV. Chromatograms were acquired with a dwell time of 100 ms when operating in either SIM or MRM modes. The monitored ions, corresponding to the protonated molecules, with their respective transitions were desipramine (267.4f72.2), nortriptyline (264.4f91.1), imipramine (281.4f86.1), amitriptyline (278.5f91.1), clomipramine (315.4f86.1), fluoxetine (310.3f44.1), fluoxetine-d5 (315.4f44.1), albendazole (266.2f234.1), mebendazole (296.2f264.1), albendazole sulfoxide (282.2f241.1), and albendazole sulfone (298.2f 266.1). For the loading capacity test of the RAM column, the monitored ions in MRM mode were shifted to the second most abundant isotopic peak, to avoid saturation of the MS detector. Fluoxetine and fluoxetine-d5 were monitored for both monoisotopic and second most abundant isotopic peaks in order to discriminate between ionization suppression, saturation of the detector, and extraction column overload. Matrix Effect Test. A Harvard Apparatus syringe pump (Holliston, MA) was used for continuous infusion experiments. To test the matrix effect on the ionization of the studied 6362

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compounds, a T-infusion was performed using a floating “T” connection of low dead volume.36 One 25 ng/mL solution containing all pharmaceutical drug standards in the mobile phase was infused at 1 µL/min. The rest of the system was operated in an ordinary way, with the separation pump delivering mobile phase at 5 µL/min. RESULTS AND DISCUSSION Development of Capillary RAM Columns. The protein exclusion capability of extraction columns prepared with ADSC18, SPS-C18, and BSA-C18 stationary phases were tested by direct injection of raw plasma without any analytical column in the system. The exclusion profiles of ADS and BSA extraction columns were compared to the profile of one flow injection of biological fluid using a fused silica capillary replacing the RAM precolumn. The peak detected using FIA had the same intensity of those peaks eluted after the proteins exclusion, meaning that the concentrations of the measured proteins on the peak maximum were equal for all peaks. Therefore, no significant amount of proteins was retained into the RAM columns. Moreover, the peak tailing returned to the baseline level after 2 min, showing that the whole exclusion of proteins lasted for a short time after the injection. The SPS column with 5-µm particle size was not able to handle the injection of such an amount of plasma. One single 1-µL injection of plasma generated clogging, the backpressure increased, and even backflushing the column with water did not completely solve the problem. The relatively small particles of the SPS column did not allow feasible injections of plasma under the same conditions as for the ADS and BSA columns; thus, the SDS column was excluded from further studies. Extraction columns were first built using a glass fiber filter as frit. This approach has been successfully used in the preparation of analytical and extraction columns for clean samples;27 however, its use in RAM columns for plasma injections resulted in clogging of the column inlet. A similar column construction was described by Petersson coupling solid-phase extraction to capillary electrophoresis, and the same problems with plasma samples were observed.37 In this work, even though no remaining particulate material was present in the plasma sample after centrifugation or filtering, column clogging took place after 5 to 8 diluted plasma injections. As the packing material used in the extraction column was proper for biological matrix, it was assumed that some part of the sample matrix was retained on the inlet frit causing its obstruction. This assumption is supported by the observation made by Yu and co-workers who showed the adsorption of proteins on the frit surface.38 Probably, some kind of protein, such as fibrins, for instance, may be retained on the glass fiber filter resulting in a premature clogging of the precolumn inlet. To avoid this obstacle, new “fritless” column hardware was developed, replacing the fiber filter by a short segment of fused silica capillary to hold the 25-µm ADS particles inside the column. The detail in Figure 1 shows the two options of column hardware evaluated during the extraction column construction. At the “fritless” column, the 25-µm particles were efficiently held inside the column (36) Bonfiglio, R.; King, R. C.; Olah, T. V.; Merkle, K. Rapid Commun. Mass Spectrom. 1999, 13, 1175-1185. (37) Petersson, M.; Wahlund, K.-G.; Nilsson, S. J. Chromatogr. A 1999, 841, 249-261. (38) Yu, Z. X.; Westerlund, D.; Boos, K. S. J. Chromatogr. B 1997, 704, 53-62.

by means of a short segment fused silica capillary with adequate dimensions. The short capillary, tightly positioned after the packed bed and contiguously contacted by the thicker connecting capillary, was able to hold the particles inside the extraction column in a very efficient way. Five columns utilizing this “fritless” approach were used their whole lifetime in backflush mode. Each column bed was inspected using a microscope and no indication of missing particles or void space could be observed in any column. The extraction columns, except the fiber filter fritted columns, were able to handle up to 60 injections of plasma samples, plus uncountable injections of aqueous solutions and urine samples. Columns prepared employing the two types of hardware were compared, considering their extraction efficiency, and both came out with the same extraction profile. The BSA and ADS columns were compared in different aspects, looking at the best option for the development of the RAM-LC-MS/MS capillary system. First, the backpressure performance of the columns was considered. Since the particle size used in the preparation of a BSA column was 10 µm, a larger internal diameter (320 µm) of that column resulted roughly in the same backpressure compared to a ADS column made of smaller internal diameter (200 µm). Reduction in the internal diameter of the BSA column is predicted to result in an inconvenient increase of column backpressure. Comparison of both columns under the same extraction conditions revealed similar recovery yields. Regarding the usability of the columns, the long term use of the BSA column resulted in leakage caused by some fragility in the epoxy resin gluing. On the other hand, none of the tested ADS columns demonstrated such fragility. The observed problems with the BSA columns were related to the relatively large space between the connection capillary of 200 µm o.d. and the column tubing of 320 µm i.d. Another disadvantage observed with the BSA column was its larger dead volume, although it did not contribute to any significant peak broadening; however, it resulted in a larger disturbance of the MS baseline. Since a predominantly aqueous solution is employed in the cleanup step, the solvent plug contained in the extraction column reaches the detector after the column switching. Therefore, larger aqueous plugs resulted in more intense baseline disturbances. Moreover, 10-µm particles could not be used in the “fritless” approach. One last consideration is the fact that the RAM-BSAC18 approach involves in situ derivatization of the stationary phase, an additional step with respect to the simple packing of ADS particles inside one column. Those reasons guided the efforts of this work toward the development of a system using ADS columns. Of course, the development of BSA-C18 particles with a larger particle size could be considered, but that would not fit into the scope of this report. The epoxy resin employed in the column hardware was tested as a source of interference in the analysis. A piece of resin was added to one vial containing mobile phase (70% ACN) and sonicated for 30 min. The supernatant was injected into the MS in scan mode, and the most intense MS peaks between 50 and 1000 m/z were identified. Afterward a new column was conditioned using a mobile phase with the same composition, and its effluent was electrosprayed into the MS. The five most intense interference MS peaks (m/z 191.0, 359.5, 422.0, 512.0, 540.0) of the epoxy resin were monitored in SIM mode. The initial baseline

Figure 2. Evaluation of peak focusing using column switching and single column injection. Trace 1, FIA (60 nL); trace 2, column switching (500 nL); trace 3, column switching (1.9 µL); trace 4, column switching (3.8 µL); trace 5, single-column (60 nL); trace 6, FIA (500 nL).

showed the presence of all the peaks, but after 5 min it started to decrease in signal and no remaining signal could be acquired after 30 min. The same MS peaks were monitored during the normal use of the columns in backflush approach, and no trace of interference was observed. Development of the Column Switching Approach. Analytical columns, 50 mm length, were prepared, and their chromatographic efficiencies were evaluated based on the reduced plate height (h) measurements at single-column mode. Values as low as 2.5 were obtained for the most retained compounds. An extracolumn effect was partially hindering the efficiency although such columns were still considered to provide adequate performance. However, the ability of the column switching system in retaining and improving the efficiency of the chromatographic system was considered critical and therefore was evaluated. As can be seen in Figure 2, comparison of trace 1 (flow injection of 60 nL, indicating the band dispersion in the lines from the injection to the detection) with traces 2-4 (different injection volumes onto the RAM column) shows the ability of the RAM column to efficiently focus the analytes. For comparison, one 60 nL injection (trace 5 in Figure 2) in single-column mode without on-column focusing is included, as well as one flow injection of 500 nL (trace 6 in Figure 2) demonstrating the impact of a large volume injection on the peak width without proper focusing. All the injections in the column switching mode demonstrated the same profile, proving efficient focusing up to 3.8 µL (largest volume tested) in the injection volume. Furthermore the peak widths obtained with the column switching approach were about one-half the width of those obtained with a 60 nL injection in single-column mode. The column switching system built for this work can be operated in two modes, named backflush and foreflush. In the backflush mode better peak profiles are expected, as the analytes can be focused on the top of the precolumn and then be directly eluted to the analytical column. This avoids the need to travel through the whole length of the poorly efficient precolumn. On the other hand, in foreflush mode the precolumn will provide a better protection of the analytical column. In this study a dramatic Analytical Chemistry, Vol. 79, No. 16, August 15, 2007

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Figure 3. Influence of column switching mode (backflush × foreflush) on the peak symmetry and efficiency. Panel A: AHs in foreflush mode; panel B: AHs in backflush mode (for AHs, peaks 1-4 are albendazole sulfoxide, albendazole sulfone, mebendazole, and albendazole, respectively); panel C: ADs in foreflush mode; panel D: ADs in backflush mode (for ADs, peaks 1-6 are desipramine, fluoxetine, nortriptyline, imipramine, amitriptyline, and clomipramine, respectively).

loss of efficiency was observed, in a general way, for the foreflush mode as shown in Figure 3 (panels a and c compared to panels b and d). The compounds were grouped according to their pharmaceutical characteristics, observing that the group presenting a more basic character (panel c), formed by the antidepressants, was most affected by the foreflush mode. This observation suggests that “active” sites are present in the RAM or the transfer lines. The antihelmintics were not so severely affected concerning the peak width; however, the two metabolites (1 and 2 in panel a) were almost completely lost. In both switching modes (panels a and b) the same extraction/cleanup procedure was followed, indicating that the loss of the two least-retained compounds occurs after the valve switching. ESI suppression would be one way to explain the reduction in response. Since they are weakly retained, they can be displaced through the extraction column during the cleanup and eluted close to the void volume (containing a highly aqueous plug of solvent and possible interference). Further work in this study was done in the backflush mode in order to obtain the best system performance. 6364

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Loading Capacity of the ADS Capillary Column. The saturation capacity of one 1-cm ADS column was evaluated with MRM measurements. Eleven different compounds (10 standards and 1 deuterated standard) were injected at the same time in increased concentrations. Saturation effects due to MS detector overload were minimized by monitoring the second most intense isotopic peak for each compound. For fluoxetine, both (mono and second most intense isotopic peaks) were evaluated in order to discriminate saturation due to column and detector overload. A linear trend in the loading profile of the column for all the 11 compounds was found until 500 pg of loaded mass/compound before saturation of the extraction starts to take place. Extrapolation of linearity of the trend line to intercept the saturation level resulted in one value of approximately 1.6 ng/compound. Assuming the packing density as approximately 0.7, the column content was ca. 220 µg. So, the linear limit was around 25 µg/g of packing material, and the calculated maximum amount is 80 µg/g corresponding to 87 and 278 nmol/g, respectively. This translates to an upper limit in concentration of 500 ng/mL for each compound utilizing 1 µL injection volume. The developed method uses plasma

samples diluted 1:2 with water, so the column could be loaded with concentrations up to 1.5 µg/mL, under the studied conditions, without reaching the upper linear limit of the ADS column. Saturation of the MS detector ion counting was not observed when the signals from the loading profile of the monoisotopic and second most intense isotopic peak were compared. Matrix Effect on the Electrospray Ionization and Extraction. The matrix effect in this RAM-LC-ESI-MS/MS approach could take place in two different places, the ESI source, causing ionization suppression, and the RAM extraction, causing breakthrough of the analytes. Figure 4 shows a summary of the results obtained in the ionization suppression tests. As the column switching system directs a plug of predominantly aqueous solvent to the ESI, an initial suppression of the baseline signal, seen in Figure 4, should be expected as normal for a short time after the valve switching. Moreover, the comparison of TIC profiles for water (trace 2), plasma (trace 3), and urine (trace 4) injections did not demonstrate important differences after the required time for the aqueous plug to leave the system. Also in Figure 4, trace 5, the MRM transition of the first eluted peak of each drug group, eluted just after the disturbance caused by the aqueous plug, is depicted. The dynamic procedure involved in the extraction performed by RAM was discussed elsewhere.39 It can be summarized as two coexistent equilibriums: one between the drug and the binding site of the proteins and another between the drug and the retention site of the stationary phase. However, because of the characteristics of the RAM the equilibriums can be displaced, benefitting the drug extraction. The RAM is especially designed to avoid the matrix effect caused by the protein and other hydrophilic macromolecules. Even though, some unspecific adsorption of proteins can occur inside the column, and small endogenous interference can compete in the interaction with the retention site. This matrix effect was evaluated, calculating the relative recovery of the biological fluid with respect to the aqueous one. For the ADs group, no important matrix effect was observed, and usually the relative recoveries for those compounds were between 80 and 120%. For the AHs group, some effects were observed for the metabolites, and a slightly lower recovery was also related to the urine matrix with respect to plasma. That can be explained by the fact that the AHs present a lower affinity for the ADS column, especially the very polar metabolites. Urine is known to have a much lower content of proteins compared to plasma; however, smaller endogenous compounds can be present, as well as higher concentration of salts. That can explain the fact that urine matrix has a larger influence on analytes having a lower interaction with the stationary phase. The cleanup mobile phase can also affect the overall performance of the extraction but it is more related to the absolute recovery of the analytes. For instance, the high concentration of organic modifier can involve a breakthrough of the analytes during the cleanup whatever the introduced matrix. Improved analyte extraction in average of 38%, possibly due to an ion paring effect, and backpressure reduction were achieved by the use of a low concentration volatile electrolyte (ammonium acetate/ammonia 20 mM) at pH 7.4, instead of water. Usually after one injection of plasma the backpressure presented (39) Hermansson, J.; Grahn, A.; Hermansson, I. J. Chromatogr. A 1998, 797, 251-263.

Figure 4. Evaluation of matrix effect on the ionization efficiency of the ESI-MS. Panel A: TIC; panel B: desipramine MRM; panel C: albendazole sulfoxide MRM. Runs 1-5 are no injection, water injection, diluted plasma injection (1:2), diluted urine injection (1:2), and standard injection, respectively.

an increasing trend, dropping after some time. With the electrolyte, the rate of increasing pressure was lower and the pressure started to drop earlier. The extraction was also affected (analyte dependent) by the amount of ACN in the cleanup mobile phase. For the metabolites of AHs, use of ACN resulted in reduced recovery. However, for most of the tested compounds, 10% of ACN did not influence recovery. Analytical Chemistry, Vol. 79, No. 16, August 15, 2007

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Figure 5. Representative chromatogram of the separation and MRM MS detection of 10 drugs and metabolites using the described approach and within a time window of less than 2.5 min. Peaks 1-10 are, respectively, albendazole sulfoxide, albendazole sulfone, mebendazole, desipramine, fluoxetine, albendazole, nortriptyline, imipramine, amitriptyline, and clomipramine.

Applicability of the Developed System. Two sets of instruments were tested during the development of the RAM-LC-MS approach, keeping the same column switching valve as the core of the system. Besides some intrinsic differences between the employed LC systems (i.e., line backpressure and flow rate limits), the overall performance of the system was the same for the analysis of the equal samples. In this work, adequate LC-MS/MS separation, shown in Figure 5, of 10 different polar basic drugs was obtained in a short analysis time (8 min in total), including the sample preparation step. In further work, a validation of such applications will be fully demonstrated and discussed. However, to prove the merit of this work, some figures are presented. Detection limits between 0.08 and 1.5 ng/mL were obtained for all evaluated compounds, LOQ (limit of quantitation) values being equal to 1 ng/mL for ADs and ranging from 1 to 5 ng/mL for AHs. Precision and reproducibility were evaluated as relative standard deviation (RSD) with five replicates at three levels and on three different days (intraday and interdays precision) showing results lower than 20% at the LOQ level and lower than 14.5% at the other levels. Accuracy measured in the same scheme resulted in values closer than 20% at the LOQ level and closer than 13.1% at the other levels. Recovery has been already mentioned in the previous section, and it is adequate to the studied levels. Linearity was between LOQ and 500 ng/mL for AHs and between LOQ and 250 ng/mL for ADs with correlation coefficients (r) higher than 0.996 for all the compounds. One important point to consider is that the required injected sample volume for obtaining those results was 1 µL of biofluid (diluted 1:2 with water). This means that such detectability was achieved by injecting only approximately 333 nL of raw sample, while most of other methods in the literature currently describe the need to load between 100 µL and 1 mL in order to reach similar detection levels. The low ng/mL quantification levels and the linearity range obtained in this work are adequate for therapeutic drug monitoring (TDM) and pharmacokinetic/bioequivalence (PK/BE) applications.40-44 The lowest (40) Preskorn, S. H.; Dorey, R. C.; Jerkovich, G. S. Clin. Chem. 1988, 34, 822828.

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level required in PK/BE studies, for the studied compounds, is about 1 ng/mL for fluoxetine45 and albendazole43 and higher for some other drugs.46 For TDM applications, the required limits are higher, and many methods in the literature report about 10 ng/mL or more as LOQ.47-51 These levels, presented here, result from a 15 year old MS system detecting loaded masses of about 0.1 pg (between few femtomoles and hundreds of attomols). With a state of the art instrument we expect to achieve between 10and 100-fold better response. Carryover phenomena were observed at a level of approximately 0.3% of previously injected samples and that can be associated with the low inertia of the valves used in this study. The use of chemically inert valves and even some more washing steps52 implemented by other chromatographic pumps/autoinjectors can improve that aspect, if needed. In a previous work, carryover was not observed using a cheminert valve.35 Recently, Bakhtiar53 reviewed quantification problems related to small drug analysis in biofluids and addressed some strategies on solving carryover problems. Moreover, if increased throughput is necessary, two extraction columns in parallel (with a 10-port valve) can be used. Also, in very demanding applications an offline 96-well simple protein precipitation could be implemented, reducing the online cleanup time and prolonging the lifetime of the extraction columns, but still avoiding ion suppression phenomenon. Of course, the use of monolithic columns31 or UPLC columns54 in the analytical dimension has been reported as another way to further shorten the analysis time. The approach developed in this work turned out to offer a good possibility for the field of biomolecules analysis in biofluids, joining the advantages of miniaturization in LC and MS. In contrast with the chip technology that is a nice promise for the future, the capillary and nano LC technology has already proven to be trustworthy in its present state. CONCLUSIONS A new approach coupling RAM-LC-ESI-MS/MS in a fully capillary scale has been developed for the simultaneous and online (41) Ivandini, T. A.; Sarada, B. V.; Terashima, C.; Rao, T. N.; Tryk, D. A.; Ishiguro, H.; Kubota, Y.; Fujishima, A. J. Electroanal. Chem. 2002, 521, 117-126. (42) Raggi, M.; Mandrioli, R.; Casamenti, G.; Volterra, V.; Desiderio, C.; Fanali, S. Chromatographia 1999, 50, 423-427. (43) Chen, X.; Zhao, L.; Xu, H.; Zhong, D. J. Pharm. Biomed. Anal. 2004, 35, 829-836. (44) Bonato, P. S.; Lanchote, V. L.; Takayanagui, O. M. J. Chromatogr. B 2003, 783, 237-245. (45) Vlase, L.; Imre, S.; Leucuta, S. Talanta 2005, 66, 659-663. (46) Yoshida, H.; Hidaka, K.; Ishida, J.; Yoshikuni, K.; Nohta, H.; Yamaguchi, M. Anal. Chim. Acta 2000, 413, 137-145. (47) Raggi, M. A.; Mandrioli, R.; Casamenti, G.; Bugamelli, F.; Volterra, V. J. Pharm. Biomed. Anal. 1998, 18, 193-199. (48) Frahnert, C.; Rao, M. L.; Grasmader, K. J. Chromatogr. B 2003, 794, 3547. (49) Kollroser, M.; Schober, C. Ther. Drug Monit. 2002, 24, 537-544. (50) Aymard, G.; Livi, P.; Pham, Y. T.; Diquet, B. J. Chromatogr. B 1997, 700, 183-189. (51) Duverneuil, C.; de la Grandmaison, G. L.; de Mazancourt, P.; Alvarez, J. C. Ther. Drug Monit. 2003, 25, 565-573. (52) Asakawa, Y.; Ozawa, C.; Osada, K.; Kaneko, S.; Asakawa, N. J. Pharm. Biomed. Anal. 2007, 43, 683-690. (53) Bakhtiar, R.; Majumdar, T. K. J. Pharmacol. Toxicol. Methods 2007, 55, 262-278. (54) de Villiers, A.; Lestremau, F.; Szucs, R.; Gelebart, S.; David, F.; Sandra, P. J. Chromatogr. A 2006, 1127, 60-69.

analysis of drugs in small volumes of untreated biological fluids (only 1:2 dilution and centrifugation). The developed capillary precolumns using RAM-ADS particles have shown adequate exclusion of proteins and completely eliminated the matrix effect on the electrospray ionization. Also, effective focalization of the analytes in the precolumn has been observed and permitted higher injection volumes in the column switching mode than that allowed for the direct injection one. The developed fritless column hardware has allowed repetitive injections of raw biological fluids and has been able to hold at least pressures of 400 bar, without any leakage. The use of a low concentration volatile electrolyte at physiological pH has shown to be better than using pure water during the clean-up step. ACN has also benefitted the clean up efficiency of the mobile phase, at lower concentrations; still it has shown a compound-dependent influence on the amount of extraction of different analytes. The loading capacity of a 1-cm precolumn has been evaluated in the simultaneous extraction of different compounds and showed a wide linear dynamic range; therefore, the limiting parameter in the detection dynamic range is the response linearity of the MS system. The developed approach has been evaluated for the separation and selective detection of 10

drugs and metabolites. Preliminary results have shown an adequate analysis in 8 min with quantifiable detection reaching values as low as 1 ng/mL. It is important to highlight that only approximately 333 nL of sample has been used to reach a low ng/mL level of detection, while traditional scale methods have been consuming between 100 µL and 1 mL of sample volume for achieving a similar performance. ACKNOWLEDGMENT A. J. Santos-Neto thanks CAPES Brazilian grant agency for financial support (PDEE program, scholarship BEX 3802/05-1). The support of the Swedish Research Council (grants 621-20025261, 629-2002-6821, 621-2005-5379 (J.B.)) is acknowledged. We acknowledge Alessandra M. Monteiro for help in constructing some of the columns tested in this work, and Professor Demerval de Carvalho for supplying some analytical standards.

Received for review April 5, 2007. Accepted June 12, 2007. AC070671G

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