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Synthesis and evaluation of a novel chiral derivatization reagent for resolution of carboxylic acid enantiomers by RP-HPLC
Microchemical Journal 135 (2017) 213–220
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Synthesis and evaluation of a novel chiral derivatization reagent for resolution of carboxylic acid enantiomers by RP-HPLC Lu-Ping Li a,1, Mei-Na Jin a,1, Qing Shi a, Chun-Yan Xu a, Ying-Zi Jiang a, Yong-Ill Lee b,⁎, Jun Zhe Min a,⁎ a Key Laboratory for Natural Resource of Changbai Mountain & Functional Molecules, Ministry of Education, College of Pharmacy Yanbian University and Department of Pharmacy, Affiliated Western Hospital, Yanbian University, Yanji 133002, Jilin Province, China b Department of Chemistry, Changwon National University, Changwon 641-773, Republic of Korea
a r t i c l e
i n f o
Article history: Received 1 September 2017 Accepted 1 September 2017 Available online 14 September 2017 Keywords: Triphenylphosphine Derivatization reagent Chiral carboxylic acid Enantiomeric separation HPLC
a b s t r a c t A novel derivatization reagent, N-[1-Oxo-5-(triphenylphosphonium)pentyl]-(S)-3-aminopyrrolidine (OTPA), with triphenylphosphine (TPP) as a basic structure carrying a permanent positive charge was developed for the enantiomeric separation of chiral carboxylic acids by high-performance liquid chromatography (HPLC). OTPA reacted with the carboxylic acids at 40 °C within 90 min in the presence of 1-ethyl-3-(3dimethylaminopropyl) carbodiimide (EDC) and 1-hydroxybenzotriazole (HOBt). The degree of epimerization (racemization) during the derivatization reaction was negligible. The separability of the diastereomers was evaluated in terms of their resolution value (Rs). The Rs values of the derivatives of non-steroidal anti-inflammatory drugs (NSAIDs), which were selected as the representative carboxylic acids, were completely separated by reversed-phase chromatography using an ODS (4.6 mm × 150 mm I.D., 5.0 μm) column. The resolution Rs values were 1.54–2.23 for the evaluated carboxylic acids and the OTPA-derivatization was also effective for the enantiomeric separation of chiral carboxylic acids. The calibration curves (r2 N 0.9971) were linear over the concentration range of 0.0125–1.25 mM for each enantiomer of ketoprofen (KET), and naproxen (NAP), 0.05–1.0 mM for each enantiomer of ibuprofen (IBU), 2-phenylpropionic acid (PPA), and loxoprofen (LOX), and 0.05–1.25 mM for each enantiomer of PBA. The limit of detection (S/N = 3) for each of the enantiomers of the NSAIDs and chiral carboxylic acid enantiomers was 1.4–7.6 μmol/L. The inter-day and intra-day assay precisions were all b6.77% and the mean recoveries (%) of the NSAIDs and chiral carboxylic acids from the spiked human plasma were 95.27– 101.12%. The derivatization followed by HPLC-UV enabled the separation and detection of NAP in human plasma with simple pretreatment. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Several pharmaceuticals such as non-steroidal anti-inflammatory drugs (NSAIDs, e.g., naproxen (NAP), ibuprofen (IBU), loxoprofen (LOX), and ketoprofen (KET)) are chiral carboxylic acids which are used widely for the treatment of various rheumatic and inflammatory diseases [1]. Moreover, some endogenous carboxylic acids are chiral compounds. For example, 3-hydroxypalmitic acid (3-HPA) is known as the most reliable biochemical indicator for long-chain 3-hydroxyacyl CoA dehydrogenase (LCHAD) deficiency [2]. Therefore, the chiral separation of chiral carboxylic acids is important for the investigations of the absorption, distribution, metabolism, exclusion, and the differences in biological effects, such as the pharmacological and toxicological properties [3,4].
⁎ Corresponding authors. E-mail addresses: [email protected] (Y.-I. Lee), [email protected] (J.Z. Min). 1 These authors contributed equally to this work (co-first author).
http://dx.doi.org/10.1016/j.microc.2017.09.009 0026-265X/© 2017 Elsevier B.V. All rights reserved.
HPLC is the most common instrumental technique for chiral carboxylic acid analysis in various methods. Although the enantiomeric separation of some chiral compounds is achieved by LC using a chiral stationary phase (CSP) column, these columns are comparatively expensive and sometimes applicable only in the normal-phase mode, which is not always suitable for the analysis of biological samples [5–9]. In contrast, although a diastereomeric method needs to be combined with derivatization procedures, it has the advantages of selectivity, sensitivity, and versatility for the determination of optical purity or identity. Therefore, methods based on diastereomeric derivatization using a chiral reagent are expected to be more practical for investigating the chiral compounds in biological samples. Lately, diastereomeric derivatization procedures for the reversedphase LC separation and ESI-MS/MS detection of carboxylic acid enantiomers have been actively examined [10]. Several chiral labeling reagents for carboxylic acids, i.e., 2-picolylamine (PA) [11], (S)anabasine [12], (S)-1-(2-pyrrolidinylmethyl)pyrrolidine (PMP) [13], (S)-1-(4-dimethylaminophenylcarbonyl)-3-aminopyrrolidine [14], (S)-3-amino-1-(3-pyridylthiocarbamoyl) pyrrolidine (PyT-N)
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[15], (S)-N-pyrrolidine-2-carboxylic acid N-(pyridine-2-yl)amide (PCP2) [16], and (S)-1-(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidin-3amine (DMT-3(S)-Apy) [17] have been developed in our laboratory. Although these reagents listed here were useful chiral derivatization reagents for the analysis of carboxylic acid enantiomers using LC-ESI-MS, they may not be useful with LC-UV because these reagents have relatively low molar absorptivity. Moreover, even though LC-MS can provide highly sensitive detection, because the instrumentation is expensive, it is unlikely to become popular [18]. Contrary to this, the use of tagging substances with reagents that afford structures capable of absorbing in the UV or VIS regions is the most popular means of derivatization, because almost all laboratories possess a UV–VIS detector and the analysts are proficient in manipulating it. The selective UV detection of chiral carboxylic acids can be successfully carried out using derivatization reagents such as 1-phenylethylamine (PEA) [19], 1-(1-naphtyl)ethylamine (NEA) [20], α-methyl-4-nitrobenzylamine [21], 1-(dimethylamino-1-naphtyl)ethylamine (DANE) [22], which are the most popular reagents for the derivatization, (S)(−)- or (R)(+)-NEA [23], and (S)-1-methyl-4-(5-(3-aminopyrrolidin1-yl)-2,4-dinitro-phenyl)piperazine (APy-PPZ) [24]. The detection of other chiral carboxylic acids has also been performed by derivatization reagents such as 4-(N,N-dimethylaminosulfonyl)-7-piperazino-2,1,3benzoxadiazole (DBD-PZ) [25]. However, the number of chiral reagents for both LC–UV determination and LC-MS analysis is highly limited. Based upon these considerations, we developed a novel chiral derivatization reagent OTPA capable of reacting efficiently with carboxylic acid enantiomers to form diastereomers for both MS detection and UV detection. Evaluated in terms of separation efficiency and detection sensitivity by LC-UV, the enantioselective analyses of NSAIDs in human plasma are also described by the subsequent derivatization. 2. Experimental 2.1. Materials and reagents (4-Carboxybutyl) triphenylphosphine (CTPP), triethylamine (TEA), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), 1-hydroxybenzotriazole hydrate (HOBt), and loxoprofen (LOX) were purchased from TCI (Tokyo, Japan). Nhydroxysuccinimide (NHS) was obtained from Wako Pure Chemicals (Osaka, Japan). Dicyclohexylcarbodiimide (DCC), (S)-3-(tertbutoxycarbonylamino)pyrrolidine (PR-Boc-N), sodium bicarbonate, ketoprofen (KET), and (S)-KET were obtained from Shanghai Jing Pure Chemical Reagents, Ltd. (Shanghai, China). 2-Phenylpropionic acid (PPA), (S)-PPA was obtained from J&K (Beijing, China). 2Phenylbutyric acid (PBA) was obtained from damas-beta (Shanghai, China). (S)-PBA, ibuprofen (IBU), (S)-IBU, naproxen (NAP), and (S)NAP were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Methanol (MeOH), acetonitrile (ACN), formic acid (FA), and trifluoroacetic acid (TFA) were of HPLC reagent grade (Fisher, USA). All other chemicals were of analytical reagent grade and were used without further purification. Deionized and distilled water (H 2 O) was used throughout the study (Unique-R20 Multi-functional ultra-pure water system, Research Scientific Instruments Co., Xiamen, China). 2.2. HPLC-UV and UPLC-ESI-MS conditions The UPLC-ESI-MS/MS analysis was performed using a Xevo TM TQ-D triple quadrupole mass spectrometer (Waters, Milford, MA) connected to an ACQUITY ultra-performance liquid chromatograph (UPLC H-class, Waters). The ACQUITY UPLC BEH C18 column (1.7 μm, 100 mm × 2.1 mm i.d.; Waters) was used at a flow rate of 0.4 mL/min and at a temperature of 40 °C. Mobile phases A and B consisted of 0.1% FA in water and 0.1% FA in acetonitrile, respectively, and the total run time was 20 min. The gradient steps were as follows: 0–20 min from 10%–70% solvent B. The injection volume was
2 μL. The process of synthesizing OTPA was analyzed by UPLC-ESIMS/MS in the positive ion mode unless otherwise stated, and the multiple reaction monitoring mode (MRM) using a switching ionization mode. The detection conditions were a capillary voltage of 3.00 kV; sample cone voltage of 10 V; source temperature of 120 °C; desolvation gas flow of 1000 L/h; cone gas flow of 150 L/h; nebulizer gas flow of 7.0 L/h; collision gas flow of 0.15 mL/min; collision energy of 15 eV; collision cell exit potential of 5 V; and desolvation temperature of 500 °C. Analytical software (MassLynx, version 4.1) was used for system control and data processing. The analysis of the derivatization of carboxylic acid enantiomers was performed using a liquid chromatograph (HITACHI 1000, Japan), 1110 pump, 1210 auto sample, 1310 column oven, and 1410 UV detector. A Kaseisorb ODS-2000 column (150 × 4.6 mm I.D., 5.0 μm, GL Sciences) was used at a flow rate of 1.0 mL/min and at a temperature of 40 °C. Isocratic elution was performed using mobile phases A and B. The run time was b22.0 min. The UV detection wavelength was determined by HPLC-DAD (HITACHI 5430, Japan). The injection volume was 10 μL. Analytical software (Primaide) was used for system control and data processing.
2.3. Synthesis of OTPA as a basic structure with permanently positively charged novel chiral carboxylic acid derivatization reagents To 40 mL of an ACN solution of CTPP (443.31 mg, 1.0 mmol) and NHS (115.03 mg, 1.0 mmol) was added DCC (226.8 mg, 1.1 mmol). After stirring for 16 h at room temperature, the precipitate was washed with H2O (3.0 mL), and then removed by way of filtration. The filtrate was evaporated to dryness to give TPP-NHS. Then, 140 μL TEA was added to the mixed solution of TPP-NHS (540 mg, 1.0 mmol) and PR-Boc-N (186.2 mg, 1.0 mmol) in 20 mL of dichloromethane. The reaction mixture was stirred at 40 °C for 15 h. After evaporation of the solvent, the residue was dissolved in saturated sodium bicarbonate solution (15 mL) and extracted with dichloromethane (50 mL, three times). The organic layer was dried over Na2SO4 and evaporated in vacuum. The residue was dissolved in dichloromethane (2 mL), after which the resulting residues were subjected to silica-gel column chromatography using dichloromethane/methanol (25/1, v/v) as the mobile phase. The TPP-PR-Boc-N were obtained as light yellow powders (yield, 59%). The compound with the Boc group was eliminated with TFA (2 mL), and the solution was then neutralized with 28% ammonia (2 mL). After drying in vacuum, OTPA was obtained as an adhesive oil (yield, 84%). LCESI-MS: m/z 431.35 [M + H]+. 1H NMR in CDCl3 (TMS): 1.27 ppm (s, 4H, \\CH2\\), 1.85 ppm (s, 2H, \\NH2), 2.24–2.43 ppm (m, 4H, \\CH2\\), 3.34 ppm (s, 3H, \\CH2\\), 4.00 ppm (m, 1H, \\CH2\\), 5.62 ppm (s, 4H,\\CH2\\), 7.67–7.84 ppm (m, 15H, Ar-H). 2.4. Derivatization of carboxylic acid enantiomers Freshly prepared solutions of EDC (10 mM) in ACN (100 μL), HOBt (10 mM) in ACN (100 μL), and chiral carboxylic acids (1 mM) in ACN (100 μL) were vigorously mixed with OTPA (10 mM) in ACN (100 μL). The reaction mixtures were stored at 40 °C for 90 min. After removal of the solvent, the resulting residues were dissolved in the mobile phase, and then an aliquot (10 μL) was subjected to HPLC-UV. 2.5. Separation and detection of the resulting diastereomers The efficiency of OTPA as the derivatization reagents for chiral carboxylic acids was evaluated by the retention factor (k′) separation factor (α), resolution value (Rs) and the limit of detection (LOD). The limits of detection (signal-to-noise ratio of 5, S/N = 5) were calculated by comparison of the UV signal intensities of the diastereomer and baseline noise.
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Fig. 1. UPLC-ESI-MS spectra and structures of OTPA. OTPA: N-[1-Oxo-5-(triphenylphosphonium)pentyl]-(S)-3-aminopyrrolidine.
2.6. Validation of the method 2.6.1. Calibration curve preparation A 10-μL aliquot of each of the solutions of the diastereomers of the chiral carboxylic acids ACN (each 0.0125–1.25 mM) was introduced to the HPLC-UV system. The calibration curves were obtained by plotting the peak area vs. the concentration of the analytes. The precision (coefficient of variation, CV, %) for each concentration was also calculated from the five replicated determinations. 2.6.2. Accuracy and precision of intra-day and inter-day assays The accuracy (%) and precision (CV) based on the intra-day and inter-day assays were determined using the diastereomers of the standard chiral carboxylic acids. These parameters were evaluated using three different concentrations in the range of 0.1–1.0 mM. The determinations were repeated three times within one day and between days. The accuracy (%) at each concentration was calculated from the calibration curves obtained from Section 2.6.1. The precision (CV, %) for each concentration was also calculated from the mean and standard deviation (SD) values for six replicated determinations.
2.6.3. Limit of detection and limit of quantitation The limit of detection (LOD) was defined as the calculated concentration at the signal-to-noise ratio of 3 and 10 (S/N = 3). The standard solutions were diluted to form a series of concentrations, then subjected to the HPLC-UV system, as described in Section 2.6.1. The LOD of each of the diastereomers was calculated from a comparison of the noise level and the peak height on the appropriate mass chromatogram on which the target diastereomers were detected.
2.7. Determination of NSAIDs (NAP, IBU, LOX, KET) and R, S-PBA, PPA spiked to human plasma We obtained 5.0-mL samples of human plasma from six healthy volunteers (three men and three women in the age group 24–40 years) treated at the Affiliated Western Hospital, Yanbian University. All patients provided written informed consent before entry into the study. The human plasma experiments were conducted according to the guidelines of the Ethical Committee for Human Experimentation at Yanbian University. The samples were centrifuged at 3000 rpm for 10 min to obtain the plasma.
Fig. 2. Chemical structures of chiral carboxylic acids. * indicates an asymmetric carbon.
Fig. 3. Derivatization reaction of chiral carboxylic acids with OTPA.
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R,S-chiral carboxylic acids 100 μL (0.8 μM–8 mM) were added to 50 μL of each of the plasma samples obtained from the healthy volunteers. Then, 200 μL can was added using vortex oscillation to remove the
protein. The extracted supernatant was evaporated to dryness under reduced pressure. The residue was dissolved in 0.1 mL ACN, and reacted with OTPA (10 mM) in 100 μL ACN in the presence of 100 μL of each ACN solution of EDC (10 mM) and HOBt (10 mM). After standing for 90 min at 40 °C, the reaction solution was filtered through a membrane filter (0.45 μm), and an aliquot (10 μL) was subjected to HPLC. Each peak on the chromatogram was monitored with the UV detector. The recovery (%) and precision (CV, %) of the five concentration sets (n = 5) were calculated from the calibration curve obtained by the described method. The HPLC–UV conditions are the same as those listed in Section 2.2. 3. Results and discussion 3.1. Syntheses of the chiral derivatization reagents A derivatization technique using a suitable reagent was adopted to obtain an efficient chiral separation and sensitive detection in reversed-phase chromatography using an ODS column [11,17,26]. The derivatization method is effective for the separation and detection of the chiral carboxylic acids, as tested in this study. A novel chiral derivatization reagent incorporating a triphenylphosphine moiety in its structure was developed for the enantioseparation and detection of carboxylic acids by the HPLC-UV system. Substituted aminopyrrolidine has been reported to be a useful skeleton for chiral derivatization reagents [22, 24,27,28]. TPP was introduced for LC-ESI-MS quantification of amines and amino acids [29]. This derivatization procedure enhanced the detection of amino acids due to the introduction of the ionized positively charged TPP group. Recently, we utilized TPP as a derivatization reagent to detect oligosaccharides using LC-ESI–MS [30]. This derivatization procedure enhanced the UV detection responses of oligosaccharides owing to the introduction of the triphenylphosphine structure. First, TPP reacted with N-hydroxysuccinimide and was esterized to form TPP-NHS and then the chiral derivatization reagent OTPA was synthesized from the reaction of TPP-NHS and PR-Boc-N at room temperature. The Boc-group was easily removed by the addition of TFA. The chemical structures were identified by NMR and MS instrumental analyses (Fig. 1). Because the synthetic procedures are very mild, no possible racemization could occur during the reactions. Indeed, no peak of the opposite isomer due to racemization appeared on the chromatograms. They were considered to be over 99% pure because starting materials with an enantiomeric excess of over 98% were used. Moreover, the reagents were stable for at least 6 months when stored at −20 °C. 3.2. Derivatization of NSAIDs and R,S-PBA, PPA
Fig. 4. Time courses of the derivatization reaction of R,S-KET, R,S-NAP, and R,S-PPA with OTPA at 40 °C.
Chiral carboxylic acids are usually labeled with a primary amine in the presence of activation reagents such as dicyclohexyl carbodiimide. The condensation reaction usually proceeds under mild conditions such as a temperature of 40 °C. In this study, the combination of EDC and HOBt was used for condensation of the OTPA and chiral carboxylic acids. The structures of the six chiral carboxylic acids that were tested and their derivatization reactions are shown in Figs. 2 and 3. The OTPA reacted with the R,S-KET, NAP, and PPA to produce the corresponding diastereomers with an amide group in the presence of the activation reagents (EDC and HOBt) [16]. The reaction time for the derivatization was first optimized with R,SKET, R,S-NAP, and R,S-PPA at 40 °C, and then the amounts of derivatized products and KET, NAP, and PPA that remained non-derivatized were monitored by HPLC-UV. As shown in Fig. 4, the amounts of derivatives that were formed gradually increased as the reaction times became longer. The peak areas of the derivatives reached a plateau after a 90 min of reaction and subsequently remained constant for a period of 2.0 h. The time courses of the derivatization reactions are almost comparable for both enantiomers of KET, NAP, and PPA. The results show that no observable difference in the reactivity of the derivatization reactions of these enantiomers was found. The shapes of the plots obtained for the
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Fig. 5. HPLC-UV chromatograms of separation of carboxylic acid enantiomers. A: NAP; B: IBU; C:LOX; D:KET; E:PBA; F:PPA.
reactions of the other reagents were similar. Thus, the reaction conditions for the labeling of the chiral carboxylic acids were determined as 40 °C for 90 min. 3.3. Separation of carboxylic acid enantiomers as OTPA-derivatives In this study, we attempted the separation of carboxylic acid enantiomers as OTPA-derivatives using a conventional reversed-phase column. The separation of a pair of enantiomers of OTPA after tagging with six chiral carboxylic acids was tried using the isocratic elution of a water–acetonitrile mixture containing 0.1% HCOOH. For the separation, an anti-pressurized column packed with ODS (4.6 mm × 150 mm I.D., 5.0 μm) was used for the separation of the carboxylic acid enantiomers by HPLC-UV. Fig. 5 shows the HPLC-UV chromatograms of the separation of carboxylic acid enantiomers. Each pair of diastereomers was clearly separated by the reversed-phase chromatography. The values of k′, α, and Rs of the OTPA-derivatives are shown in Table 1. As seen in Table 1, the use of derivatization
Table 1 Separability of diastereomers derived from chiral reagents. Carboxylic acid compounds
k′
NAP IBU LOX KET PBA PPA
9.959 (S) 15.59 (S) 7.69 (1) 10.27 (S) 16.39 (S) 14.77 (S)
a b
Rs = 2 × (t2 − t1)/(W1 + W2). Mobile phase: 0.1% FA in H2O/CH3CN.
12.01 (R) 18.53 (R) 8.47 (2) 11.56 (R) 17.82 (R) 16.60 (R)
α
Rsa
Mobile phaseb
1.21 1.19 1.10 1.13 1.09 1.12
2.23 2.07 1.55 1.72 1.54 1.77
65/35 62/38 65/35 64/36 69/31 71/29
enabled the satisfactory separation of the diastereomers within 20 min; the resolution (Rs) values for all the derivatives exceeded 1.54. Thus, the OTPA-derivatization was useful for the enantiomeric separation of chiral carboxylic acids. Based upon these observations, the conditions presented in Section 2.2 were ultimately selected for the separation of the carboxylic acid enantiomers.
3.4. Validation of the proposed method The calibration curves were obtained using five different concentrations. The determination at each concentration was repeated five times. A good calibration curve was obtained for each of the carboxylic acid enantiomers. Table 2 shows the calibration characteristics and detection limits of the six carboxylic acid enantiomers. Linear calibration curves were obtained for each carboxylic acid enantiomer (r2 N 0.997). The detection limits (S/N = 3) were 1.4–7.6 μmol/L. To evaluate the present method, the precision (CVs, %) was determined. The precision for three different concentrations were evaluated using intra-day and inter-day assays. As shown in Table 3, the CVs of the intra-day and inter-day determinations were 0.54–4.02% and 0.57–6.77%, respectively. The accuracy of the procedure was evaluated by spiking the human plasma with a known concentration of carboxylic acid enantiomeric standard. The recovery was established by analyzing a spiked standard solution of a known concentration of carboxylic acid enantiomers on the HPLC-UV system. As shown in Table 4, the mean recoveries (%) of carboxylic acid enantiomers from the spiked human plasma were in the range 95.27–101.12%. Furthermore, the precisions (CV) were within 0.65–8.02%. The good linearity, sensitivity, and precision demonstrated that the present method is applicable for human plasma analyses.
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Table 2 Calibration curves of chiral carboxylic acid compounds by the proposed method. Carboxylic acid compounds
Calibration range (mM)
Linear equation
Linearity (R2)
CV% (n = 5)
LOD (μmol/L)
S-KET R-KET S-NAP R-NAP S-IBU R-IBU S-PPA R-PPA LOX-1 LOX-2 S-PBA R-PBA
0.0125–1.25 0.0125–1.25 0.0125–1.25 0.0125–1.25 0.05–1.0 0.05–1.0 0.05–1.0 0.05–1.0 0.05–1.0 0.05–1.0 0.05–1.25 0.05–1.25
y = 1,289,360.68 × −29,523.1 y = 1,185,142.67 × −30,438.53 y = 618,318.39 × +11,930.97 y = 618,527.6 × +17,965.95 y = 110,012.76 × +2819.62 y = 123,376.67 × +3402.2 y = 101,171.44 × −360.68 y = 104,033.76 × +2518.1 y = 94,064.41 × −3690.61 y = 107,034.57 × −4416.54 y = 79,794.18 × −1582.28 y = 78,437.45 × −1969.16
0.9990 0.9990 0.9984 0.9971 0.9994 0.9991 0.9985 0.9973 0.9994 0.9991 0.9980 0.9984
0.23–1.41 0.58–1.66 0.35–2.75 0.63–3.45 0.67–4.01 0.51–2.58 0.94–2.55 0.40–2.98 0.18–1.58 0.64–1.55 0.43–2.93 0.36–1.47
1.4 1.4 2.2 2.2 4.0 4.0 5.6 5.6 6.3 6.3 7.6 7.6
LOD: Limit of detection.
3.5. Determination of carboxylic acid enantiomers in human plasma As examples of the bioanalysis by the present method, the separation and detection of carboxylic acid enantiomers in human plasma were performed using OTPA. As shown in Fig. 6, the R,S-NAP in the spiked human plasma was completely separated and detected without interference of any endogenous substances. Furthermore, the chromatograms in Fig. 6 A were almost the same as those of authentic R,SNAP in Fig. 6C. Consequently, the OTPA derivatization reagent can be used as a reagent for the separation and detection of a pair of carboxylic acid enantiomers. The OTPA features are preferable for targeted
Table 3 Accuracy and precision of the proposed method by intra-day and inter-day assays. Carboxylic acid compounds
Amount (mM)
Intra-day assay CV (%) (n = 6)
Inter-day assay CV (%) (n = 6)
S-KET
0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0
2.46 0.58 3.28 1.49 0.64 3.00 1.23 1.00 1.78 1.92 4.02 2.95 0.94 0.81 1.80 1.27 0.90 1.63 2.26 1.22 1.25 1.95 2.95 1.92 0.54 3.57 1.55 0.89 1.73 2.36 1.91 1.23 1.54 1.19 0.74 1.39
2.65 3.70 4.12 1.99 2.88 4.17 1.46 1.98 1.90 3.72 6.77 3.39 2.03 0.81 1.14 2.71 1.22 2.06 2.26 2.37 2.88 1.01 3.49 3.00 0.57 3.85 2.73 1.11 2.17 3.04 3.50 2.11 2.28 1.23 1.84 1.35
R-KET
S-NAP
R-NAP
S-IBU
R-IBU
S-PPA
R-PPA
LOX-1
LOX-2
S-PBA
R-PBA
carboxylic acid analyses such as metabolomics. Judging from these results, OTPA seems to be a useful chiral derivatization reagent for the determination of chiral carboxylic acids in real samples. 4. Conclusion We developed a novel derivatization reagent, OTPA, for the enantiomeric separation of chiral carboxylic acids using HPLC. The derivatization was completed at 40 °C within 90 min. A simple and practical procedure using HPLC-UV was developed for the enantiomeric determination of carboxylic acids. As a result, the diastereomers derived from the chiral carboxylic acids were satisfactorily separated by reversedphase chromatography (Rs = 1.54–2.23). Since the proposed method provides satisfactory separation and recovery, it was adapted for the determination of the enantiomers of NSAIDs in human plasma. The
Table 4 Determination of chiral carboxylic acid compounds in spiked human plasma. Carboxylic acid compounds
Spiked amount (mmol/L)
Detection amount (mmol/L)
CV (%) (n = 5)
Recovery Mean (%) recovery (%)
S-NAP
0.1 0.6 1.0 0.1 0.6 1.0 0.1 0.6 1.0 0.1 0.6 1.0 0.1 0.6 1.0 0.1 0.6 1.0 0.1 0.6 1.0 0.1 0.6 1.0 0.1 0.6 1.0 0.1 0.6 1.0
0.101 ± 0.0010 0.586 ± 0.0073 0.994 ± 0.0218 0.104 ± 0.0015 0.590 ± 0.0069 1.005 ± 0.0270 0.090 ± 0.0018 0.595 ± 0.0213 0.969 ± 0.0566 0.098 ± 0.0035 0.585 ± 0.0274 0.988 ± 0.0348 0.089 ± 0.0038 0.600 ± 0.0208 0.972 ± 0.0144 0.091 ± 0.0031 0.597 ± 0.0184 0.975 ± 0.0176 0.093 ± 0.0065 0.628 ± 0.0494 0.996 ± 0.0225 0.090 ± 0.0045 0.623 ± 0.0500 0.991 ± 0.0140 0.102 ± 0.0007 0.595 ± 0.0093 0.984 ± 0.0135 0.097 ± 0.0007 0.618 ± 0.0098 0.989 ± 0.0203
1.01 1.25 2.19 1.42 1.17 2.69 1.95 3.59 5.85 3.52 4.69 3.52 4.28 3.47 1.48 3.43 3.07 1.80 7.01 7.86 2.26 5.00 8.02 1.41 0.65 1.55 1.37 0.74 1.59 2.05
101.10 97.61 99.42 104.50 98.37 100.5 90.00 99.11 96.86 98.11 97.52 98.80 88.58 100.00 97.21 90.72 99.52 97.52 92.84 104.7 99.58 89.67 103.8 99.11 102.0 99.25 98.38 96.60 102.9 98.88
R-NAP
S-IBU
R-IBU
S-PPA
R-PPA
S-PBA
R-PBA
S-KET
R-KET
99.37
101.12
95.32
98.14
95.27
95.92
99.03
97.51
99.88
99.47
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Fig. 6. HPLC-UV Chromatograms of the diastereomers obtained from R,S-NAP in spiked human plasma. (A) R,S-NAP standards, (B) Blank plasma, (C) R,S-NAP in spiked plasma.
present method has sufficient specificity and practicality for the analysis of human plasma samples. Furthermore, the method also seems to be applicable for the enantioselective determination of carboxylic acids in various biological specimens. Research aimed at the simultaneous determination of chiral carboxylic acids in biological fluids is currently underway in our laboratory.
Acknowledgments The present research was supported by the National Natural Science Foundation of China (81360487, 81660594), and the Science and Technology Development Project of Jilin Province of China (20160101207JC).
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Microchemical Journal journal homepage: www.elsevier.com/locate/microc
Synthesis and evaluation of a novel chiral derivatization reagent for resolution of carboxylic acid enantiomers by RP-HPLC Lu-Ping Li a,1, Mei-Na Jin a,1, Qing Shi a, Chun-Yan Xu a, Ying-Zi Jiang a, Yong-Ill Lee b,⁎, Jun Zhe Min a,⁎ a Key Laboratory for Natural Resource of Changbai Mountain & Functional Molecules, Ministry of Education, College of Pharmacy Yanbian University and Department of Pharmacy, Affiliated Western Hospital, Yanbian University, Yanji 133002, Jilin Province, China b Department of Chemistry, Changwon National University, Changwon 641-773, Republic of Korea
a r t i c l e
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Article history: Received 1 September 2017 Accepted 1 September 2017 Available online 14 September 2017 Keywords: Triphenylphosphine Derivatization reagent Chiral carboxylic acid Enantiomeric separation HPLC
a b s t r a c t A novel derivatization reagent, N-[1-Oxo-5-(triphenylphosphonium)pentyl]-(S)-3-aminopyrrolidine (OTPA), with triphenylphosphine (TPP) as a basic structure carrying a permanent positive charge was developed for the enantiomeric separation of chiral carboxylic acids by high-performance liquid chromatography (HPLC). OTPA reacted with the carboxylic acids at 40 °C within 90 min in the presence of 1-ethyl-3-(3dimethylaminopropyl) carbodiimide (EDC) and 1-hydroxybenzotriazole (HOBt). The degree of epimerization (racemization) during the derivatization reaction was negligible. The separability of the diastereomers was evaluated in terms of their resolution value (Rs). The Rs values of the derivatives of non-steroidal anti-inflammatory drugs (NSAIDs), which were selected as the representative carboxylic acids, were completely separated by reversed-phase chromatography using an ODS (4.6 mm × 150 mm I.D., 5.0 μm) column. The resolution Rs values were 1.54–2.23 for the evaluated carboxylic acids and the OTPA-derivatization was also effective for the enantiomeric separation of chiral carboxylic acids. The calibration curves (r2 N 0.9971) were linear over the concentration range of 0.0125–1.25 mM for each enantiomer of ketoprofen (KET), and naproxen (NAP), 0.05–1.0 mM for each enantiomer of ibuprofen (IBU), 2-phenylpropionic acid (PPA), and loxoprofen (LOX), and 0.05–1.25 mM for each enantiomer of PBA. The limit of detection (S/N = 3) for each of the enantiomers of the NSAIDs and chiral carboxylic acid enantiomers was 1.4–7.6 μmol/L. The inter-day and intra-day assay precisions were all b6.77% and the mean recoveries (%) of the NSAIDs and chiral carboxylic acids from the spiked human plasma were 95.27– 101.12%. The derivatization followed by HPLC-UV enabled the separation and detection of NAP in human plasma with simple pretreatment. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Several pharmaceuticals such as non-steroidal anti-inflammatory drugs (NSAIDs, e.g., naproxen (NAP), ibuprofen (IBU), loxoprofen (LOX), and ketoprofen (KET)) are chiral carboxylic acids which are used widely for the treatment of various rheumatic and inflammatory diseases [1]. Moreover, some endogenous carboxylic acids are chiral compounds. For example, 3-hydroxypalmitic acid (3-HPA) is known as the most reliable biochemical indicator for long-chain 3-hydroxyacyl CoA dehydrogenase (LCHAD) deficiency [2]. Therefore, the chiral separation of chiral carboxylic acids is important for the investigations of the absorption, distribution, metabolism, exclusion, and the differences in biological effects, such as the pharmacological and toxicological properties [3,4].
⁎ Corresponding authors. E-mail addresses: [email protected] (Y.-I. Lee), [email protected] (J.Z. Min). 1 These authors contributed equally to this work (co-first author).
http://dx.doi.org/10.1016/j.microc.2017.09.009 0026-265X/© 2017 Elsevier B.V. All rights reserved.
HPLC is the most common instrumental technique for chiral carboxylic acid analysis in various methods. Although the enantiomeric separation of some chiral compounds is achieved by LC using a chiral stationary phase (CSP) column, these columns are comparatively expensive and sometimes applicable only in the normal-phase mode, which is not always suitable for the analysis of biological samples [5–9]. In contrast, although a diastereomeric method needs to be combined with derivatization procedures, it has the advantages of selectivity, sensitivity, and versatility for the determination of optical purity or identity. Therefore, methods based on diastereomeric derivatization using a chiral reagent are expected to be more practical for investigating the chiral compounds in biological samples. Lately, diastereomeric derivatization procedures for the reversedphase LC separation and ESI-MS/MS detection of carboxylic acid enantiomers have been actively examined [10]. Several chiral labeling reagents for carboxylic acids, i.e., 2-picolylamine (PA) [11], (S)anabasine [12], (S)-1-(2-pyrrolidinylmethyl)pyrrolidine (PMP) [13], (S)-1-(4-dimethylaminophenylcarbonyl)-3-aminopyrrolidine [14], (S)-3-amino-1-(3-pyridylthiocarbamoyl) pyrrolidine (PyT-N)
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[15], (S)-N-pyrrolidine-2-carboxylic acid N-(pyridine-2-yl)amide (PCP2) [16], and (S)-1-(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidin-3amine (DMT-3(S)-Apy) [17] have been developed in our laboratory. Although these reagents listed here were useful chiral derivatization reagents for the analysis of carboxylic acid enantiomers using LC-ESI-MS, they may not be useful with LC-UV because these reagents have relatively low molar absorptivity. Moreover, even though LC-MS can provide highly sensitive detection, because the instrumentation is expensive, it is unlikely to become popular [18]. Contrary to this, the use of tagging substances with reagents that afford structures capable of absorbing in the UV or VIS regions is the most popular means of derivatization, because almost all laboratories possess a UV–VIS detector and the analysts are proficient in manipulating it. The selective UV detection of chiral carboxylic acids can be successfully carried out using derivatization reagents such as 1-phenylethylamine (PEA) [19], 1-(1-naphtyl)ethylamine (NEA) [20], α-methyl-4-nitrobenzylamine [21], 1-(dimethylamino-1-naphtyl)ethylamine (DANE) [22], which are the most popular reagents for the derivatization, (S)(−)- or (R)(+)-NEA [23], and (S)-1-methyl-4-(5-(3-aminopyrrolidin1-yl)-2,4-dinitro-phenyl)piperazine (APy-PPZ) [24]. The detection of other chiral carboxylic acids has also been performed by derivatization reagents such as 4-(N,N-dimethylaminosulfonyl)-7-piperazino-2,1,3benzoxadiazole (DBD-PZ) [25]. However, the number of chiral reagents for both LC–UV determination and LC-MS analysis is highly limited. Based upon these considerations, we developed a novel chiral derivatization reagent OTPA capable of reacting efficiently with carboxylic acid enantiomers to form diastereomers for both MS detection and UV detection. Evaluated in terms of separation efficiency and detection sensitivity by LC-UV, the enantioselective analyses of NSAIDs in human plasma are also described by the subsequent derivatization. 2. Experimental 2.1. Materials and reagents (4-Carboxybutyl) triphenylphosphine (CTPP), triethylamine (TEA), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), 1-hydroxybenzotriazole hydrate (HOBt), and loxoprofen (LOX) were purchased from TCI (Tokyo, Japan). Nhydroxysuccinimide (NHS) was obtained from Wako Pure Chemicals (Osaka, Japan). Dicyclohexylcarbodiimide (DCC), (S)-3-(tertbutoxycarbonylamino)pyrrolidine (PR-Boc-N), sodium bicarbonate, ketoprofen (KET), and (S)-KET were obtained from Shanghai Jing Pure Chemical Reagents, Ltd. (Shanghai, China). 2-Phenylpropionic acid (PPA), (S)-PPA was obtained from J&K (Beijing, China). 2Phenylbutyric acid (PBA) was obtained from damas-beta (Shanghai, China). (S)-PBA, ibuprofen (IBU), (S)-IBU, naproxen (NAP), and (S)NAP were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Methanol (MeOH), acetonitrile (ACN), formic acid (FA), and trifluoroacetic acid (TFA) were of HPLC reagent grade (Fisher, USA). All other chemicals were of analytical reagent grade and were used without further purification. Deionized and distilled water (H 2 O) was used throughout the study (Unique-R20 Multi-functional ultra-pure water system, Research Scientific Instruments Co., Xiamen, China). 2.2. HPLC-UV and UPLC-ESI-MS conditions The UPLC-ESI-MS/MS analysis was performed using a Xevo TM TQ-D triple quadrupole mass spectrometer (Waters, Milford, MA) connected to an ACQUITY ultra-performance liquid chromatograph (UPLC H-class, Waters). The ACQUITY UPLC BEH C18 column (1.7 μm, 100 mm × 2.1 mm i.d.; Waters) was used at a flow rate of 0.4 mL/min and at a temperature of 40 °C. Mobile phases A and B consisted of 0.1% FA in water and 0.1% FA in acetonitrile, respectively, and the total run time was 20 min. The gradient steps were as follows: 0–20 min from 10%–70% solvent B. The injection volume was
2 μL. The process of synthesizing OTPA was analyzed by UPLC-ESIMS/MS in the positive ion mode unless otherwise stated, and the multiple reaction monitoring mode (MRM) using a switching ionization mode. The detection conditions were a capillary voltage of 3.00 kV; sample cone voltage of 10 V; source temperature of 120 °C; desolvation gas flow of 1000 L/h; cone gas flow of 150 L/h; nebulizer gas flow of 7.0 L/h; collision gas flow of 0.15 mL/min; collision energy of 15 eV; collision cell exit potential of 5 V; and desolvation temperature of 500 °C. Analytical software (MassLynx, version 4.1) was used for system control and data processing. The analysis of the derivatization of carboxylic acid enantiomers was performed using a liquid chromatograph (HITACHI 1000, Japan), 1110 pump, 1210 auto sample, 1310 column oven, and 1410 UV detector. A Kaseisorb ODS-2000 column (150 × 4.6 mm I.D., 5.0 μm, GL Sciences) was used at a flow rate of 1.0 mL/min and at a temperature of 40 °C. Isocratic elution was performed using mobile phases A and B. The run time was b22.0 min. The UV detection wavelength was determined by HPLC-DAD (HITACHI 5430, Japan). The injection volume was 10 μL. Analytical software (Primaide) was used for system control and data processing.
2.3. Synthesis of OTPA as a basic structure with permanently positively charged novel chiral carboxylic acid derivatization reagents To 40 mL of an ACN solution of CTPP (443.31 mg, 1.0 mmol) and NHS (115.03 mg, 1.0 mmol) was added DCC (226.8 mg, 1.1 mmol). After stirring for 16 h at room temperature, the precipitate was washed with H2O (3.0 mL), and then removed by way of filtration. The filtrate was evaporated to dryness to give TPP-NHS. Then, 140 μL TEA was added to the mixed solution of TPP-NHS (540 mg, 1.0 mmol) and PR-Boc-N (186.2 mg, 1.0 mmol) in 20 mL of dichloromethane. The reaction mixture was stirred at 40 °C for 15 h. After evaporation of the solvent, the residue was dissolved in saturated sodium bicarbonate solution (15 mL) and extracted with dichloromethane (50 mL, three times). The organic layer was dried over Na2SO4 and evaporated in vacuum. The residue was dissolved in dichloromethane (2 mL), after which the resulting residues were subjected to silica-gel column chromatography using dichloromethane/methanol (25/1, v/v) as the mobile phase. The TPP-PR-Boc-N were obtained as light yellow powders (yield, 59%). The compound with the Boc group was eliminated with TFA (2 mL), and the solution was then neutralized with 28% ammonia (2 mL). After drying in vacuum, OTPA was obtained as an adhesive oil (yield, 84%). LCESI-MS: m/z 431.35 [M + H]+. 1H NMR in CDCl3 (TMS): 1.27 ppm (s, 4H, \\CH2\\), 1.85 ppm (s, 2H, \\NH2), 2.24–2.43 ppm (m, 4H, \\CH2\\), 3.34 ppm (s, 3H, \\CH2\\), 4.00 ppm (m, 1H, \\CH2\\), 5.62 ppm (s, 4H,\\CH2\\), 7.67–7.84 ppm (m, 15H, Ar-H). 2.4. Derivatization of carboxylic acid enantiomers Freshly prepared solutions of EDC (10 mM) in ACN (100 μL), HOBt (10 mM) in ACN (100 μL), and chiral carboxylic acids (1 mM) in ACN (100 μL) were vigorously mixed with OTPA (10 mM) in ACN (100 μL). The reaction mixtures were stored at 40 °C for 90 min. After removal of the solvent, the resulting residues were dissolved in the mobile phase, and then an aliquot (10 μL) was subjected to HPLC-UV. 2.5. Separation and detection of the resulting diastereomers The efficiency of OTPA as the derivatization reagents for chiral carboxylic acids was evaluated by the retention factor (k′) separation factor (α), resolution value (Rs) and the limit of detection (LOD). The limits of detection (signal-to-noise ratio of 5, S/N = 5) were calculated by comparison of the UV signal intensities of the diastereomer and baseline noise.
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Fig. 1. UPLC-ESI-MS spectra and structures of OTPA. OTPA: N-[1-Oxo-5-(triphenylphosphonium)pentyl]-(S)-3-aminopyrrolidine.
2.6. Validation of the method 2.6.1. Calibration curve preparation A 10-μL aliquot of each of the solutions of the diastereomers of the chiral carboxylic acids ACN (each 0.0125–1.25 mM) was introduced to the HPLC-UV system. The calibration curves were obtained by plotting the peak area vs. the concentration of the analytes. The precision (coefficient of variation, CV, %) for each concentration was also calculated from the five replicated determinations. 2.6.2. Accuracy and precision of intra-day and inter-day assays The accuracy (%) and precision (CV) based on the intra-day and inter-day assays were determined using the diastereomers of the standard chiral carboxylic acids. These parameters were evaluated using three different concentrations in the range of 0.1–1.0 mM. The determinations were repeated three times within one day and between days. The accuracy (%) at each concentration was calculated from the calibration curves obtained from Section 2.6.1. The precision (CV, %) for each concentration was also calculated from the mean and standard deviation (SD) values for six replicated determinations.
2.6.3. Limit of detection and limit of quantitation The limit of detection (LOD) was defined as the calculated concentration at the signal-to-noise ratio of 3 and 10 (S/N = 3). The standard solutions were diluted to form a series of concentrations, then subjected to the HPLC-UV system, as described in Section 2.6.1. The LOD of each of the diastereomers was calculated from a comparison of the noise level and the peak height on the appropriate mass chromatogram on which the target diastereomers were detected.
2.7. Determination of NSAIDs (NAP, IBU, LOX, KET) and R, S-PBA, PPA spiked to human plasma We obtained 5.0-mL samples of human plasma from six healthy volunteers (three men and three women in the age group 24–40 years) treated at the Affiliated Western Hospital, Yanbian University. All patients provided written informed consent before entry into the study. The human plasma experiments were conducted according to the guidelines of the Ethical Committee for Human Experimentation at Yanbian University. The samples were centrifuged at 3000 rpm for 10 min to obtain the plasma.
Fig. 2. Chemical structures of chiral carboxylic acids. * indicates an asymmetric carbon.
Fig. 3. Derivatization reaction of chiral carboxylic acids with OTPA.
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R,S-chiral carboxylic acids 100 μL (0.8 μM–8 mM) were added to 50 μL of each of the plasma samples obtained from the healthy volunteers. Then, 200 μL can was added using vortex oscillation to remove the
protein. The extracted supernatant was evaporated to dryness under reduced pressure. The residue was dissolved in 0.1 mL ACN, and reacted with OTPA (10 mM) in 100 μL ACN in the presence of 100 μL of each ACN solution of EDC (10 mM) and HOBt (10 mM). After standing for 90 min at 40 °C, the reaction solution was filtered through a membrane filter (0.45 μm), and an aliquot (10 μL) was subjected to HPLC. Each peak on the chromatogram was monitored with the UV detector. The recovery (%) and precision (CV, %) of the five concentration sets (n = 5) were calculated from the calibration curve obtained by the described method. The HPLC–UV conditions are the same as those listed in Section 2.2. 3. Results and discussion 3.1. Syntheses of the chiral derivatization reagents A derivatization technique using a suitable reagent was adopted to obtain an efficient chiral separation and sensitive detection in reversed-phase chromatography using an ODS column [11,17,26]. The derivatization method is effective for the separation and detection of the chiral carboxylic acids, as tested in this study. A novel chiral derivatization reagent incorporating a triphenylphosphine moiety in its structure was developed for the enantioseparation and detection of carboxylic acids by the HPLC-UV system. Substituted aminopyrrolidine has been reported to be a useful skeleton for chiral derivatization reagents [22, 24,27,28]. TPP was introduced for LC-ESI-MS quantification of amines and amino acids [29]. This derivatization procedure enhanced the detection of amino acids due to the introduction of the ionized positively charged TPP group. Recently, we utilized TPP as a derivatization reagent to detect oligosaccharides using LC-ESI–MS [30]. This derivatization procedure enhanced the UV detection responses of oligosaccharides owing to the introduction of the triphenylphosphine structure. First, TPP reacted with N-hydroxysuccinimide and was esterized to form TPP-NHS and then the chiral derivatization reagent OTPA was synthesized from the reaction of TPP-NHS and PR-Boc-N at room temperature. The Boc-group was easily removed by the addition of TFA. The chemical structures were identified by NMR and MS instrumental analyses (Fig. 1). Because the synthetic procedures are very mild, no possible racemization could occur during the reactions. Indeed, no peak of the opposite isomer due to racemization appeared on the chromatograms. They were considered to be over 99% pure because starting materials with an enantiomeric excess of over 98% were used. Moreover, the reagents were stable for at least 6 months when stored at −20 °C. 3.2. Derivatization of NSAIDs and R,S-PBA, PPA
Fig. 4. Time courses of the derivatization reaction of R,S-KET, R,S-NAP, and R,S-PPA with OTPA at 40 °C.
Chiral carboxylic acids are usually labeled with a primary amine in the presence of activation reagents such as dicyclohexyl carbodiimide. The condensation reaction usually proceeds under mild conditions such as a temperature of 40 °C. In this study, the combination of EDC and HOBt was used for condensation of the OTPA and chiral carboxylic acids. The structures of the six chiral carboxylic acids that were tested and their derivatization reactions are shown in Figs. 2 and 3. The OTPA reacted with the R,S-KET, NAP, and PPA to produce the corresponding diastereomers with an amide group in the presence of the activation reagents (EDC and HOBt) [16]. The reaction time for the derivatization was first optimized with R,SKET, R,S-NAP, and R,S-PPA at 40 °C, and then the amounts of derivatized products and KET, NAP, and PPA that remained non-derivatized were monitored by HPLC-UV. As shown in Fig. 4, the amounts of derivatives that were formed gradually increased as the reaction times became longer. The peak areas of the derivatives reached a plateau after a 90 min of reaction and subsequently remained constant for a period of 2.0 h. The time courses of the derivatization reactions are almost comparable for both enantiomers of KET, NAP, and PPA. The results show that no observable difference in the reactivity of the derivatization reactions of these enantiomers was found. The shapes of the plots obtained for the
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Fig. 5. HPLC-UV chromatograms of separation of carboxylic acid enantiomers. A: NAP; B: IBU; C:LOX; D:KET; E:PBA; F:PPA.
reactions of the other reagents were similar. Thus, the reaction conditions for the labeling of the chiral carboxylic acids were determined as 40 °C for 90 min. 3.3. Separation of carboxylic acid enantiomers as OTPA-derivatives In this study, we attempted the separation of carboxylic acid enantiomers as OTPA-derivatives using a conventional reversed-phase column. The separation of a pair of enantiomers of OTPA after tagging with six chiral carboxylic acids was tried using the isocratic elution of a water–acetonitrile mixture containing 0.1% HCOOH. For the separation, an anti-pressurized column packed with ODS (4.6 mm × 150 mm I.D., 5.0 μm) was used for the separation of the carboxylic acid enantiomers by HPLC-UV. Fig. 5 shows the HPLC-UV chromatograms of the separation of carboxylic acid enantiomers. Each pair of diastereomers was clearly separated by the reversed-phase chromatography. The values of k′, α, and Rs of the OTPA-derivatives are shown in Table 1. As seen in Table 1, the use of derivatization
Table 1 Separability of diastereomers derived from chiral reagents. Carboxylic acid compounds
k′
NAP IBU LOX KET PBA PPA
9.959 (S) 15.59 (S) 7.69 (1) 10.27 (S) 16.39 (S) 14.77 (S)
a b
Rs = 2 × (t2 − t1)/(W1 + W2). Mobile phase: 0.1% FA in H2O/CH3CN.
12.01 (R) 18.53 (R) 8.47 (2) 11.56 (R) 17.82 (R) 16.60 (R)
α
Rsa
Mobile phaseb
1.21 1.19 1.10 1.13 1.09 1.12
2.23 2.07 1.55 1.72 1.54 1.77
65/35 62/38 65/35 64/36 69/31 71/29
enabled the satisfactory separation of the diastereomers within 20 min; the resolution (Rs) values for all the derivatives exceeded 1.54. Thus, the OTPA-derivatization was useful for the enantiomeric separation of chiral carboxylic acids. Based upon these observations, the conditions presented in Section 2.2 were ultimately selected for the separation of the carboxylic acid enantiomers.
3.4. Validation of the proposed method The calibration curves were obtained using five different concentrations. The determination at each concentration was repeated five times. A good calibration curve was obtained for each of the carboxylic acid enantiomers. Table 2 shows the calibration characteristics and detection limits of the six carboxylic acid enantiomers. Linear calibration curves were obtained for each carboxylic acid enantiomer (r2 N 0.997). The detection limits (S/N = 3) were 1.4–7.6 μmol/L. To evaluate the present method, the precision (CVs, %) was determined. The precision for three different concentrations were evaluated using intra-day and inter-day assays. As shown in Table 3, the CVs of the intra-day and inter-day determinations were 0.54–4.02% and 0.57–6.77%, respectively. The accuracy of the procedure was evaluated by spiking the human plasma with a known concentration of carboxylic acid enantiomeric standard. The recovery was established by analyzing a spiked standard solution of a known concentration of carboxylic acid enantiomers on the HPLC-UV system. As shown in Table 4, the mean recoveries (%) of carboxylic acid enantiomers from the spiked human plasma were in the range 95.27–101.12%. Furthermore, the precisions (CV) were within 0.65–8.02%. The good linearity, sensitivity, and precision demonstrated that the present method is applicable for human plasma analyses.
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Table 2 Calibration curves of chiral carboxylic acid compounds by the proposed method. Carboxylic acid compounds
Calibration range (mM)
Linear equation
Linearity (R2)
CV% (n = 5)
LOD (μmol/L)
S-KET R-KET S-NAP R-NAP S-IBU R-IBU S-PPA R-PPA LOX-1 LOX-2 S-PBA R-PBA
0.0125–1.25 0.0125–1.25 0.0125–1.25 0.0125–1.25 0.05–1.0 0.05–1.0 0.05–1.0 0.05–1.0 0.05–1.0 0.05–1.0 0.05–1.25 0.05–1.25
y = 1,289,360.68 × −29,523.1 y = 1,185,142.67 × −30,438.53 y = 618,318.39 × +11,930.97 y = 618,527.6 × +17,965.95 y = 110,012.76 × +2819.62 y = 123,376.67 × +3402.2 y = 101,171.44 × −360.68 y = 104,033.76 × +2518.1 y = 94,064.41 × −3690.61 y = 107,034.57 × −4416.54 y = 79,794.18 × −1582.28 y = 78,437.45 × −1969.16
0.9990 0.9990 0.9984 0.9971 0.9994 0.9991 0.9985 0.9973 0.9994 0.9991 0.9980 0.9984
0.23–1.41 0.58–1.66 0.35–2.75 0.63–3.45 0.67–4.01 0.51–2.58 0.94–2.55 0.40–2.98 0.18–1.58 0.64–1.55 0.43–2.93 0.36–1.47
1.4 1.4 2.2 2.2 4.0 4.0 5.6 5.6 6.3 6.3 7.6 7.6
LOD: Limit of detection.
3.5. Determination of carboxylic acid enantiomers in human plasma As examples of the bioanalysis by the present method, the separation and detection of carboxylic acid enantiomers in human plasma were performed using OTPA. As shown in Fig. 6, the R,S-NAP in the spiked human plasma was completely separated and detected without interference of any endogenous substances. Furthermore, the chromatograms in Fig. 6 A were almost the same as those of authentic R,SNAP in Fig. 6C. Consequently, the OTPA derivatization reagent can be used as a reagent for the separation and detection of a pair of carboxylic acid enantiomers. The OTPA features are preferable for targeted
Table 3 Accuracy and precision of the proposed method by intra-day and inter-day assays. Carboxylic acid compounds
Amount (mM)
Intra-day assay CV (%) (n = 6)
Inter-day assay CV (%) (n = 6)
S-KET
0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0
2.46 0.58 3.28 1.49 0.64 3.00 1.23 1.00 1.78 1.92 4.02 2.95 0.94 0.81 1.80 1.27 0.90 1.63 2.26 1.22 1.25 1.95 2.95 1.92 0.54 3.57 1.55 0.89 1.73 2.36 1.91 1.23 1.54 1.19 0.74 1.39
2.65 3.70 4.12 1.99 2.88 4.17 1.46 1.98 1.90 3.72 6.77 3.39 2.03 0.81 1.14 2.71 1.22 2.06 2.26 2.37 2.88 1.01 3.49 3.00 0.57 3.85 2.73 1.11 2.17 3.04 3.50 2.11 2.28 1.23 1.84 1.35
R-KET
S-NAP
R-NAP
S-IBU
R-IBU
S-PPA
R-PPA
LOX-1
LOX-2
S-PBA
R-PBA
carboxylic acid analyses such as metabolomics. Judging from these results, OTPA seems to be a useful chiral derivatization reagent for the determination of chiral carboxylic acids in real samples. 4. Conclusion We developed a novel derivatization reagent, OTPA, for the enantiomeric separation of chiral carboxylic acids using HPLC. The derivatization was completed at 40 °C within 90 min. A simple and practical procedure using HPLC-UV was developed for the enantiomeric determination of carboxylic acids. As a result, the diastereomers derived from the chiral carboxylic acids were satisfactorily separated by reversedphase chromatography (Rs = 1.54–2.23). Since the proposed method provides satisfactory separation and recovery, it was adapted for the determination of the enantiomers of NSAIDs in human plasma. The
Table 4 Determination of chiral carboxylic acid compounds in spiked human plasma. Carboxylic acid compounds
Spiked amount (mmol/L)
Detection amount (mmol/L)
CV (%) (n = 5)
Recovery Mean (%) recovery (%)
S-NAP
0.1 0.6 1.0 0.1 0.6 1.0 0.1 0.6 1.0 0.1 0.6 1.0 0.1 0.6 1.0 0.1 0.6 1.0 0.1 0.6 1.0 0.1 0.6 1.0 0.1 0.6 1.0 0.1 0.6 1.0
0.101 ± 0.0010 0.586 ± 0.0073 0.994 ± 0.0218 0.104 ± 0.0015 0.590 ± 0.0069 1.005 ± 0.0270 0.090 ± 0.0018 0.595 ± 0.0213 0.969 ± 0.0566 0.098 ± 0.0035 0.585 ± 0.0274 0.988 ± 0.0348 0.089 ± 0.0038 0.600 ± 0.0208 0.972 ± 0.0144 0.091 ± 0.0031 0.597 ± 0.0184 0.975 ± 0.0176 0.093 ± 0.0065 0.628 ± 0.0494 0.996 ± 0.0225 0.090 ± 0.0045 0.623 ± 0.0500 0.991 ± 0.0140 0.102 ± 0.0007 0.595 ± 0.0093 0.984 ± 0.0135 0.097 ± 0.0007 0.618 ± 0.0098 0.989 ± 0.0203
1.01 1.25 2.19 1.42 1.17 2.69 1.95 3.59 5.85 3.52 4.69 3.52 4.28 3.47 1.48 3.43 3.07 1.80 7.01 7.86 2.26 5.00 8.02 1.41 0.65 1.55 1.37 0.74 1.59 2.05
101.10 97.61 99.42 104.50 98.37 100.5 90.00 99.11 96.86 98.11 97.52 98.80 88.58 100.00 97.21 90.72 99.52 97.52 92.84 104.7 99.58 89.67 103.8 99.11 102.0 99.25 98.38 96.60 102.9 98.88
R-NAP
S-IBU
R-IBU
S-PPA
R-PPA
S-PBA
R-PBA
S-KET
R-KET
99.37
101.12
95.32
98.14
95.27
95.92
99.03
97.51
99.88
99.47
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Fig. 6. HPLC-UV Chromatograms of the diastereomers obtained from R,S-NAP in spiked human plasma. (A) R,S-NAP standards, (B) Blank plasma, (C) R,S-NAP in spiked plasma.
present method has sufficient specificity and practicality for the analysis of human plasma samples. Furthermore, the method also seems to be applicable for the enantioselective determination of carboxylic acids in various biological specimens. Research aimed at the simultaneous determination of chiral carboxylic acids in biological fluids is currently underway in our laboratory.
Acknowledgments The present research was supported by the National Natural Science Foundation of China (81360487, 81660594), and the Science and Technology Development Project of Jilin Province of China (20160101207JC).
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