laser induced fluorescence detection using quinidine-based monolithic columns.

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Enantioseparation of N-derivatized amino acids by micro-liquid chromatography/laser induced fluorescence detection using quinidine-based monolithic columns Huihui Wu a,1 , Qiqin Wang a,b,1 , Meng Ruan a , Kun Peng a , Peijie Zhu a , Jacques Crommen a,c , Hai Han a,∗ , Zhengjin Jiang a,∗ a Department of Pharmacy and Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine & New Drug Research, Jinan University, Guangzhou 510632, China b Department of Analytical Chemistry, Physical Chemistry and Chemical Engineering, Faculty of Biology, Environmental Sciences and Chemistry, University of Alcalá, Ctra. Madrid—Barcelona, Km. 33.600, Alcalá de Henares, 28871 Madrid, Spain c Laboratory of Analytical Pharmaceutical Chemistry, Department of Pharmaceutical Sciences, University of Liege, CHU B36, B-4000 Liege, Belgium

a r t i c l e

i n f o

Article history: Received 17 October 2015 Received in revised form 6 December 2015 Accepted 9 December 2015 Available online xxx Keywords: Carbamoylated quinidine Monolithic column Self-assembled LIF detector Enantioseparation NBD-derivatized amino acids

a b s t r a c t A novel carbamoylated quinidine based monolith, namely poly(O-9-[2-(methacryloyloxy)ethylcarbamoyl]-10,11-dihydroquinidine-co-ethylene dimethacrylate (poly(MQD-co-EDMA)), was prepared for the micro-LC enantioseparation of N-derivatized amino acids. The influence of the mobile phase composition, including the organic modifier proportion, the apparent pH and the buffer concentration, on the enantioresolution of N-derivatized amino acids was systematically investigated. Satisfactory column performance in terms of permeability, efficiency and reproducibility was obtained in most cases. The majority of the enantiomers of the tested N-protected amino acids, including 3,5-DNB, 3,5-DClB, FMOC, 3,5-DMB, p-NB, m-ClB, p-ClB and B derivatives, could be baseline separated on the poly(MQD-co-EDMA) monolithic column within 25 min. A self-assembled laser induced fluorescence (LIF) detector was employed to improve sensitivity when analyzing 7-nitro-2,1,3-benzoxadiazole (NBD) derivatives of amino acids. Ten NBD-derivatized amino acids, including arginine and histidine whose enantioseparation on quinidine carbamate based CSPs has not been reported so far, were enantioresolved on the poly(MQD-co-EDMA) monolith column. It is worth noting that the d-enantiomers of NBD-derivatized amino acids eluted first, except in the case of glutamic acid. The LOD values obtained with the LIF detector were comparable to those reported using conventional LC-FL methods. The prepared poly(MQD-co-EDMA) monolithic column coupled with the LIF detector opens up interesting perspectives to the determination of trace d-amino acids in biological samples. © 2015 Elsevier B.V. All rights reserved.

1. Introduction

Abbreviations: AIBN, 2,2 -azobisisobutyronitrile; B, benzoyl; m-ClB, m-chlorobenzoyl; p-ClB, p-chlorobenzoyl; CSPs, chiral stationary phases; 3,5-DClB, 3,5-dichlorobenzoyl; 3,5-DMB, 3,5-dimethoxybenzoyl; 3,5-DNB, 3,5-dinitrobenzoyl; EDMA, ethylene dimethacrylate; FL, fluorescence; FMOC, 9-fluorenylmethoxycarbonyl; HEMA, 2-hydroxyethyl methacrylate; LIF, laser induced fluorescence; ␥-MAPS, 3-(trimethoxysilyl)-propyl methacrylate; MQD, p-NB, O-9-[2-(methacryloyloxy)-ethylcarbamoyl]-10,11-dihydroquinidine; p-nitrobenzoyl; NBD-F, 4-fluoro-7-nitro-2,1,3-benzoxadiazole. ∗ Corresponding authors. E-mail addresses: [email protected] (H. Han), [email protected] (Z. Jiang). 1 These authors contributed equally to this work.

It has been assumed that amino acids were essentially present in higher animals as l-forms, and the antipodes, d-forms, were very rare and had a minor role in the regulation of physiological functions. However, with the development in analytical sciences, several important d-amino acids such as d-serine (d-Ser) [1], daspartic acid (d-Asp) [2], d-glutamic acid (d-Glu) [3], d-proline (d-Pro) [4] and d-alanine (d-Ala) [5] have been found in mammals including human beings and have been demonstrated to be naturally occurring physiologically active substances or biomarkers. For example, d-Asp is naturally present in various endocrine tissues of mammals and was reported to play significant physiological roles in hormonal synthesis and regulation [6]. d-Ser is an endogenous co-agonist of N-methyl-d-aspartate receptors. It was reported to

http://dx.doi.org/10.1016/j.jpba.2015.12.011 0731-7085/© 2015 Elsevier B.V. All rights reserved.

Please cite this article in press as: H. Wu, et al., Enantioseparation of N-derivatized amino acids by micro-liquid chromatography/laser induced fluorescence detection using quinidine-based monolithic columns, J. Pharm. Biomed. Anal. (2015), http://dx.doi.org/10.1016/j.jpba.2015.12.011

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regulate synaptic plasticity and cerebellar functions [7]; and d-Ser or the d/l-Ser ratio can be a potential new biomarker for acute kidney injury [1] and amyotrophic lateral sclerosis (ALS) [8]. Therefore, it is important to develop analytical methods for the quantification of d-amino acids in biological samples. Chiral LC methods have become the mainstream analytical approach [3–5,9]. However, in many cases, relatively long analytical times were required in order to obtain a satisfactory enantiomeric resolution of amino acids due to the fact that the l-forms always exist in large amounts in most samples from mammalians compared to the trace d-forms [3,10,11]. Therefore, sensitive, rapid and highly efficient enantioselective strategies with emphasis on enantiomer elution order are highly desired for the LC analysis of d-amino acids in biological samples. Various chiral stationary phases (CSPs) have been widely used for the enantioseparation of amino acids, such as those containing cyclodextrins [12], cinchona alkaloids [13,14] or macrocyclic antibiotics [15] as chiral selectors. Especially, CSPs in which carbamoylated quinine or quinidine are immobilized on silica particles have become popular because of their excellent enantioselectivity toward various kinds of N-derivatized amino acids [13,14]. The amino acid enantiomers present in biological samples were usually separated on quinidine-based CSPs in Nderivatized forms using various derivatization reagents such as 9-fluorenylmethoxycarbonyl (FMOC) [16] or 3,5-dinitrobenzoyl (3,5-DNB) chlorides [14]. This could enhance the detection sensitivity and selectivity as well as the intermolecular interactions between the amino acids and the chiral selector. Nevertheless, in the case of 3,5-DNB or FMOC derivatization, the quantification of trace d-amino acids on the quinidine based CSPs is often difficult because the large amount of their l-form always elute first [17], and therefore affect sensitivity and accuracy. In recent years, 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) is widely used in HPLC analysis of free d-amino acids in mammalian tissues [18,19] because it can easily react with secondary amine and primary amine groups with high specificity and generate intense fluorescent derivatives [20]. In particular, using NBD-F as a derivative reagent and fluorescent detection, the trace d-amino acids could be determined in fmol level [3]. Microscale separation has exhibited several advantages, such as increased sensitivity and reduced sample and solvent consumption, so that it has gained much attention in proteomics to detect low abundance peptides and proteins. However, there are not so many types of chiral capillary columns and the selection of a suitable chiral column is rather difficult. Recently, quinidine-based chiral monolithic columns exhibited some interesting features in terms of enantioselectivity, column efficiency and short analysis time for the separation of amino acid enantiomers using both CEC and micro-LC modes [17,21]. Using O-9-[2-(methacryloyloxy)-ethylcarbamoyl]10,11-dihydroquinidine (MQD) as chiral selector, Wang et al. [17,22] re-optimizedthe preparation of the poly(MQD-co-HEMAco-EDMA) monolithic columns which had been firstly prepared by Lämmerhofer et al. [21,23] for CEC. High selectivity and resolution were obtained for the enantiomers of N-derivatized amino acids in themicro-LC mode. However, to the best of our knowledge, such monolithic columns have never been coupled with fluorescence (FL) detection and their enantioseparation ability for NBD-derivatized amino acids has never been reported so far. In this study, carbamoylated quinidine based poly(MQD-coEDMA) monolithic columnswere prepared without addition of the co-monomer 2-hydroxyethyl methacrylate (HEMA). The composition of the polymerization mixture was optimized in order to obtain satisfactory column permeability and efficiency in the micro-LC mode. The mobile phase composition, including the organic modifier proportion, the buffer concentration and the apparent pH, was also optimized in order to achieve high enantioresolution for N-

derivatized amino acids. 44 N-benzoylated or FMOC-derivatized amino acids were enantioseparated on this type of monolithic column. Enantioselectivity (˛) and enantioresolution (Rs ) values obtained on the poly(MQD-co-EDMA) monolithic column were compared to those achieved on the poly(MQD-co-HEMA-co-EDMA) monolithic column. Moreover, a self-assembled LIF detector [24] was employed in order to further improve the detection sensitivity in amino acid analysis. Ten NBD-derivatized proteinogenic amino acids including arginine (Arg) and histidine (His) were enantioseparated on the poly(MQD-co-EDMA) monolith. Their enantioselective analysis using FL detection was also systemically investigated.

2. Experimental 2.1. Chemicals and materials 3-(Trimethoxysilyl)-propyl methacrylate (␥-MAPS), methanol (MeOH), ethylene dimethacrylate (EDMA), 2,2 azobisisobutyronitrile (AIBN), 1-dodecanol, cyclohexanol, acetonitrile (ACN), ammonium formate, formic acid, ammonium acetate, acetic acid, chloroform, trifluoroacetic acid (TFA), boric acid were purchased from Aladdin Chemicals (Shanghai, China). The nine derivatization reagents, including p-nitrobenzoyl (p-NB) chloride, m-chlorobenzoyl (m-ClB) chloride, p-chlorobenzoyl (p-ClB) chloride, benzoyl (B) chloride, 3,5-dinitrobenzoyl (3,5DNB) chloride, 3,5-dimethoxybenzoyl (3,5-DMB) chloride, 3,5-dichlorobenzoyl (3,5-DClB) chloride, FMOC chloride, and all racemic amino acids (leucine (Leu), methionine (Met), Ala, threonine (Thr), valine (Val), Ser, isoleucine (Ile), phenylalanine (Phe), tryptophan (Trp), Pro, asparagine (Asn), glutamine (Gln), Arg, His, Glu, Asp and cysteine (Cys)) were obtained from Energy Chemical (Shanghai, China). NBD-F was supplied by J&K Chemical (Beijing, China). All N-derivatized amino acids were synthesized according to the literature except for NBD-derivatized amino acids. NBD-derivatized amino acids were synthesized according to Refs. [3,4,19]. In brief, to 10 ␮L of a 1.8 mM amino acid solution, 10 ␮L of 200 mM borate buffer (pH 9.5) and 10 ␮L of 20 mM NBD-F in acetonitrile were added. After standing for 4 min at 65 ◦ C in a water bath, 95 ␮L of 2% trifluoroacetic acid solution was added to stop the reaction, and the solutionwas injected after diluted with 2% trifluoroacetic acid. Distilled water was filtered through a 2 ␮m membrane before use. The fused-silica capillaries (375 ␮m O.D. × 100 ␮m I.D.) were obtained from Ruifeng Chromatography Ltd. (Yongnian, Hebei, China).

2.2. Instrumentation All micro-LC–UV experiments were carried out on a selfassembled HPLC system that consisted of a DiNa nano isocratic pump (Tokyo, Japan), a Valco four-port injection valve with 20 nL internal loop (Houston, TX, USA), and a Shimadzu SPD-15C UV detector (Kyoto, Japan) with a lab-made on-column detection system. A LIF detector was self-assembled according to previous report (Fig. 1) [24]. All scanning electron microscopy (SEM) experiments were performed using an ultra-high 165 resolution Hitachi S-4800 SEM (Tokyo, Japan) at an acceleration voltage of 1 kV. A Jinghong DK-S22 water bath (Shanghai, China) was used for thermally initiated copolymerization. A Sartorius PB-10 pH meter (Göttingen, Germany) was employed to adjust the pH of the mobile phase. UV data acquisition and handing were performed using a Unimicro TrisepTM Workstation 2003 (Shanghai, China), while FL data acquisition and processing were conducted using a LabView program written in house (www.ni.com). All chromatograms were converted to a text file and redrawn using Microcal Origin 7.5.


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Table 1 Optimization of the composition of the polymerization mixture used for the preparation of the poly(MQD-co-EDMA) monolithic columns. Column

H1 H2 H3 H4 H5 H6 H7 H8

Monomers (%, w/w)

Porogens (%, w/w)

MQD

EDMA

Dodecanol

Cyclohexanol

45 45 45 50 52.5 55 50 50

55 55 55 50 47.5 45 50 50

77 77 77 77 77 77 82 72

23 23 23 23 23 23 18 28

Monomers:Porogens (%, w/w)

Back-pressure (Mpa)

Theoretical plates (m−1 )

20 15 10 15 15 15 15 15

9.3 0.5 0 1.0 2.5 8.7 1.1 2.4

10,424 15,011 0 18,306 17,856 4442 14,502 15,686

80 85 90 85 85 85 85 85

Conditions: column dimensions: 160 mm × 100 ␮m I.D.; mobile phase, H2 O/ACN (60/40, v/v); UV detection wavelength: 214 nm; flow rate: 0.6 ␮L/min; injection volume: 20 nL; sample: naphthalene.

2.3. Chromatographic conditions Unless otherwise stated, the mobile phase for the enantioseparation of 3,5-DClB, 3,5-DNB, 3,5-DMB, p-NB, m-ClB, p-ClB,B and NBD-derivatized amino acids was a mixture of 0.1 M ammonium formate/ACN (20/80; v/v), while the enantioseparation of FMOCderivatized amino acids was performed using a mixture of 0.1 M ammonium formate/ACN (50/50; v/v). The apparent pH of the mobile phase was adjusted to the desired value by adding formic acid. The flow rate was 1 ␮L/min. All stock solutions of NBDderivatized amino acids werediluted with 2% trifluoroacetic acid solution, while the other N-derivatized amino acids were dissolved in MeOH to reach a final concentration around 1 mg/mL unless otherwise stated. Both the mobile phase and the sample solutions were subjected to a filtration through 0.22 ␮m membrane before use. UV detection was carried out at a wavelength of 254 nm, while NBDderivatized amino acids were detected using the LIF detector at a excitation wavelength of 470 nm and an emission wavelength of 530 nm, using on-column detection.

functional monomer (MQD), the crosslinker (EDMA), the binary porogenic mixture (1-dodecanol and cyclohexanol) and the initiator AIBN (1% (w/w) with respect to the total amount of monomer) was mixed ultrasonically into a homogenous solution in a 2 mL vial [17]. The composition of the polymerization mixture was optimized in order to obtain satisfactory permeability and efficiency, as detailed in Table 1. After sonication and bubbling with nitrogen for 5 min, the polymerization mixture was transferred into a 25 cm long capillary. The filled capillary was sealed with GC septa and submerged into a water bath at 65 ◦ C for 12 h. The resulting monolithic column was then rinsed with methanol to remove the unreacted compounds and solvents. A 2–3 mm detection window was created at a distance of 4 cm from the end of the column using a thermal wire stripper. Finally, the column was cut to a total length of 20 cm with an effective length of 16 cm. A 3–5 mm length of the capillary containing the polymer was kept for scanning electron microscopy (SEM) analysis [25].

3. Results and discussion 2.4. Preparation of the poly(MQD-co-EDMA) monolithic columns In order to provide anchoring sites for thepolymeric bulk, the capillaries were treated with ␥-MAPS using a method described elsewhere [17]. The polymerization mixture consisting of the

3.1. Preparation and optimization ofthe poly(MQD-co-EDMA) monolithic columns In our recent studies [17], the poly(MQD-co-HEMA-co-EDMA) monolithic column was systemically re-optimized according to

Fig. 1. Schematic diagram of the micro-LC/LIF system.


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Table 2 Permeability of the poly(MQD-co-EDMA) monolithic column. Mobile phase

Relative polaritya

Viscosity (×10−3 Pa s)

Permeability K (×10−13 m2 )

ACN MeOH ACN/water (50/50,v/v)

0.460 0.762 –

0.369 0.544 0.820

1.170 1.016 0.719

a

Relative polarity data were obtained from http://virtual.yosemite.cc.ca.us/smurov/orgsoltab.htm; viscosity data of pure solvents were obtained from Ref. [25,26].

Lämmerhofer et al. [21,23]. with some modifications. This monolithic column exhibited good enantioselectivity and column efficiency in the micro-LC mode for a wide range of N-derivatized amino acids. Furthermore, through comparative experiments between the poly(MQD-co-HEMA-co-EDMA) andpoly(MQD-coGMA-co-EDMA) monoliths, Lämmerhofer et al. observed that the polarity of the polymeric surface could affect the enantioselectivity of the final quinidine based monolithic column in the CEC mode. In order to further study the effect of the co-monomer HEMA on the enantioseparation ability of quinidine-based monolithic columns in the micro-LC mode, a novel poly(MQD-co-EDMA) monolithic column was prepared and optimized. It is well known that the permeability of a monolithic column can vary significantly with minor changes of the composition of the polymerization mixture [17]. As shown in Table 1, the porogen weight fraction was varied from 80% (column H1) to 90% (column H3), while the ratios of the binary porogenic mixture (77/23, w/w) and MQD/EDMA (45/55, w/w) were kept constant. The results indicate that the content of porogens has a dramatic influence on column permeability (Table 1). The column H1 prepared with 80% porogens was too tight to pump through, while the mixture containing 90% porogens could not be polymerized. Column H2 exhibited a suitable backpressure and the highest column efficiency, and therefore a porogen weight fraction of 85% was selected for all further experiments. It was also observed that the EDMA content could affect the column performance. As the weight fraction of EDMA in the monomer mixture decreased from 55% (Column H2) to 47.5% (Column H5), the backpressure increased from 0.5 to 2.5 MPa while the theoretical plate number remained almost constant. A further decrease of the EDMA weight content to 45% (Column H6) caused a significant decrease in column efficiency and a clear increase of backpres-

sure. As depicted in Fig. 2a and b, SEM experiments of column H4 indicate that the monolith is composed of spherical units agglomerated into larger clusters interdispersed by large-pore channels, which is a typical characteristic of monolithic structures. When the EDMA fraction in the polymerization mixture was increased, it was found that the microglobules became bigger for column H2 (Fig. 2c), while the opposite effect was observed for column H6 (Fig. 2d). An EDMA weight fraction of 50% was selected for further studies, since column H4 exhibited a suitable backpressure and a higher column efficiency (18,306 theoretical plates/m at a linear flow rate of 1.1 mm/s using naphthalene as test compound) when compared to columns H2 and H6. The influence of the porogenic mixture composition (1dodecanol/cyclohexanol) was also investigated by varying the weight content of cyclohexanol from 18% to 28%, while the proportions of the other components were kept constant. As can be seen in Table 1, no significant changes in backpressure and column efficiency were observed over the studied cyclohexanol weight content range. Based on these optimization experiments, a polymerization mixture comprising 15% monomers (MQD/EDMA, 50/50, w/w) and 85% porogens (dodecanol/cyclohexanol, 77/23, w/w) was selected for all further studies. 3.2. Permeability and reproducibility of the poly(MQD-co-EDMA) monolithic columns According to Bristow and Knox [25,26], the permeability K of a column can be expressed as follows: K=

uL P

Fig. 2. Scanning electron microphotographs of monolithic columns with different EDMA contents: (a) and (b) column H4; (c) column H2; (d) column H6.




where L is the length of the column, u is the linear velocity of the mobile phase, is the dynamic viscosity of the eluent, and P is the pressure drop across the column. Toluene was selected as deadtime marker when ACN and MeOH were used as mobile phases. When using water/ACN (50/50, v/v) as mobile phase, thiourea was selected as dead-timemarker. As shown in Table 2, relatively similar permeability values were observed when different solvents were pumped through the poly(MQD-co-EDMA) monolithic column. These results indicate that the monolith does not swell or shrink in solvents with different polarities and it is suited for LC applications. Furthermore, the reproducibility of the poly(MQD-co-EDMA) monolith was also evaluated through the relative standard deviations (RSDs) of k1 , k2 , ˛ and Rs for m-ClB-Leu. A mixture of 0.1 M ammonium formate/ACN (20/80, v/v, apparent pH 6.0) was used as mobile phase. The RSD values for run-to-run, day-to-day, column-to-column and batch-to-batch reproducibilities were in the ranges from 1.09% to 2.59%, 0.89% to 3.22%, 1.12% to 2.89% and 1.07% to 3.14%, respectively, which shows the excellent stability of the poly(MQD-co-EDMA) monolithic column and the satisfactory reproducibility of different batches of capillary columns.

3.3. Effect of mobile phase composition on the enantioseparation of N-derivatized amino acids The enantioseparation conditions, such as the organic modifier content, the buffer concentration and the apparent pH of the mobile phase, were also optimized. Two N-derivatized amino acids, i.e. mClB-Leu and p-ClB-Leu, were selected as test analytes. The effect of the ACN content on k and Rs was investigated by varying the proportion of ACN in the mobile phase from 70% to 90%. As shown in Table S1, the higher the ACN content in the mobile phase, the lower the k and Rs values of the analytes. Slightly higher Rs values were obtained with a mobile phase containing 70% ACN, but considering both the retention times and the Rs values of the chiral samples, an ACN content of 80% was chosen for further experiments. The apparent pH is another important parameter in the optimization of the enantioseparation of N-derivatized amino acids. Different apparent pH values were tested (5.0, 6.0, and 7.0) in a mobile phase made of 0.1 M ammonium formate/ACN (20/80, v/v). As shown in Table S2, both k, as well as ˛ and Rs values of m-ClB-Leu (pKa = 3.81) and p-ClB-Leu (pKa = 3.54) increased with increasing apparent pH from 5.0 to 6.0. According to a previous report [17], this behavior is certainly related to the fact that within this apparent pH range, the quinidine stationary phase remains fully positively charged while the negative charge of the two N-derivatized amino acids increases with increasing apparent pH, which enhances the electrostatic interactions and hence enantioselectivity. However, the opposite effect was observed for k, ˛ and Rs values in the range from 6.0 to 7.0. Therefore, an apparent pH 6.0 was considered as the most suitable for further experiments. The influence of the buffer concentration in the mobile phase on k, ˛ and Rs was also evaluated by varying the ammonium formate concentration from 0.05 to 0.15 M, while the other separation conditions were kept constant (Table S3). No significant influence on enantioselectivity for both m-ClB-Leu and p-ClB-Leu were observed over this concentration range. However, k values increased with decreasing concentration of ammonium formate. These results might indicate that the contribution of electrostatic interactions to the retention of these acidic analytes increases with decreasing concentration of the competing anion formate [27]. A 0.1 M ammonium formate was chosen because it represents the best compromise between enantioresolution and analysis time.

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Table 3 Enantioseparation of amino acid derivatives. Sample

k1

k2

˛

Rs

3,5-DNB-Ilea 3,5-DNB-Vala 3,5-DNB-Trpa 3,5-DNB-Leua 3,5-DNB-Phea 3,5-DNB-Meta 3,5-DNB-Thra 3,5-DNB-Alaa 3,5-DNB-Sera 3,5-DClB-Ilea 3,5-DClB-Vala 3,5-DClB-Phea 3,5-DClB-Leua 3,5-DClB-Trpa 3,5-DClB-Meta 3,5-DClB-Thra 3,5-DClB-Cysa 3,5-DClB-Alaa 3,5-DClB-Sera FMOC-Ileb FMOC-Valb FMOC-Pheb FMOC-Cysc FMOC-Trpb FMOC-Leub FMOC-Serb FMOC-Metb FMOC-Alac 3,5-DMB-Leua 3,5-DMB-Meta 3,5-DMB-Alaa p-NB-Leua p-NB-Meta p-NB-Alaa m-ClB-Leua m-ClB- Thra m-ClB-Alaa m-ClB-Meta p-ClB-Leua p-ClB-Meta p-ClB-Alaa B-Leua B-Meta B-Thr

2.69 2.78 4.13 3.04 3.92 4.00 3.71 2.83 3.82 4.12 3.03 4.24 3.38 5.48 5.16 4.45 6.84 4.33 4.80 7.21 6.53 10.00 8.41 3.03 6.69 2.75 8.41 5.21 4.46 4.79 2.38 2.41 2.72 2.41 2.23 2.43 2.59 3.01 2.25 2.79 2.72 1.68 2.20 2.04

16.88 12.64 15.48 12.27 14.16 13.89 10.74 7.63 9.16 13.02 8.76 10.68 8.75 12.94 12.67 9.94 15.40 8.32 9.04 9.80 8.79 12.41 10.66 4.04 8.15 3.36 10.31 5.98 10.41 9.77 3.95 3.65 4.28 3.35 3.83 3.81 3.69 4.77 3.38 4.08 3.58 2.33 2.80 2.45

6.27 4.55 3.75 4.04 3.61 3.48 2.89 2.69 2.40 3.16 2.90 2.52 2.59 2.36 2.45 2.23 2.25 1.92 1.88 1.36 1.35 1.24 1.27 1.33 1.22 1.22 1.23 1.15 2.33 2.04 1.66 1.51 1.58 1.39 1.72 1.57 1.43 1.59 1.50 1.46 1.32 1.38 1.27 1.20

9.30 7.84 8.26 8.38 7.24 7.65 7.59 6.53 5.64 5.92 5.64 6.82 5.17 4.46 5.76 5.81 3.92 4.19 4.47 1.95 1.64 1.04 1.47 1.49 1.03 1.18 1.31 0.84 4.94 4.78 3.33 2.94 3.15 2.43 3.66 2.97 2.94 3.43 2.67 2.67 2.10 1.74 1.78 1.29

N1 /m 5092 6459 6189 6545 6038 7400 9987 10248 9365 5452 7302 7485 6351 5616 6281 8993 5300 7461 7880 4954 3261 3116 4313 4274 3815 6529 4553 6247 4044 4336 7389 10,815 8135 10,621 9603 8834 11,677 9018 8629 8634 10,116 7835 11,712 12,778

N2 /m 7141 6273 6057 6554 4132 7399 5757 6049 7988 3468 3153 8673 4131 3374 7159 9533 3164 5976 5104 5026 4377 2509 4924 4475 3195 5876 5301 4198 4355 7141 6542 8723 8728 10,222 8328 9370 12,555 9517 7890 8872 11,131 6114 10,689 8889

Conditions: column: 160 mm × 100 ␮m I.D. poly(MQD-co-EDMA) monolithic column; mobile phase. a 0.1 M ammonium formate/ACN (20/80, v/v) (apparent pH 6.0). b 0.1 M ammonium formate/ACN (50/50, v/v) (apparent pH 6.0). c 0.1 M ammonium formate/ACN (60/40, v/v) (apparent pH 6.0); UV detection wavelength: 254 nm; flow rate: 1 ␮L/min; injection volume: 20 nL.

3.4. Enantioseparation of amino acids derivatives 3.4.1. Enantioseparation of N-derivatized amino acids In order to systemically evaluate the enantioseparation ability of the poly(MQD-co-EDMA) monolithic column in the micro-LC mode, the behavior of 44 FMOC-derivatized or N-benzoylated amino acids was studied (Table 3). The structures of the different N-protecting groups and amino acids tested in this study are listed in Fig. S1. Unless otherwise stated, the enantiomers of all N-derivatized amino acids examined were separated under the optimized conditions (0.1 M ammonium formate/ACN (20/80, v/v), apparent pH 6.0). The results (k1 , k2 , ˛, Rs , N1 and N2 ) obtained for the 44 N-derivatized amino acids (Leu, Met, Thr, Ala, Trp, Phe, Ser, Ile, Val and Cys) on the poly(MQD-co-EDMA) monolithic column are summarized in Table 3. Under the selected conditions, 36 out of the 44 analytes could be baseline enantioseparated (Rs > 1.5), and the enantiomers of the other 8 analytes could be partially separated (0.84 ≤ Rs < 1.5) on this monolithic column. Fig. S2 shows representative chromatograms obtained under the selected conditions.


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Fig. 3. Enantioselectivy (˛) values for N-derivatized amino acids. Comparison between results obtained with the poly(MQD-co-EDMA) and poly(MQD-co-EDMA-co-HEMA) monolithic columns. The ˛ values with the poly(MQD-co-HEMA-co-EDMA) monolith were obtained from Ref. [17].

In order to investigate the effect of the co-monomer HEMA on the enantioseparation ability of the quinidine based monolith, the ˛ values of the 44 N-derivatized amino acids obtained on both the poly(MQD-co-EDMA) and poly(MQD-co-HEMA-co-EDMA) monoliths were comparatively studied. As shown in Fig. 3, similar ˛ values for FMOC, 3,5-DMB, p-NB, m-ClB, p-ClB and B-derivatized amino acids were observed on both columns, while a slightly higher enantioselectivity was obtained for 3,5-DNB and 3,5-DClBderivatized amino acids on the poly(MQD-co-HEMA-co-EDMA) monolith (Fig. 3). The reason for these small differences is still not clear. However, it proves that the effect of the co-monomer HEMA on enantioselectivity in quinidine based monoliths is not crucial. The novel poly(MQD-co-EDMA) monolithic column prepared in this study is also well suited for the enantioseparation of N-derivatized amino acids in the micro-LC mode. In order to confirm the enantiomer elution order of these N-derivatized amino acids, enantioenriched 3,5-DClB-Met and 3,5-DNB-Ala were chosen as analytes. As shown in Fig. S3, the l-forms of both 3,5-DClB-Met and 3,5-DNB-Ala elute first. The same enantiomer elution order was observed on the poly(MQDco-HEMA-co-EDMA) monolith. 3.4.2. Enantioseparation of NBD-derivatized amino acids on the poly(MQD-co-EDMA) monolith Although the poly(MQD-co-EDMA) monolith exhibits good enantioselectivity for FMOC-derivatized or N-benzoylated amino acids, the l-forms of these N-derivatized amino acids always elute before the d-forms on this column (Fig. S3) and consequently the detectability remains an unavoidable issue for the analysis of trace d-amino acids in biological fluids. It has been reported that for the majority of NBD-derivatized amino acids, the l-forms elute before their d-forms on commercially available quinine based packed columns [4]. In order to reverse the enantiomer elution order and increase the detection sensitivity on the poly(MQDco-EDMA) monolithic column, NBD-F was chosen as fluorescent derivatization reagent for amino acids in this research. Ten NBDderivatized amino acids (Ala, Pro, Phe, Ser, Asn, Gln, Arg, His, Glu and Asp), the d-forms of which were reported to haveidentified physiological functions or could be potential biomarkers [2–9], were tested on the poly(MQD-co-EDMA) monolith using the

Table 4 Enantioseparation of NBD-derivatized amino acids. Sample

NBD-dl-Alaa NBD-dl-Proa NBD-dl-Phea NBD-dl-Sera NBD-dl-Asna NBD-dl-Glna NBD-dl-Arga NBD-dl-Hisa NBD-dl-Glub NBD-dl-Aspb

t (min) D

L

24.80 7.37 25.28 24.81 17.77 16.80 4.41 11.13 8.73 19.90

28.01 9.66 27.84 27.89 20.64 20.19 5.06 12.08 4.72 22.83

kD

kL

˛

Rs

8.42 3.50 12.88 8.59 9.19 8.57 0.61 2.52 3.11 5.45

9.64 4.89 14.29 9.78 10.83 10.50 0.85 2.82 1.22 6.40

1.14 1.40 1.11 1.14 1.18 1.22 1.39 1.12 2.54 1.17

1.32 1.69 0.56 1.30 1.54 1.95 1.51 0.85 4.28 1.30

Conditions: column: 160 mm × 100 ␮m I.D. poly(MQD-co-EDMA); mobile phase. a 0.1 M ammonium formate/ACN (20/80, v/v) (apparent pH 6.0). b 0.1 M ammonium formate/ACN (20/80, v/v) (apparent pH 3.0); fluorescence detection: excitation wavelength: 470 nm, emission wavelength: 530 nm; flow rate: 1 ␮L/min; injection volume: 20 nL.

optimized mobile phase composition (cf. Section 3.4.1) and fluorescence detection. A self-assembled LIF detector was employed for these experiments (Fig. 1). The results (k1 , k2 , ˛, Rs , N1 and N2 ) are listed in Table 4. All tested NBD-derivatized amino acids could be enantioseparated within 28 min, with ˛ values ranging from 1.11 to 2.54. Among them, Pro, Asn, Gln, Arg and Glu could be baseline separated (Rs > 1.50). It is worth noting that NBD-His and NBDArg enantiomers, which had not been reported to be separated on commercially available quinine or quinidine-based packed column, could be separated (˛ = 1.12 and 1.39, Rs = 0.85 and 1.51, respectively) on the poly(MQD-co-EDMA) monolithic column. This suggests that His and Arg enantiomers could be determined in biological samples on this column using NBD-F derivatization. The elution orders of NBD-derivatized amino acids were also confirmed by using the enantiomeric excess samples. Typical chromatograms are shown in Fig. 4. For all tested NBD-derivatized amino acids except NBD-Glu, d-forms eluted before l-forms (Table 4). This elution order for NBD-derivatized amino acid enantiomers, observed on the poly(MQD-co-EDMA) monolithic column is consistent with the results obtained on quinine or quinidinebased packed columns [3,4]. This provides an opportunity to




7

Fig. 5. Determination of NBD-D-Arg in the presence of a large excess of NBD-l-Arg. Conditions: column: 160 mm × 100 ␮m I.D. poly(MQD-co-EDMA); mobile phase, 0.1 M ammonium formate/ACN (20/80, v/v) (apparent pH 6.0); fluorescence detection, excitation wavelength: 470 nm, emission wavelength: 530 nm; flow rate: 1 ␮L/min; injection volume: 20 nL; sample: NBD-d-Arg (180 nmol/L) + NBD-l-Arg (70 ␮mol/L).

Fig. 4. Elution order of NBD-derivatized amino acid enantiomers on the poly(MQDco-EDMA) monolithic column. Conditions: column: 160 mm × 100 ␮m I.D. poly(MQD-co-EDMA); mobile phase, (a) 0.1 M ammonium formate solution/ACN (20/80, v/v) (apparent pH 6.0) for NBD-Gln, NBD-Pro, and NBD-Arg; (b) 0.1 M ammonium formate/ACN (20/80, v/v) (apparent pH 3.0) for NBD-Glu; fluorescence detection:

improve the detection limits for trace d-amino acids in biological fluids. It is also worth noting that a longer time (40 min) is necessary for achieving the enantioseparation of NBD-derivatized amino acids on a conventional chiral packed column, compared to that needed (28 min) on the poly(MQD-co-EDMA) monolithic column. This faster enantioseparation could be a potential advantage for the application of this monolithic column in a two-dimensional LC system for the analysis of d-amino acids. The various derivatization methods, reported until now for the LC determination of amino acids, show different detection sensitivities. For example, the LODs for N-ɑ-(5-fluoro-2,4-dinitrophenyl)-(d or l)-valine amide (FDNP-Val-NH2 ) derivatized Asp and Glu were 5-10 pmol using UV detection [16,28]. For Glu and Asp derivatized with o-phthalaldehyde (OPA), the reported LODs were 10 fmol [29] and 20–500 fmol [29,30], respectively, using fluorescence detection. Using NBD-F derivatization and MS detection, the LOD values for Asp and Glu were around 500 fmol [31]. The use of fluorescence detection can improve significantly the detection sensitivity for NBD-derivatized amino acids. Using a commercially available FL detector, the LOD values were 2–9.1 fmol for NBD-Asp and 2–17.8 fmol for Glu [3], and 7.1 and 12.1 fmol for NBD-His and Arg, respectively. In the present work, by employing a self-assembled LIF detector, the observed LOD (S/N = 3) and LOQ (S/N = 10) values for NBD-glutamine were 3.2 and 9.6 fmol, respectively. This leads to a gain in sensitivity of about 3000 folds compared to the commonly used LC–UV method which provides LOD and LOQ values for FMOCGln of 10 and 30 pmol, respectively. The LOD values for the other three baseline separated NBD-derivatized amino acids (His, Glu and Asp) were 12.9, 2.6 and 1.3 fmol, respectively. The relative limits of detection (RLOD) and quantification (RLOQ) for the d-enantiomer using this method were also determined by spiking small amounts of d-Arg into a large excess of l-Arg. In this case, a RLOD of 0.17% for NBD-d-Arg could be obtained, corresponding to an injected amount of 2.4 fmol in the presence of 1.4 pmol NBD-l-Arg, as well as a RLOQ of 0.25%, corresponding to an injected amount of 3.6 fmol NBD-dArg in the presence of 1.4 pmol NBD-l-Arg (Fig. 5 ). The detection sensitivity obtained with this method is higher than that observed

excitation wavelength: 470 nm, emission wavelength: 530 nm; flow rate: 1 ␮L/min; injection volume: 20 nL.




8

in most reported methods and the injection volume is only 20 nL, which is beneficial in the case of limited biological samples. These results indicate that the poly(MQD-co-EDMA) monolithic column combined with fluorescent detection could be applied to the determination of trace d-amino acids in the presence of large amounts of their l-forms.

[6] [7]

[8]

4. Conclusion [9]

In this research, a novel quinidine based monolith (poly(MQDco-EDMA)), which does not contain the co-monomer HEMA, was prepared. For the enantioseparation of N-benzoylated and FMOCderivatized amino acids, the optimized poly(MQD-co-EDMA) monolithic column showssimilar selectivity to that obtained with the poly(MQD-co-HEMA-co-EDMA) monolith, which indicates that the co-monomer HEMA has no significant influence on the enantiorecognition ability of the quinidine based monolith. The poly(MQD-co-EDMA) monolithic column also shows satisfactory enantioseparation performance for NBD-derivatized amino acids in terms of analysis time and enantiomer elution order, which makes it an suitable choice for determining d-amino acids in the presence of a large excess of their l-forms. By using the poly(MQD-co-EDMA) monolithic column, the enantioseparation of NBD-derivatized amino acids can be performed in a shorter time than that needed on quinine or quinidine-based packed columns, and therefore the whole analytical process can be accelerated. By employing a self-assembled LIF detector, the LOD values for NBD-derivatized amino acids were measured at the fmol level, and the RLOD and RLOQ values for NBD-d-Arg were as low as 0.17% and 0.25%, respectively. In conclusion, the combination of the poly(MQD-co-EDMA) monolithic column with LIF detection opens up interesting prospects for the determination of d-amino acids in biological samples. Acknowledgments We gratefully appreciate the financial support from the National Natural Science Foundation of China (Grants: 81273477 and 81202499). Qiqin Wang gratefully acknowledges funding from the “Excellent doctoral climb program” within Jinan University. We gratefully thank Prof. Xiayan Wang from Beijing University of Technology for the technical support for the self-assembled LIF detector.

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

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.jpba.2015.12.011.

[22]

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