Journal of Chromatography A, 1323 (2014) 163–173

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Non-aqueous capillary electrophoresis for the analysis of acidic compounds using negative electrospray ionization mass spectrometry Grégoire Bonvin, Julie Schappler, Serge Rudaz ∗ School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Bd d’Yvoy 20, 1211 Geneva 4, Switzerland

a r t i c l e

i n f o

Article history: Received 17 September 2013 Received in revised form 30 October 2013 Accepted 1 November 2013 Available online 13 November 2013 Keywords: Capillary electrophoresis CE-MS NACE Negative ESI Sheathless interface Sheath liquid interface

a b s t r a c t Non-aqueous capillary electrophoresis (NACE) is an attractive CE mode, in which water solvent of the background electrolyte (BGE) is replaced by organic solvent or by a mixture of organic solvents. This substitution alters several parameters, such as the pKa , permittivity, viscosity, zeta potential, and conductivity, resulting in a modification of CE separation performance (i.e., selectivity and/or efficiency). In addition, the use of NACE is particularly well adapted to ESI-MS due to the high volatility of solvents and the low currents that are generated. Organic solvents reduce the number of side electrochemical reactions at the ESI tip, thereby allowing the stabilization of the ESI current and a decrease in background noise. All these features make NACE an interesting alternative to the aqueous capillary zone electrophoresis (CZE) mode, especially in combination with mass spectrometry (MS) detection. The aim of this work was to evaluate the use of NACE coupled to negative ESI-MS for the analysis of acidic compounds with two available CE-MS interfaces (sheath liquid and sheathless). First, NACE was compared to aqueous CZE for the analysis of several pharmaceutical acidic compounds (non-steroidal anti-inflammatory drugs, NSAIDs). Then, the separation performance and the sensitivity achieved by both interfaces were evaluated, as were the impact of the BGE and the sample composition. Finally, analyses of glucuronides in urine samples subjected to a minimal sample pre-treatment (“dilute-and-shoot”) were performed by NACE-ESI-MS, and the matrix effect was evaluated. A 20- to 100-fold improvement in sensitivity was achieved using the NACE mode in combination with the sheathless interface and no matrix effect was observed regardless of the interfaces. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Non-aqueous capillary electrophoresis (NACE) is an attractive CE mode that consists of using organic solvents as the background electrolyte (BGE) medium. The use of an organic BGE in CE has been the subject of several reviews [1–4], and it offers numerous interesting characteristics, such as different relative permittivity (εr ), as well as acido-basic and solvation properties, leading to a significant modification of the degree of ionization and the protonation equilibrium. The pKa and zeta-potential ( pot ) of solutes (i.e., analytes and ions constituting the BGE) and silanol groups are strongly affected by the nature of the BGE medium, resulting in changes in the electrophoretic mobility (eff ), electroosmotic flow mobility (EOF ) leading to a change in selectivity and sometimes improve the separation of certain analytes [1]. The low solvation property and permittivity of organic solvents also introduces supplemental interactions such as heteroassociation, homoassociation, and ion-pairing interactions, which can result in additional and

∗ Corresponding author. Tel.: +41 22 379 63 36; fax: +41 22 379 68 08. E-mail address: [email protected] (S. Rudaz). 0021-9673/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2013.11.011

different selectivity [1,3]. This feature can even confer electrophoretic mobility on initially weakly and/or non-charged analytes [5–9]. In addition, the low polarity of organic solvents can sometimes enhance the dissolution of hydrophobic and poorly water-soluble compounds, as well as improve the stability of water-sensitive compounds [10], allowing their analysis without any degradation. However, from a CE-side, the asserted advantages of non-aqueous solvents over aqueous solvents are generally overestimated [11,12] and NACE should be regarded as a complementary and alternative mode to the aqueous CZE. NACE is a tailored mode for coupling CE with electrospray ionization (ESI) mass spectrometry (MS) and has been used for the analysis of pharmaceutical compounds [13–27], structurally related alkaloids in plants [28–39], and chiral compounds [40–43]. In general, the use of highly volatile organic BGEs with low surface tensions improves the formation of easily evaporable droplets, increasing ionization efficiency while ensuring a stable spray over a wide range of voltages. Moreover, the absence of water reduces the number of side electrochemical reactions, thus stabilizing the ESI current. All these properties are particularly well adapted to the negative ionization mode. Additionally, the deprotonation process in negative ESI can be further improved by using BGEs that are

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composed of high gas-phase basicity (GB) solvents such as acetonitrile (GB = 748.0 kJ/mol [44]), methanol (GB = 724.5 kJ/mol [44]), or a mixture of them. In CE, the coupling to ESI-MS is mainly achieved with two configurations [45,39]. The first configuration is the sheath liquid interface in which the spray is assisted by a nebulizing gas and an additional sheath liquid. The latter is delivered at a few ␮L/min (1–10 ␮L/min) through a stainless steel needle and is mixed with the CE effluent at the capillary tip. Consequently, the ESI operates in electrospray mode and behaves as a concentration-sensitive detector. The sheath liquid and the BGE (i.e., composition and pH) have a direct impact on ionization, evaporation, the ESI current, and the spray stability. In NACE mode, the sheath liquid setup was found to be robust and yielded very good results in terms of sensitivity, selectivity, and stability [1–4]. Nevertheless, CE performance (i.e. separation efficiency and resolution) decreased with the use of poor viscosity solvents due to the suction effect that was generated by the nebulizing gas and sheath liquid [46,47]. Consequently, relatively long capillaries with small internal diameters (I.D.) are required to overcome this issue [26]. Interestingly, the loss of sensitivity observed in aqueous CZE because of a dilution with the sheath liquid could be an advantage in NACE. The water in the sheath liquid exhibits higher solvation properties, thus allowing the suppression of covalent and ionic interactions between analytes and ions that can hamper the ionization process [48,49]. The second configuration consists of a porous sheathless interface, initially developed by Moini et al. [50], that functions without pneumatic assistance. In this case, the ESI operates in the nanospray mode because the flow rate at the tip is in the range of nL/min. The BGE composition is the key parameter that influences both ionization and the spray stability. This interface acts as a concentration-sensitive detector at flow rates above 10–15 nL/min and as a mass-sensitive detector below this range [51]. Consequently, the composition and pH of the BGE determine whether ESI acts as a mass- or concentrationsensitive detector. This interface has yielded very promising results for the analysis of pharmaceutical compounds [52–55], peptides [51,56–58], and proteins [59–61], but only in aqueous CZE or as an infusion device. To our knowledge, the sheathless interface has never been evaluated in NACE-ESI-MS in the negative ionization mode. The purpose of this work was to evaluate the potential of NACE for the analysis of representative acidic compounds using both interfaces (sheath liquid and sheathless). First, the NACE mode was compared to the aqueous CZE mode for the analysis of pharmaceutical acidic compounds (non-steroidal anti-inflammatory drugs, NSAIDs). Then, the separation performance and sensitivity were evaluated. A special focus was placed on the impact of the BGE and sample compositions because one major issue in NACE is the compatibility of the organic BGE with the sample solvent or the matrix in which the analytes are dissolved. A change in this composition can affect the homogeneity and the local electric field of the analyte zone, as well as the pKa of the analytes and their interactions with the dissolution solvent, which can drastically change the method performance. Analyses of glucuronides in urine samples were eventually performed by NACE-ESI-MS after a simple urine dilution (i.e., the “dilute-and-shoot” approach). Because this sample pre-treatment method is non-selective, the matrix effect was evaluated for both interfaces.

2. Materials and methods 2.1. Chemicals Acetonitrile (ACN), methanol (MeOH), propan-2-ol (iPrOH), acetic acid glacial, ammonium acetate were ULC/MS grade

from Biosolve BV (Volkenswaard, The Netherlands). Ammonium hydroxide and triethylamine (TEA) solution were of analytical grade from Sigma–Fluka (Buchs, Switzerland). Mefenamic acid (Mef), flufenamic acid (Flu), diclofenac (Dic), ibubrofen (Ibu), suprofen (Sup), and indomethacin (Ind) were purchased from Sigma–Aldrich (Steinheim, Germany). Morphine-3␤-glucuronide (M3G), codeine-6-␤-glucuronide (C6G), naloxone6-␤-glucuronide (N6G), and ethyl-␤-glucuronide (EthG) reference solutions at 1 mg/mL in MeOH were obtained from Lipomed AG (Arlesheim, Switzerland). 2.2. BGE and sample preparation 2.2.1. BGEs In CZE mode, separations were carried out with aqueous BGEs at different concentrations of acetic acid (25, 50, and 75 mM) buffered with ammonium hydroxide at different pH (8.5, 9.0, and 9.5). pH values were measured with a SevenMulti pH meter (MettlerToledo, Schwerzenbach, Switzerland). In NACE mode, separations were carried out with organic BGEs constituted of different concentrations of ammonium acetate (5, 10, and 15 mM) dissolved in either ACN-MeOH 80:20 (v/v) or ACNMeOH 60:40 (v/v). 2.2.2. Standard samples The non-steroidal anti-inflammatory drugs (NSAIDs) stock standard solutions were prepared by dissolving each reference compound in MeOH to obtain a concentration of 1 mg/mL. NSAID standard solutions at desired concentrations were prepared by diluting stock solutions in water or in different proportion of ACNMeOH. Glucuronide standard solutions were prepared at desired concentration by diluting each reference solution in ACN-MeOH 60:40 (v/v). Each solution was stored in sealed glass vial at 4 ◦ C. 2.2.3. Urine samples Urine samples were prepared by pooling six blank urines collected from healthy non-drug consumers and then stored in polypropylene tubes at −20 ◦ C. The pooled urine samples were defrosted at ambient temperature and centrifuged at 11,180 × g (rotating radius = 10 cm) for 2 min (Heragus Biofuge 17 DS, SepaTech, Engen, Germany). The supernatant was filtered through a 0.45 ␮m Nylon fiber (BGB Analytik AG, Böckten, Switzerland) and spiked at the desired concentration with glucuronide reference solutions. The spiked urines were finally diluted 20-fold in ACNMeOH 60:40 (v/v) and directly injected in the CE capillary (for matrix effect quantitative determination) or infused (for matrix effect qualitative determination). 2.2.4. Matrix effect 2.2.4.1. Quantitative determination. The matrix effect (ME) was quantified using a method previously described by Matuszewski et al. [62]. Two types of samples were required. Sample A consisted of a mixture of all glucuronides at 5 ␮g/mL in water and was diluted 20-fold in ACN-MeOH 60:40 (v/v) prior to injection. Sample B consisted of blank pooled urine diluted 20-fold in ACN-MeOH 60:40 (v/v) and was spiked with a mixture of all glucuronides at 250 ng/mL prior to injection. The ME was calculated by comparing peak areas normalized by analytes’ migration time of sample B vs. sample A. Each sample was injected twice (n = 2) and analyzed by NACE-ESI-MS with both interfaces. 2.2.4.2. Qualitative determination. The matrix effect was determined using the sheath liquid interface as a post-capillary infusion device [63]. A blank pooled urine was spiked with a mixture of all glucuronides reference solution at 5 ␮g/mL and diluted 20-fold in

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ACN-MeOH 60:40 (v/v) prior to injection. A mixture of the same glucuronides at 5 ␮g/mL dissolved in iPrOH-H2 O-NH4 OH 49.5:49.5:1 (v/v/v) was continuously infused at 4 ␮L/min via the sheath liquid device. Analyses were performed in duplicate (n = 2).

2.3. Instrumentation 2.3.1. CE CE analysis was carried out with a ProteomeLabTM PA 800 plus Protein Characterization System (Beckman Coulter, Brea, CA, USA) equipped with a temperature controlled autosampler and a power supply that was capable of delivering up to 30 kV. Experiments were performed at 25 ◦ C in normal polarity mode with the anode at the inlet and the cathode at the outlet. A constant voltage of 30 kV, with an initial ramping of 3000 V/s (10 s), was applied during the analysis. With the sheath liquid interface, the separation was performed in an uncoated fused silica capillary (BGB Analytik AG, Böckten, Switzerland) with an I.D. of 50 ␮m, an O.D. of 365 ␮m, and a total length of 100 cm. Prior to each sample injection, the capillary was rinsed at 6 bar for 1.5 min with fresh BGE. Samples diluted in organic solvent were hydrodynamically injected at100 mbar for 9 s (equivalent to 2.0% of the capillary length). Samples diluted in water were hydrodynamically injected at 100 mbar for 25 s (equivalent to 2.0% of the capillary length). Spiked urine samples diluted in organic solvent were hydrodynamically injected at 50 mbar for 9 s (equivalent to 1.0% of the capillary length). A BGE post-plug, equivalent to 0.5% of the capillary length, was implemented after each sample injection. With the sheathless interface, the separation was performed in a prototype uncoated FS capillary with a porous tip (Beckman Coulter, Brea, California, USA) and with an I.D. of 30 ␮m, an O.D. of 150 ␮m, and a total length of 100 cm. Prior to each sample injection, the capillary was rinsed at 6 bar for 3 min with fresh BGE. Samples diluted in organic solvent were hydrodynamically injected at 250 mbar for 10 s (equivalent to 2.0% of the capillary length). Spiked urine samples diluted in organic solvent were hydrodynamically injected at 125 mbar for 10 s (equivalent to 1.0% of the capillary length). A BGE post-plug, equivalent to 0.5% of the capillary length, was implemented after each sample injection. It should be noted that the viscosities of each tested BGE were taken into account to adjust the injected volume. The viscosities () of the organic BGEs were experimentally determined by measuring the counter-pressure generated by the different BGE infused at 2 mL/min in a 0.124 mm × 200 cm tube. Then the different viscosities were calculated by the Poiseuille–Hagen equation. Their values were equal to 0.36 mPa s regardless of the electrolyte concentration.

2.3.2. Interfaces 2.3.2.1. Sheath liquid interface. CE was coupled to ESI-MS via a commercial sheath liquid interface equipped with a stainless steel needle (Agilent, Palo Alto, CA, USA). Different sheath liquid solutions were tested, i.e., iPrOH-H2 O-NH4 OH 49.5:49.5:1 (v/v/v), iPrOH-TEA 25 mM 50:50 (v/v), and iPrOH-organic BGE (10 mM ammonium acetate diluted in ACN-MeOH 80:20 (v/v)) 50:50 (v/v). Sheath liquid consisting of iPrOH-H2 O-NH4 OH 49.5:49.5:1 (v/v/v) was eventually selected because it resulted in the best ESI current stability and ionization independent of the nature of the BGE. The other sheath liquids generated very unstable ESI currents, substantial noise, and/or strong ionization suppression. The sheath liquid flow rate (delivered by a syringe pump) was set to 3 ␮L/min. The ESI voltage was set between −4400 V and −4500 V to reach a stable ESI current ranging from 31 to 39 nA. The nebulizing gas pressure

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was set to 4 psi (27,779 Pa), and the drying gas flow rate and temperature were set to 4 L/min and 250 ◦ C, respectively.

2.3.2.2. Sheathless interface. CE was coupled to ESI-MS via a prototype sheathless nanospray interface developed by Beckman Coulter. The end of the separation capillary was etched (3–4 cm), resulting in an O.D. of approximately 40 ␮m. The capillary was inserted into a grounded ESI needle until ∼5–8 mm of the porous tip protruded. The ESI needle, made of stainless steel, was filled with ammonium acetate 75 mM (pH = 9.0) providing the electrical contact. The etched capillary-ESI needle set was inserted into a special housing designed by Beckman Coulter that permits the protection of the protruding tip and its proper installation near the MS entrance. The latter was modified with a gas diverter and a nanoelectrospray shield (Bruker Daltonics GmbH, Bremen, Germany). The interface positioning was optimized according to a previous study [52]. The ESI voltage was set between −1200 V and −1300 V to reach a stable ESI current ranging from 31 to 39 nA. The nebulizing gas was switched off, and the drying gas flow rate and temperature were set to 0.3 L/min and 350 ◦ C, respectively.

2.3.3. MS MS measurements were performed with a single quadrupole Agilent Series 1100 MSD (Agilent, Palo Alto, CA, USA). MS detection was carried out in selected ion monitoring (SIM) mode for the negative molecular ion [M−H]− (except for Sup where the [M−COOH]− fragment was followed). For both interfaces and CE modes, the optimal fragmentor voltage values were obtained by direct infusion through the CE capillary of each selected compound into the mass spectrometer. The limits of detection (LODs) were expressed as the concentration where the signal-to-noise ratio was equal to 3 (S/N = 3) and calculated from the respective extracted ion electropherograms.

3. Results and discussion The present work evaluated the usefulness of non-aqueous capillary electrophoresis (NACE) for the analysis of acidic compounds in negative ESI-MS. Experiments were conducted with two CEMS interface configurations, namely the sheath liquid interface and the sheathless interface. The performance of NACE in terms of separation, efficiency, and sensitivity was first evaluated with nonsteroidal anti-inflammatory drugs (NSAIDs) as model compounds. In a second set of experiments, the separation of glucuronides was performed by NACE-ESI-MS in standard solutions and in urine samples to emphasize the strengths of NACE-ESI-MS for biological analysis.

3.1. NACE-ESI-MS with the sheath liquid interface In a first set of experiments, NSAIDs were selected as model compounds (Fig. 1) because they represent an important pharmacological class of drugs that are commonly used for their analgesic and antipyretic properties. The set of NSAIDs that was used for the present work was composed of mefenamic acid (Mef), flufenamic acid (Flu), diclofenac (Dic), ibuprofen (Ibu), suprofen (Sup), and indomethacin (Ind), which can only be detected by MS in negative ionization mode (ESI− ) due to the presence of a single ionizable carboxylic group. Aqueous CZE and NACE were compared in terms of separation performance i.e., selectivity, peak width (expressed as full width at half maximum, FWHM), efficiency (N), resolution, and sensitivity with the sheath liquid interface. A special focus was placed on the BGE and sample compositions for optimal NACE.

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Fig. 1. Selected non-steroidal antiflammatory drugs (NSAIDs) and glucuronides. The pKa values (* basic pKa ) were calculated using Advanced Chemistry Development (ACD/Labs) Software V11.02 (© 1994–2013 ACD/Labs).

3.1.1. Investigation of the BGE Several aqueous BGEs at different ammonium acetate concentrations (25, 50, and 75 mM) and pH values (8.5, 9.0, and 9.5) were tested in aqueous CZE mode with the sheath liquid interface. The best results were achieved using a 50 mM ammonium acetate buffer at pH 8.5 (Fig. 2A), although an insufficient separation was achieved. To improve the separation selectivity, NACE was selected, and the influence of the organic solvent proportion and electrolyte concentration in the BGE was evaluated. The choice of the organic solvents is of utmost importance and was made based on two specific properties: (i) sufficiently high εr / and εr 2 / values to guarantee appropriate selectivity and separation efficiency with a short analysis time and (ii) low surface tension (i.e., good volatility) and high gas-phase basicity (GB) to, respectively, ensure optimal evaporation and deprotonation during the ESI process. Mixtures of ACN and MeOH were selected to fulfill these requirements. MeOH possesses amphiprotic properties, showing the ability to form hydrogen bonds, whereas ACN is classified as a dipolar aprotic solvent and can modulate the solvation of analytes and their interactions with the BGE. Both solvents have been used in NACEUV to separate pharmaceutical drugs [15–27], including NSAIDs [64]. Among the different organic mixtures that were evaluated, two BGEs that were composed of 10 mM ammonium acetate in ACN-MeOH 80:20 (v/v) and 60:40 (v/v) gave the best results in terms of selectivity and were selected for further investigation. For both BGEs, a complete separation of NSAIDs was obtained (n = 3). A strong EOF was generated toward the CE cathode, while NSAIDs migrated in the opposite direction (toward the anode) and were thus detected after the EOF. The BGE composed of ACN-MeOH 60:40 (v/v) exhibited the best apparent selectivity due to the higher proportion of MeOH, that allowed a better solvation of the ammonium and acetate ions [65]. Thus additional interactions via hydrogen bonding with the analytes, as well as ion-pairing interactions were improved. With this proportion, the eff of each compound was increased (ca. 3–99%) and the EOF was decreased (ca. 39%). Therefore, the analysis time was increased (ca. 1.5- 2 fold) leading to a peak width increase (ca. 2- 3-fold) compared to the ACNMeOH 80:20 (v/v) BGE. The latter was selected due to a significant increase in EOF and decrease in eff , resulting in a shorter analysis time that decreased analyte diffusion into the low-viscosity BGE.

For the selected NSAIDs, the pKa value in aqueous solution is between 3.67 and 4.47 (Fig. 1) so anionic migration may still be possible in organic solvents but the addition of appropriate electrolytes at a given concentration plays an important role to have supplementary selectivity. Indeed, it directly influences the ion-pairing interactions, as well as the EOF and eff . Ammonium acetate was selected as the supporting electrolyte because of its MS compatibility but also due to its ability to create these interactions. Different concentrations of ammonium acetate (5, 10, and 15 mM in ACNMeOH 80:20 (v/v)) were tested (n = 3). As expected, the increase in the ammonium acetate concentration reduced significantly the EOF , (about 16% from 5 mM to 15 mM) leading to larger peak widths (between 11 and 33% from 5 mM to 15 mM) and longer analysis times (between 1.5 and 2 min from 5 mM to 15 mM). In addition, with an higher concentration of electrolytes, the eff values of each analyte were lowered (between 1 and 19% from 5 mM to 15 mM) and closely migrating compounds such as Dic and Mef, which exhibited significant decrease of eff (i.e., 19% and 13%) were no longer resolved. The modification on EOF and eff was essentially due to the increase of ammonium and acetate ions, which reduced  pot (wall and ion) as well as affected the heteroassociation and ion-pairing interactions [65]. It should be noted that for minimizing the modification of the BGE composition due to evaporation issues and improving the robustness of the system, the vials were changed every two analyses. With this procedure, no change of the volume in the vial was noticed. As presented in Fig. 2B, the best result was obtained with 5 mM ammonium acetate, demonstrating a strong improvement of the selectivity and a decrease in the analysis time while maintaining high separation efficiency (N = 135 000–200 000) and small peak widths (FWHM = 1.8–3.0 s). The comparison of the aqueous CZE and NACE modes under their optimal conditions (Fig. 2A and B) showed an important selectivity enhancement, which was essentially due to the ability of the organic BGE to generate additional interactions (via heteroassociation, homoassociation, and ion-pairing) between the electrolytes and the analytes. This selectivity improvement was enhanced by the changes in eff and EOF resulting from the different solvation properties and viscosities produced by the presence of ACN and MeOH in the BGE. The resolution could be further improved with the use of the sheathless interface (Fig. 2C) as it will be discussed in Section 3.2.

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Fig. 2. Investigation of the BGE. CE-MS electropherograms in negative ESI obtained for selected NSAIDs (dissolved at 1 ␮g/mL in ACN-MeOH 60:40 (v/v)) with the sheath liquid interface in (A) Aqueous CZE mode; BGE: ammonium acetate 50 mM, pH 8.5 and (B) NACE mode; BGE: ammonium acetate 5 mM in ACN-MeOH 80:20 (v/v). (C) CE-MS electropherograms in negative ESI obtained for selected NSAIDs (dissolved at 1 ␮g/mL in ACN-MeOH 60:40 (v/v)) with the sheathless interface in NACE mode; BGE: ammonium acetate 5 mM in ACN-MeOH 80:20 (v/v).

3.1.2. Effect of the sample dissolution solvent The compatibility of organic BGEs with the solvent or the matrix in which the analytes are dissolved is a key parameter in NACE [1–4]. Compatibility mismatch in terms of solvent or ionic strength between sample and BGE zones can greatly change the homogeneity of the local electric field and reduce the separation performance. Consequently, various compositions of the sample dissolution solvent were tested, i.e., ACN 100%, ACN-MeOH 80:20 (v/v), ACN-MeOH 60:40 (v/v), ACN-MeOH 30:70 (v/v), and MeOH 100% (n = 3), with the BGE previously selected (i.e., 5 mM ammonium acetate in ACN-MeOH 80:20 (v/v)). The best results were achieved with a sample dissolution solvent that was composed of ACN-MeOH 60:40 (v/v). This solvent provided acceptable peak width (FWHM = 2.1–2.8 s) and high

separation efficiency (N = 171 000–267 000) and led to peak width and efficiency improvements of 13–49% and 62–530%, respectively, compared to ACN 100% (FWHM = 3.4–4.5 s and N = 42 000–96 000). As shown in Fig. 3A–E, percentages of ACN above 80% significantly enlarged the peaks, hampering the complete resolution of some NSAIDs (e.g., Ind and Sup). Additionally, as the proportion of MeOH in the dissolution solvent increased, the migration time of each analyte increased until they reached a plateau. This effect was the direct result of the important decrease in the EOF, which was not balanced by the increase in the electrophoretic mobility of the analytes (Fig. S-1A and C). These changes in EOF and eff suggested that the presence of MeOH in the sample decreased the electric field strength in the sample zone by modifying the local  pot (wall and ion). As observed by several studies [66–68] a difference of electric field

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Fig. 3. Effect of the sample dissolution solvent. NACE-MS electropherograms in negative ESI obtained with the sheath liquid interface and the sheathless interface for selected NSAIDs dissolved at 1 ␮g/mL in (A) ACN 100%, (B) ACN-MeOH 80:20 (v/v), (C) ACN-MeOH 60:40 (v/v), (D) ACN-MeOH 30:70 (v/v), and (E) MeOH 100%; BGE: ammonium acetate 5 mM in ACN-MeOH 80:20 (v/v).

strength between the sample zone and the BGE generates a discrepancy of the local EOF and electrophoretic velocities resulting in a modification of the global mobilities. Additionally, a high difference in local electric field creates dispersion that can potentially explain the decrease of separation efficiency when high percentage of ACN is present in the sample zone. Analogous results were obtained with the BGE that was composed of ACN-MeOH 60:40 (v/v), suggesting that the effect of the dissolution solvent is similar for BGEs composed of a mixture of ACN and MeOH. Consequently, ACN-MeOH 60:40 (v/v) was considered the optimal sample dissolution solvent that offered the best performance in terms of peak width and resolution. 3.1.3. Sensitivity A significant increase in intensity was observed with NACE-MS compared to its aqueous CZE-MS counterpart, leading to a 1- to 10-fold improvement of the LOD (Table 1). It should be noted that LODs as low as to 10 ng/mL were achieved using a single quadrupole MS instrument. The good sensitivity that was obtained mainly resulted from several cumulative effects of the organic BGE on the whole electrospray process. First, the concentration of salts was lower in NACE than in aqueous CZE, which potentially reduced ion

suppression effects. Second, the lower surface tension of MeOH and ACN compared to water enabled a stable spray due to improved droplet evaporation and ion desolvation. Last, ACN and MeOH exhibits higher GB than water, aiding the deprotonation of the carboxylic groups. Typically, an increase in signal intensity was observed when a higher proportion of ACN was present in the BGE, and the ACN-MeOH 80:20 (v/v) BGE provided a sensitivity

Table 1 Limits of detection (LODs) obtained for selected NSAIDs (dissolved in ACN-MeOH 60:40 (v/v)) with the sheath liquid and sheathless interfaces in aqueous CZE mode; BGE: ammonium acetate 50 mM, pH 8.5 and NACE mode; BGE: ammonium acetate 5 mM in ACN-MeOH 80:20 (v/v).

Ibuprofen Suprofen Indometahcin Diclofneac Mefenamic acid Flufenamic acid

CZE

NACE

Sheath liquid (ng/mL)

Sheath liquid (ng/mL)

Sheathless (ng/mL)

20 100 50 100 20 10

20 50 10 10 10 10

1 1 1 5 1 1

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improvement of a factor of 6 compared to the ACN-MeOH 60:40 (v/v) BGE. It is worth mentioning that the global increase of intensity between NACE and aqueous CZE was not related to a decrease of the background noise, as it can be observed on the Fig. 2A and B insets. Consequently, the improvement of sensitivity seemed to be primarily related to an enhancement of the ionization efficiency. In terms of overall sensitivity, Ibu and Flu exhibited similar LODs, while for Mef and Sup, a 2-fold improvement was observed. The experimental conditions were much more efficient for the detection of Ind and Dic, which showed a 5- to 10-fold improvement by the use of NACE conditions. It should be noted that this sensitivity improvement occurred with the same sheath liquid, supporting the observation that the BGE composition had a considerable impact, whereas its proportion was relatively low compared to the sheath liquid at the tip. 3.2. NACE-ESI-MS with the sheathless interface In the absence of sheath liquid, the BGE is the critical element of both separation and ionization. Consequently, the use of organic BGEs appears particularly well adapted to the sheathless interface because organic solvents stabilize the cone-jet mode, facilitate the desolvation process, and enhance deprotonation (in the case of negative ESI), resulting in a global improvement of the sensitivity and robustness of the electrospray process. Moreover, the absence of a suction effect can improve the separation efficiency. 3.2.1. Investigation of the BGE With the optimal ACN-MeOH proportion previously determined (i.e., 80:20 (v/v)), a significant decrease of the signal intensity was observed with the sheathless interface (Fig. 4A) when the electrolyte concentration increased (from 5 to 15 mM). This behavior was attributed to the increase in the potential interactions (such as heteroassociation, homoassociation, and ion-pairing) between the components (analytes and electrolytes) of the BGE that could hamper the deprotonation during ionization. It should be noted that this effect was not related to a mass-sensitive behavior of the sheathless interface because the capillary flow rates were relatively high at the tip due to the presence of a strong EOF. For example, at 15 mM (the lowest EOF value), a flow rate of 131 nL/min was measured, which corresponded to the concentration-sensitive domain of the sheathless interface [51]. Interestingly, no modification of the signal intensity was observed with the sheath liquid configuration (Fig. 4B). As postulated by Posch et al. [48], the additional liquid, due to high solvation properties, circumvented the previously mentioned interactions, thus decreasing the suppression effects of high electrolyte concentrations. 3.2.2. Effect of the sample dissolution solvent A set of experiments similar to those that were conducted with the sheath liquid interface was performed, and the best results was also obtained with ACN-MeOH 60:40 (v/v) as the sample dissolution solvent. This composition guaranteed good performance with the sheathless interface (FWHM = 1.3–2.6 s and N = 211 000–457 000) and led to improvements of 13–49% and 62–530% in terms of peak width and separation efficiency, respectively, compared to ACN 100% (FWHM = 2.2–3.1 s and N = 62 000–152 000). As for its sheath liquid counterpart, a percentage of ACN above 80% decreased the resolution (Fig. 3A–E). Furthermore, the same effects on eff and EOF were observed (Fig. S-1B and C), with the only difference being that higher EOFs were obtained with the sheathless configuration (see Section 3.2.3 for a detailed discussion). Interestingly, both interfaces displayed the same mean values for the peak width decrease (−30% vs.

169

−32% in sheathless and sheath liquid, respectively) and separation efficiency increase (225% vs. 208% in sheathless and sheath liquid, respectively), supporting the conclusion that the performance enhancement was independent of the interface.

3.2.3. Efficiency, selectivity, and sensitivity Despite the presence of a possible stacking effect due to the lower quantities (ca. 3-fold) that were injected in the sheathless configuration because of the smaller capillary I.D., the separation efficiency with this interface was significantly enhanced, as depicted in Fig. 2C. The latter provided separation efficiencies between 210 000–340 000 and peak widths between 1.2 and 2.4 s, corresponding to global improvements of 35–145% and 13–49%, respectively, compared to the sheath liquid interface. This excellent performance resulted from the absence of pneumatic assistance that could induce peak broadening, which was particularly significant with the low-viscosity BGEs that were present in NACE. In addition, the smallest distance between the ESI tip and the MS entrance in the sheathless configuration (1 mm vs. 4 cm in sheath liquid) could also reduce peak dispersion. Finally, the fact that narrow capillary was used with the sheathless interface could certainly help to further increase the separation efficiency as previously observed by Iko et al. [69]. A slight difference in the migration times was noticed, which could possibly be explained by the difference in capillary I.D. between the two configurations. According to Petsev et al. [70], EOF is accelerated as the capillary I.D. is reduced, which was evidenced by the higher EOF value obtained for the sheathless interface (i.e., 1.28 × 10−7 m2 /V s vs. 1.14 × 10−7 m2 /V s for sheathless and sheath liquid, respectively). This trend was confirmed by independent CE-UV experiments (n = 6), in which a 12.7% increase in the EOF was measured with a capillary I.D. that was reduced from 50 ␮m to 25 ␮m. Regarding sensitivity, a 2- to 50-fold improvement in the LOD was observed with the sheathless interface (Table 1) with respect to the sheath liquid configuration. For all the selected NSAIDs, the LODs were approximately 1 ng/mL, and the best sensitivity improvement was observed for the propionic acid derivatives (i.e., Ibu and Sup). Interestingly, Sup was the only ion that was decarboxylated during the ionization process but presented also the greatest improvement of LOD and intensity. This observation could indicate that the different intrinsic properties of both interfaces have an effect on this type of reaction. According to these results, the sheathless interface seemed to help the decarboxylation of the acidic group leading to improvement of intensity. However, further investigations on other acidic compounds are mandatory to confirm this hypothesis. Because the injected quantities in sheathless mode were approximately 3-fold lower than in sheath liquid mode, the ionization yield was considerably improved. This improvement could be attributed to the high charge density droplets produced by the nanospray and a better ionization efficiency provided by the sheathless interface, as already discussed elsewhere [51–61]. However, the improvement of sensitivity was not related to a background noise decrease because both interfaces presented similar background noise intensity (see Fig. 2B and C insets). A part of the sensitivity improvement could also be attributed to the higher separation efficiency experienced with the sheathless interface as abovementioned. Overall, a sensitivity improvement between 10and 100-fold was obtained by replacing aqueous CZE-ESI-MS using the sheath liquid interface with NACE-ESI-MS using the sheathless configuration. Finally regarding the precision, both interfaces exhibited similar relative standard deviations in term of migration time, area, intensity, and peak width (Table S-1A and B).

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A

B Mefenamic acid 240 m/z Ibuprofen 205 m/z Suprofen 215 m/z

700000 600000

70000

Flufenamic acid 280 m/z Indomethacin 356 m/z Diclofenac 294 m/z

Mefenamic acid 240 m/z Ibuprofen 205 m/z Suprofen 215 m/z

60000

Flufenamic acid 280 m/z Indomethacin 356 m/z Diclofenac 294 m/z

50000

500000

40000

400000

30000

300000 200000

20000

100000

10000 0

0

5 mM

10 mM

5 mM

15 mM

10 mM

15 mM

Fig. 4. Effect of the electrolyte concentration. Peak intensity as a function of the ammonium acetate concentration obtained with NACE-MS for selected NSAIDs (dissolved at 1 ␮g/mL in ACN-MeOH 60:40 (v/v)) with (A) the sheathless interface and (B) the sheath liquid interface; BGE: ACN-MeOH 80:20 (v/v).

3.3. Application to biological samples: glucuronide analysis The above-mentioned results that were obtained with NSAIDs indicate that the NACE-ESI-MS method in negative mode appears to be an appropriate analytical tool for the analysis of important phase II drug metabolites such as glucuronides. Drug metabolites are infrequently analyzed using aqueous CZE due to their amphoteric properties. Thus, the supplementary interactions (e.g., ion-pairing and heteroassociation) provided by the organic solvents can be helpful to improve selectivity. Additionally, glucuronides are present at low concentrations in urine (ng/mL range); therefore, NACE-ESI-MS with the sheathless interface could provide the benefit of increased sensitivity, particularly if simple urine dilution is used as the sample pre-treatment. Finally, because glucuronides are separated as negatively-charged species migrating after the EOF, the interferences from positive endogenous ions (e.g., salts) and compounds (e.g., amino acids and urea), commonly known as the matrix effect, should be attenuated because these positive ions would be primarily detected before the EOF. In this study, morphine-3-␤-glucuronide (M3G), codeine6-␤-glucuronide (C6G), naloxone-6-␤-glucuronide (N6G), and ethyl-␤-glucuronide (EthG) were selected as model glucuronide analytes. 3.3.1. Method development and performance In a first set of experiments, a BGE composed of 5 mM ammonium acetate in ACN-MeOH 80:20 (v/v) was tested. With the former BGE, M3G and C6G were not separated whereas N6G and EthG were completely resolved (data not shown). This result was not surprising considering that C6G and M3G have similar pKa values (see Fig. 1), which seemed to persist even in presence of organic solvents. Thus, these solvents were not able to create a significant shift between the pKa values of both species. According the previous results obtained on NSAIDs, a BGE composed of 5 mM

ammonium acetate in ACN-MeOH 60:40 (v/v) appeared a good option to improve the selectivity thanks to its ability to create supplement heteroassociation and ion pairing interactions. Moreover, glucuronides exhibit more potential hydrogen bonding sites than NSAIDs (15 vs. 5 for glucuronides and NSAIDs, respectively), so a higher proportion of MeOH could lead to a further improvement of these interactions as well as alter in a different way the solvation shell of both compounds. As shown by Fig. 5A and B, a complete separation was achieved with a BGE composed of 5 mM ammonium acetate in ACN-MeOH 60:40 (v/v). As shown in Table 2 and Table S-2A and B, both interfaces presented good sensitivity and acceptable repeatability in terms of migration time, area, peak width, and intensity with standard samples dissolved in ACN-MeOH 60:40 (v/v). The sheathless interface provided higher separation performance in terms of efficiency and peak widths, as well as lower LODs. A 10-fold gain in sensitivity was obtained for M3G and EthG, while a 20-fold increase was reached for C6G and N6G. According to the good sensitivity obtained for the standard samples, glucuronides were further analyzed in urine samples after a simple 20-fold dilution in ACN-MeOH 60:40 (v/v) (“diluteand-shoot” approach). This sample pre-treatment was selected to normalize the samples and decrease their conductivity for the best injection performance. Satisfactory separation with relatively good repeatability (Table S-2A and B) of the four glucuronides in urine was achieved (Fig. 5C) with the sheathless interface, and LODs as low as 500 ng/mL in urine for each compound (Table 2) were obtained, corresponding to an injected concentration of 25 ng/mL. Complete separation was also achieved with the sheath liquid interface (data not shown) with a lower sensitivity (ca. 20-fold). It should be noted that a smaller volume of urine samples was injected into the capillary (i.e., 1% vs. 2% for standard samples) to avoid an increase of the peak widths probably caused by the presence of water in the biological sample. Slight changes in the mobilities and

Table 2 Limits of detection (LODs) obtained for selected glucuronides with the sheath liquid and the sheathless interfaces for standard samples (compounds dissolved at 500 ng/mL in ACN-MeOH 60:40 (v/v)) and urine samples (compounds spiked at 10 ␮g/mL in urine and diluted 20-fold with ACN-MeOH 60:40 (v/v)); BGE: ammonium acetate 5 mM in ACN-MeOH 60:40 (v/v). Standard samples

Morphine 3-␤-glucuronide Ethyl-␤-glucuronide Naloxone-3-␤-glucuronide Codeine-6-␤-glucuronide

Urine samples

Sheath liquid (␮g/mL)

Sheathless (␮g/mL)

Sheath liquid (␮g/mL)

Sheathless (␮g/mL)

0.1 0.1 0.1 0.1

0.01 0.01 0.005 0.005

10 10 10 10

0.5 0.5 0.5 0.5

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171

Fig. 5. NACE-MS electropherograms in negative ESI obtained for selected glucuronides (dissolved at 500 ng/mL in ACN-MeOH 60:40 (v/v)) with (A) the sheath liquid interface and (B) the sheathless interface. (C) NACE-MS electropherogram in negative ESI with the sheathless interface obtained for selected glucuronides (spiked at 10 ␮g/mL in urine and diluted 20-fold with ACN-MeOH 60:40 (v/v)); BGE: ammonium acetate 5 mM in ACN-MeOH 60:40 (v/v).

resolution were observed with respect to the standard samples, as presented in Fig. 5B and C. This result was consistent, considering the sensitivity of NACE to the sample dissolution solvent (see Sections 3.1.2 and 3.2.2). Because good sensitivity was obtained, the “dilute-and-shoot” approach, which is particularly well adapted to the generic methods required in clinical and forensic screenings [71], could be implemented with the developed NACE-ESI-MS method. Because this simple and fast sample pre-treatment does not remove all interfering species (e.g., endogenous compounds, salts, etc.), they may alter the analyte ionization process (signal suppression or enhancement) and the method’s global performance (i.e., repeatability, sensitivity). Consequently, a complete study of the matrix effect was performed with both interfaces. 3.3.2. Matrix effect evaluation Evaluation of the matrix effect (ME) was first performed based on a quantitative approach similar to that reported by Matuszewski et al. [62]. It should be mentioned that in chromatography, the global matrix effect can be directly related to the ratio of the observed peak areas because a constant mobile phase flow rate is used; therefore, the detector is only influenced by the signal intensity [60,62]. The situation is more complex in CE because the mobilities are different between compounds. In addition, the observed peak area could be influenced by a matrix effect on

ionization (related to the peak height) and by a matrix effect on CE (related to the peak width). Therefore, because the mobilities were different between the interfaces due to a capillary I.D. discrepancy (see Section 3.2.3), the peak areas and peak widths were normalized by the migration times (tmig ). As indicated in Table 3, area recoveries for both interfaces were within the consensus cut-off values (i.e., ±25%) [62], highlighting the absence of a significant matrix effect. Nevertheless, all recovery values were positive, suggesting the presence of signal enhancement, which was not expected with the ESI source [72]. As previously mentioned, this result could be explained by the peak area variability being a function of the variations in both peak width and peak height. The comparison of the recovery values of peak heights and peak widths in Table 3 revealed that the important increase in the peak widths was not completely compensated by a decrease in the peak heights, which caused a global increase in the area recoveries (excepted for EthG, exhibiting an opposite behavior). In other words for most glucuronides, a CE matrix effect was experienced in NACE-ESI-MS due to the composition of the biological sample that affected the peak widths (see Sections 3.1.2 and 3.2.2). The matrix effect was also qualitatively evaluated thanks to the sheath liquid interface that was used as a post-capillary infusion device [63]. This procedure offered the opportunity to

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Table 3 Quantitative evaluation of the matrix effect. Recoveries expressed as the mean value of area/tmig , FWHM/tmig , and height obtained for selected glucuronides with the sheath liquid and the sheathless interfaces; BGE: ammonium acetate 5 mM in ACN-MeOH 60:40 (v/v). Sheath liquid

Morphine 3-␤-glucuronide Ethyl-␤-glucuronide Naloxone-3-␤-glucuronide Codeine-6-␤-glucuronide

Sheathless

Aera/tmig

FWHM/tmig

Height

Area/tmig

FWHM/tmig

Height

108.6% 118.7% 115.1% 109.1%

118.5% 103.1% 116.6% 124.8%

95.5% 136.2% 99.3% 95.2%

106.2% 115.2% 124.1% 120.5%

154.0% 101.3% 168.2% 132.0%

67.9% 117.3% 64.5% 98.1%

Fig. 6. Qualitative evaluation of the matrix effect. Total ion electropherogram (TIE) and extracted ion electropherogram (EIE) in negative ESI for the selected glucuronides using the sheath liquid interface as a post-capillary infusion device; BGE: ammonium acetate 5 mM in ACN-MeOH 60:40 (v/v).

characterize the matrix effect in terms of time, duration, and intensity. As illustrated in Fig. 6, significant signal suppression between 3.5 and 6.5 min occurred, while no co-migrating interfering species were detected in the analytes’ migration zone. This result was confirmed by the injection of water and diluted blank pooled urine (data not shown). The latter exhibited a similar signal suppression pattern, whereas the former displayed no ionization alteration, leading to the conclusion that the matrix effect came from endogenous interfering species. Thus, both approaches emphasized that the ionization of glucuronides was not inhibited by interfering endogenous species. This result highlights the strength of NACE-ESI-MS in the context of the analysis of acidic compounds in biological samples. It should be noted that the use of deuterated internal standards is still recommended to compensate for sample preparation, injection,

and ionization variability when a complete quantitative analysis is planned. 4. Conclusion The present work demonstrated the potential of NACE-ESI-MS in negative ionization mode with sheath liquid and sheathless interfaces for the analysis of acidic pharmaceutical compounds. In the first part of the study, a careful evaluation of the BGE in terms of organic composition and electrolyte concentration for the analysis of NSAIDs was performed, and the best results were obtained with a BGE composed of 5 mM ammonium acetate in ACN-MeOH 80:20 (v/v). The influence of the dissolution solvent was also investigated, and it had an important effect on the peak width and resolution. Forty percent MeOH in the sample was determined to be optimal to

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achieve the best performance (regardless of the interface or the BGE composition). Complete separation was achieved with NACE due to specific interactions, such as ion pairing and heteroassociation. A significant gain in sensitivity (1- to 10-fold) was also achieved due to optimal deprotonation and droplet desolvation, which were facilitated by the low salt concentration and the presence of an organic solvent in the BGE. The global performance was further improved by the use of a sheathless interface, which led to a sensitivity increase of 2- to 50-fold, as well as a decrease of 10–45% in the peak width. This enhancement was due to the specific properties of the interface, such as the production of high-density charged droplets that improved the ionization and the absence of a suction effect caused by the sheath liquid inducing peak broadening. The analysis of selected glucuronides was then performed with a BGE composed of 5 mM ammonium acetate in ACN-MeOH 60:40 (v/v), providing complete separation due to the BGE’s ability to generate more hydrogen bonding. Both interfaces exhibited good sensitivity with standard samples (LODs as low as 100 ng/mL and 5 ng/mL for the sheath liquid and sheathless interfaces, respectively). The analysis of glucuronides in urine after a 20-fold dilution was successfully performed, demonstrating the potential of NACE-ESI-MS for the direct analysis of phase II metabolites after a simple “diluteand-shoot” procedure. Finally, the evaluation of the matrix effect revealed the absence of ionization interference in the analytes’ detection window. Although NACE seemed an interesting alternative for the analysis of urine samples, the 20-fold dilution step could be problematic because some minor metabolites could be hindered due the relatively high dilution factor. In this context, pre-concentration and/or sample preparation step could be considered to further improve the method sensitivity. Conflict of interest The authors have declared no conflict of interest. Acknowledgements The authors wish to thank Dr Jean-Marc Busnel from Beckman Coulter for his technical support and his fruitful discussion. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chroma.2013. 11.011. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16]

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