Chiral liquid chromatography-mass spectrometry (LC-MS/MS) method development with β-cyclodextrin (β-CD) derivatized chiral stationary phase for the enhanced separation and determination of flurbiprofen enantiomers: Application to a stereoselective pharmacokinetic study

Article abstract of DOI:10.1039/d0nj01516d

A method was developed and validated for the enantioselective determination of flurbiprofen in rat plasma using liquid chromatography/electrospray ionization-tandem mass spectrometry under reversed-phase elution mode. Two polysaccharide derivatized chiral stationary phases and a homemade β-cyclodextrin (β-CD) derivatized based chiral column were evaluated. The latter one, a per-4-chlorophenylcarbamate-β-cyclodextrin bonded chiral stationary phase which was synthesized in our laboratory, enabled the highly sensitive detection and complete separation (resolution 2.0) of the flurbiprofen enantiomers. The assay was carried out after the solid-phase extraction procedure with C18 cartridges, and with R-(-)-ibuprofen used as the internal standard. The developed method has been validated for specificity, linearity, accuracy, precision, recovery, matrix effect, stability, carryover effect and dilution effect. The lower limit of quantification for R-flurbiprofen and S-flurbiprofen was 10 ng mL-1 in rat plasma, respectively. Linearity was confirmed in the range of 10.0-20000.0 ng mL-1 with a correlation coefficient (r2) greater than 0.996. The established method was successfully applied to a stereoselective pharmacokinetic study of flurbiprofen enantiomers in rat plasma following oral administration. This journal is

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Chiral liquid chromatography–mass spectrometry (LC–MS/MS) method development
with β-cyclodextrin (β-CD) derivatized chiral stationary phase for the enhanced
separation and determination of flurbiprofen enantiomers: Application to a
stereoselective pharmacokinetic study
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Jiayi Sun, Beibei Liu, Liangzhao Cai, Jia Yu*, Xingjie Guo *
Affiliation:
School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, Liaoning
Province, P. R. China.
Correspondence:
10 Jia Yu, Shenyang Pharmaceutical University, No. 103, Wenhua Road Shenhe District
11 110016, Shenyang, Liaoning Province, P. R. China
12 E-mail: yujia_yc@163.com Tel: +86-24-4352-0575
13 Xingjie Guo, Shenyang Pharmaceutical University, No. 103, Wenhua Road Shenhe
14 District 110016, Shenyang, Liaoning Province, P. R. China
15 E-mail: gxjhyz@gmail.com Tel: +86-24-4352-0575
16
17 Abstract
18 A method was developed and validated for the enantioselective determination of
19 flurbiprofen in rat plasma using liquid chromatography/electrospray ionization-tandem
20 mass spectrometry under reversed-phase elution mode. Two polysaccharide derivatized
21 chiral stationary phases and a homemade β-cyclodextrin derivatized based chiral
22 column were evaluated. The latter one per-4-chlorophenylcarbamate-β-cyclodextrin
23 bonded chiral stationary phase which was synthesized in our lab enabled the highly
24 sensitive detection and complete separation (resolution 2.0) of the flurbiprofen
25 enantiomers. The assay was carried out after the solid-phase extraction procedure with
26 C18 cartridges, and with R-(−)-ibuprofen used as the internal standard. The developed
27 method has been validated for specificity, linearity, accuracy, precision, recovery,
28 matrix effect, stability, carry over effect and dilution effect. The lower limit of
29 quantification for R-flurbiprofen and S-flurbiprofen was 10 ng mL-1 in rat plasma,
30 respectively. Linearity was confirmed in the range of 10.0–20000.0 ng mL-1 with a
31 correlation coefficient (r2) greater than 0.996. The established method was successfully
32 applied to a stereoselective pharmacokinetic study of flurbiprofen enantiomers in rat
33 plasma following oral administration.
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35 Key words: cyclodextrin derivatized chiral stationary phase, flurbiprofen enantiomers,
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36 LC–MS/MS, stereoselective pharmacokinetics
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38 1. Introduction
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The 2-arylpropionic acids (2-APAs) are an important group of nonsteroidal anti-
41 inflammatory drugs (NSAIDs) which are widely used for the treatment of pain and
42 inflammation in rheumatic and musculoskeletal disorders [1-2]. Epidemiological
43 researches have demonstrated that long-term use of this class of drugs can reduce
44 Alzheimer’s disease risk development and delay its onset [3-5]. In the last few decades,
45 2-APAs analyses have been the focus of intensive pharmaceutical research, since these
46 drugs possess a stereogenic center in the propionic acid moiety. Enantiomers of chiral
47 2-APAs show identical physicochemical properties but may differ in their biological
48 properties such as pharmacology, pharmacokinetics, metabolism and toxicology [6].
49 One enantiomer may be effective, while the other could be inactive or responsible for
50 side or antagonist effects, despite this, most chiral 2-APAs intended for either human
51 or animal use are still administered as racemates [7].
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Flurbiprofen (FBP), 2-(2-fluorobiphenyl-4-yl) propanoic acid, is a commercially
54 available anti-inflammatory/analgesic of the chiral 2-APAs class. Clinically, it is used
55 as a racemate for the management of pain and inflammation in patients with rheumatoid
56 arthritis and osteoarthritis [8]. Enantiospecific differences of the two enantiomers in
57 pharmacological properties have been reported after administration of FBP racemates,
58 since the S-(+) form was much more effective in cyclooxygenase inhibition which was
59 responsible for the anti-inflammatory activity and has been also related to a
60 neuroprotective function, whereas the other form performed the very undesirable side
61 effects, such as gastrointestinal irritation [9].
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Several methods have been established for the determination of racemic FBP in
64 plasma samples including high-performance thin-layer chromatography (HPTLC) [10],
65 gas chromatography with mass spectrometry [11], high-performance liquid
66 chromatography (HPLC) coupled with ultra-violet/visible (UV/Vis) [12-13],
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67 fluorescence [14-16], and tandem mass spectrometric detection [8,17-18]. However,
68 only a few HPLC methods have been reported for the stereoselective determination of
69 FBP in plasma [19-22]. In these stereoselective assays, chiral separations were obtained
70 by the use of pre-column derivatization or chiral stationary phases (CSPs). Since the
71 reported methods [19,20,22] for the determination of FBP enantiomers in plasma
72 needed a derivatization reaction with a tedious workup procedure, the HPLC method
73 by CSP emerged as a powerful method for chiral separation of FBP. Teng et al. [21]
74 have reported a stereospecific HPLC-UV analysis of FBP on a Chiralpak AD-RH
75 column, but the sensitivity of this method was poor (50 ng mL-1 for each enantiomer)
76 and the analysis time was approximately 20 min. Moreover, the commercial CSP used
77 for chromatographic separation sometimes requires specific elution mode, such as
78 normal-phase mode and reversed-phase mode, and often it is very expensive. Thus,
79 there is a need for new packing materials offering higher enantioselectivity with the
80 simplified preparation process and shortened analysis time. By far, cyclodextrin (CD)
81 derivatives have been used extensively in CSPs ascribing to their ability to
82 accommodate guest molecules through the formation of inclusion complexes. Although
83 there has emerged a series of CD CSPs developed via different ways, there still exist
84 many challenges in this area. On one hand, the development of stable and functional
85 linkages is relatively difficult. On the other hand, CD rims modification usually
86 enhances CSP's enantioselectivity toward specific categories of enantiomers while
87 diminishing its original recognition ability to some enantiomers. In other words, the
88 drugs that can be chirally separated on each CSP is limited. Hence, it is a great challenge
89 to explore novel approaches for the fabrication of more versatile CD CSPs. Recently,
90 we reported a β-CD derivatized CSP by one step amino linkage immobilized to silica
91 gel [23]. The column with per-4-chlorophenylcarbamate-β-CD bonded CSP (CPCDP)
92 exhibited more excellent chiral recognition ability for azole antifungal agents, proton
93 pump inhibitors and dihydropyridine calcium antagonists in normal phase mode than
94 its analogy, commercial 3,5-dimethylphenyl carbamate-β-CD based CSP, due to
95 different position of electro-withdrawing or electron-donating functionalities on
96 phenylcarbamate moiety. The enhanced enantioselectivities were attributed to the
97 enlarged difference in the formed temporary complexes aroused from multiple
98 intermolecular interactions between CSP and solutes, which may include inclusion
99 complexation, π–π stacking interaction, hydrogen-bonding, dipole–dipole interaction
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100 and steric hindrance.
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The present study focused on the further exhibited enhanced enantioselective
103 ability of the per-4-chlorophenylcarbamate-β-CD bonded CSP (CPCDP) towards FBP
104 enantiomers. An efficiency chiral separation method with reversed-phase was
105 developed and interfaced with a simple and reproducible LC-MS/MS method system.
106 The relatively high-resolution value was obtained with the lower LLOQ (10 ng mL-1
107 for each enantiomer) in a shorter analysis time (10 min), and the validated method was
108 successfully applied to a stereoselective pharmacokinetic study in the rat after oral
109 administration of racemic FBP.
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Fig. 1. The chemical structures of (a) FBP and (b) R-(−)-IBF (IS).
114 2. Materials and methods
115 2.1. Chemicals and consumables
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Racemic flurbiprofen (FBP) was purchased from the National Institute for Food
118 and Drug Control (Beijing, China). R-(−)-ibuprofen (IBF) was obtained from Sigma-
119 Aldrich (St. Louis, USA). Their chemical structures were shown in Fig.1. Ultrapure
120 water was purified by the Milli-Q academic water purification system (Millipore, MA,
121 USA) and was used in all experiments. Methanol (MeOH) and acetonitrile (ACN) of
122 HPLC grade were procured from Fisher Scientific (Fair Lawn, NJ, USA). Other HPLC
123 grade solvents, n-hexane (n-hex), isopropanol (IPA), diethyl ether (DEE) and ethyl
124 acetate (EtAc) were supplied by Concord Technology (Tianjing, China). Analytical-
125 grade ammonium acetate and glacial acetic acid were provided by Sigma-Aldrich (St.
126 Louis, USA). Formic acid (analytical grade) was purchased from Tianjin Heng Xing
127 Chemical Reagent Co., Ltd. (Tianjin, China). The cartridge used for SPE, Sep-pak C18
128 (1 cc/50 mg), Sep-pak NH2 (1 cc/50mg) and Oasis HLB (1 cc/30mg) were from Waters
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129 Corporation (Waters Co., Milford, MA, USA).
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131 2.2. Preparation of standards, reagents and calibration solutions
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A target amount of 25 mg racemic FBP was accurately weighed on an analytical
134 balance and dissolved with MeOH in a 25 mL amber-colored volumetric flask to make
135 a storage solution with a concentration of 1.00 mg mL-1. A methanolic stock solution
136 of R-(−)-IBF (internal standard, IS) was prepared similarly with the target concentration
137 of 0.1 mg mL-1. This solution was diluted with MeOH to make a working IS solution
138 of 5 µg mL-1. Calibration standards were obtained daily from the stock solution of FBP
139 by sequential dilution with blank rat plasma, yielding a series of final concentrations
140 namely, 0.01, 0.05, 0.10, 0.20, 1.00, 5.00, 10.0 and 20.0 µg mL-1 for R-(−)- and S-(+)-
141 FBP in plasma. Similarly, quality control (QC) samples were made from the stock
142 solution of FBP at the concentrations of 0.02 (low quality control, LQC), 1.00 (medium
143 quality control, MQC) and 16.0 µg mL-1 (high quality control, HQC) for each
144 enantiomer of FBP. These solutions were protected from light and stored at 4 °C before
145 use.
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147 2.3. Sample preparation
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FBP was extracted from plasma samples using a solid-phase extraction method
150 with Sep-pak C18 cartridge (1 cc/50 mg). Briefly, previously frozen plasma samples
151 were thawed and vortexed and 100 µL was transferred to a centrifuge tube then 10 µL
152 aliquot of the IS working solution was added followed by 10 µL of 0.1% (v) formic
153 acid. The tube was vortexed for 3 min. The cartridge was preconditioned with 3 mL of
154 MeOH and then equilibrated with 3 mL of water. Then, the sample solution was loaded
155 onto the SPE column. When the sample had eluted under gravity, the column was
156 washed with 5% (v) MeOH in water (1 mL). Vacuum was applied to the cartridge for 2
157 min to completely dry the resin. Finally, elution was carried out with 1 mL MeOH into
158 pre-labeled tubes, and the elute was evaporated to dryness under a gentle stream of
159 nitrogen at the ambient temperature. The obtained residue was reconstituted with 100
160 µLmobile phase and vortexed for 3 min. After filtering through a 0.22 µm nylon syringe
161 filter, an aliquot of 10 µL was used for injection in the chromatographic system.
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All sample analysis was carried on an Agilent (Palo Alto, CA, USA) 1200 HPLC
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166 system coupled to an Agilent 6410 triple-quadrupole mass spectrometer with
167 electrospray ionization. The enantioseparation of FBP was performed on the CDCDP
168 column, which was prepared in the lab according to our previous report [23]. Another
169 two columns for comparison, Chiralpak AD-RH and OJ-RH column (150 × 4.6 mm i.d.,
170 5 µm), coupled to a guard column (10 × 4.6 mm i.d., 5 µm), were supplied by Daicel
171 Chiral Technologies Co., Ltd (Tokyo, Japan). The column temperature was set at 20 °C.
172 An isocratic mode was used to separate and elute targeted analytes from the column.
173 The optimized enantiomeric separation was obtained with the mobile phase consisting
174 of 5 mM ammonium acetate buffer at pH 5.0 and MeOH (30:70, v/v), with a flow rate
175 of 0.5 mL min-1. The autosampler temperature was set at 4°C and the injection volume
176 was 10 µL. Detection was implemented using multiple reaction monitoring (MRM)
177 mode in the ESI negative ion mode to monitor precursor-product ion pair transitions of
178 m/z 243.0–199.1 for FBP enantiomers and m/z 205.0–161.1 for R-(−)-IBF, respectively.
179 Other instrumental parameters were set as follows: capillary voltage 3.50 kV; source
180 temperature 150 °C; drying gas temperature 500 °C; desolvation gas flow 15 L min-1;
181 nebulizer gas pressure 35 psi. Nitrogen was used as nebulizing and desolvation gas.
182 Argon was applied as collision gas.
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184 2.5 Method validation
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The validation was performed based on "Guidance for Industry: Bioanalytical
187 Method Validation" from the United States Food and Drug Administration [24].
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189 2.5.1 Specificity
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The assay selectivity was determined by analyzing extracts of multiple samples
192 (n=6) of plasma from different batches to evaluate the possibility of endogenous
193 interferences at the retention time of analytes and the IS. The peak area of the
194 endogenous components co-eluted with the compound of interest should be less than
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195 20% of the peak area of the lower limit of quantification (LLOQ) and 5% of the peak
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198 2.5.2 Linearity and LLOQ
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201 curves containing eight nonzero levels on three consecutive days. The calibration
202 curves were originated individually by using the peak area ratio of each enantiomer to
203 IS (y-axis) versus the corresponding concentration of enantiomer (x-axis) whereby a
204 least-square 1/x2-weighted linear regression. The acceptance criterion was that the
205 coefficient correlation (r2) must be more than 0.99. The LLOQ is the concentration at
206 which a method can discriminate with a signal-noise ratio greater than or equal to 5.
207 The accuracy of each spiked LLOQ sample (n=6) should be within ±20% and its
208 precision should not be greater than 20%.
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210 2.5.3 Accuracy and precision
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213 QC samples at three levels (0.02, 1.00, 16.0 µg mL-1) for each enantiomer on three
214 consecutive days. The final concentration of the spiked sample was determined by the
215 calibration curve. The accuracy was calculated through the ratio of the concentration
216 obtained from the standard curve versus the nominal concentration spiked in the blank
217 sample. The precision of intra- and inter-day were expressed as the relative standard
218 deviation (RSD), while accuracy was reported as the relative error (RE). For successful
219 method validation, the intra- and inter-day precision values were required not to exceed
220 15% and the accuracy was required to be within ±15%.
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222 2.5.4 Extraction recovery and matrix effect
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To estimate the extraction recovery of each enantiomer, the individual peak areas
225 of analytes and IS obtained from QC samples (spiked before extraction) were contrasted
226 with that achieved from unextracted samples (spiked after extraction) at three QC
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228 comparison of peak area ratios of analytes to IS in the spiked post-extraction plasma
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236 plasma samples at two QC concentration levels (LQC and HQC). The different storage
237 and handling conditions were as follows: (a) bench-top stability at room temperature
238 for 12h; (b) freeze–thaw stability after three freeze–thaw cycles at −80°C; (c) long-term
239 stability at −80°C for 120 days; (d) autosampler tray stability at 4°C for 24 h. The
240 analytes were considered to be stable when the accuracy biases were within ±15% of
241 the freshly prepared concentrations.
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243 2.5.6 Carry over effect
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246 after the high concentration standard (20000.0 µg L-1). The outcomes of the carry over
247 effect should be less than or equal to 20% of the analytes and 5% for the IS.
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249 2.5.7 Dilution effect
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252 samples were determined, which were prepared at a concentration (100.0 µg mL-1 for
253 each enantiomer) above the upper limit of quantification (ULOQ) level and diluted 10-
254 fold to a concentration (10000.0ng mL-1 for each enantiomer) within the calibration
255 curve range. The accuracy should be in the range of 85–115% of the nominal
256 concentration, and the precision should less than 15%.
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258 2.6 Application to a pharmacokinetic study
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261 Center of Shenyang Pharmaceutical University (license no. SCXK-liao-20150001).
262 The rats were allowed to adapt for five days under controlled conditions (temperature:
263 25 °C, a standard diet, free access to water) and fasted 12 h before the experiment.
264 Following the oral administration of 10 mg kg-1 of racemic FBP to six Sprague Dawley
265 rats, orbital blood samples were collected at 0.083, 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4,
266 6, 8, 12 and 24 h in heparinized tubes. The pharmacokinetic parameters of FBP
267 enantiomers were determined using DAS 2.1.1 software and the entire data were
268 expressed as mean ± standard deviation. The animal protocol was permitted by the
269 Committee on the Ethics of Animal Experiments of Shenyang Pharmaceutical
270 University (permit number, SYPU-IACUC-C2019-6-16-202).
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272 3. Results and discussion
273 3.1 Optimization of chromatographic conditions
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In the routine CSP enantioseparation, it seemed to be that organic eluents (normal
276 phase elution mode) were frequently used. But in analyses of samples from biological
277 fluids, the choice of reversed-phase mode was much more beneficial due to the low
278 sample solubility of polar species in certain mobile phases and the lack of appropriate
279 enantioselectivity in organic eluents. Besides, for LC–MS applications the reversed-
280 phase mode has better compatibility and suitability. Thus, with the aim of achieving the
281 desired stereospecific separation, the enantioseparation performances of three CSPs in
282 reversed-phase mode were tested and compared: a homemade CDCDP column
283 (immobilized with per-4-chlorophenylcarbamate-β-CD), a Chiralpak AD-RH column
284 [coated with amylose tris(3,5-dimethylphenylcarbamate)] and a Chiralpak OJ-RH
285 column [coated with cellulose tris (4-methylbenzoate)]. The latter two columns have
286 been reported to exhibit chiral recognition ability for FBP extracted from different
287 matrices [21, 25]. According to the reported results, the analysis time of FBP for
288 complete chiral separation on the Chiralpak AD-RH column was about 20 min [21],
289 while that on Chiralpak OJ-RH column was about 35 minutes [25]. The prolonged
290 retention time was not conducive to the high-throughput biological sample analysis.
291 However, on CDCDP column the analyte enantiomers were completely separated
292 within 10 min (the resolution was about 2.0, Fig. 2a) after optimization of the conditions.
293 Besides, at the similar retention time level, the resolution of FBP on Chiralpak AD-RH
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294 and Chiralpak OJ-RH were less than 1.5 [1.0 for the AD-RH (Fig. 2b) and 0.8 for the
295 OJ-RH column (Fig. 2c)]. Therefore, the CDCDP column was selected for the
296 subsequent experiment due to its enhanced enantioseparation ability towards FBP.
297
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The possible chiral separation mechanism might be based on the inclusion-
299 complexation between the CSP and the analyte [26]. Chiralpak AD-RH and OJ-RH
300 columns are polysaccharide derivatives. So, they both have the functional groups for
301 chiral interactions: methyl, carboxyl and benzene ring [27-28]. However, the
302 enantiomeric resolution of FBP on AD-RH and OJ-RH column was not the same, and
303 FBP obtained better enantioseparation on AD-RH column. A reasonable explanation
304 may be that the hydrogen-bonding effect might be an essential factor and be beneficial
305 to the spatial recognition of CSP to the FBP enantiomers. By comparing the structure
306 of CSP for AD-RH and OJ-RH columns, it can be known that there is one more amino
307 group in the structure of AD-RH column. And FBP contains electronegative benzene
308 ring group, which can form hydrogen bonds with polar groups such as amino groups
309 on the CSP. The derivatized β-CD in CDCDP column increased the hydrogen bonding
310 capability on the analyte and the analyte with bulky substituents near the chiral center
311 gave the opportunity for steric interactions also, thus resulting in enhanced and better
312 enantioseparation ability in a relatively short analysis time [24, 29].
313
314
315
316
317
318
Fig. 2. The enantioseparation of FBP on (a) CDCDP, (b) AD-RH and (c) OJ-RH
column.
Using CDCDP for the enantiomeric separation of R-(−)-FBP and S-(+)-FBP, the
319 detailed mobile phase condition optimization was studied. Initially, MeOH/ACN-water
320 without any additive in the mobile phase, the FBP enantiomers could not be separated.
321 Then, the addition of acidic additives and buffer solutions were evaluated. The
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322 experimental results showed that the composition of the mobile phase including MeOH
323 (organic phase) and formic acid or ammonium acetate buffer solution (aqueous phase)
324 significantly improved the separation of FBP. However, when formic acid was present
325 in the mobile phase, the retention time was prolonged. Then, the different
326 concentrations of ammonium acetate buffer solution were investigated to obtain a
327 satisfactory chiral separation. With the increase of buffer concentration, there was no
328 obvious difference in the resolution of FBP, however, the response of R-(−)-FBP and S-
329 (+)-FBP two enantiomers was gradually increased with the concentration of ammonium
330 acetate in the range of 2.0-10 mM. It should be noted that using MS detection, when no
331 buffer salt was present in the mobile phase, the ESI-MS signal intensity of the analyte
332 was poor. Conversely, too high a buffer concentration may cause the intensity of the
333 analyte to be dramatically suppressed. The typical MRM chromatograms, which
334 represented the effect of the mobile phase on the enantioseparation of R-(−)-FBP and
335 S-(+)-FBP, were shown in the Supplementary Fig. S1. Although the enantiomeric
336 resolution could be enhanced by further decreasing the amount of organic solvent, the
337 retention times of two enantiomers were increased as well as their detection sensitivity.
338 For the high throughput pharmacokinetic study of enantiomers, rapid analysis time and
339 good sensitivity were mainly comprehensively considered. When 70% (v) MeOH and
340 30% (v) 5 mM ammonium acetate buffer (pH adjusted to 5.0 with glacial acetic acid)
341 was used, the retention and resolution were optimal and an acceptable sensitivity was
342 obtained.
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344 3.2 Sample preparation
345
346
During sample pretreatment, the liquid–liquid extraction (LLE) and solid-phase
347 extraction (SPE) were compared in order to obtain optimum extraction and
348 preconcentration of FBP enantiomers from plasma. For each procedure, the recoveries
349 were calculated and then compared to the extraction efficiency results obtained from
350 another one. Therefore, plasma samples containing each enantiomer of FBP at
351 concentrations of 0.02, 1.00 and 16.0 µg mL-1 were analyzed, respectively. For each
352 concentration, three replicates were made.
353
In the LLE part, according to the literature [12,19,21] and the solubility of FBP in
354 different solvent systems, n-hex-EtAc (60:40, v/v), n-hex-DEE (80:20, v/v) and
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355 trimethylpentane-IPA (95:5, v/v) are effective in extracting the FBP from plasma with
356 good recoveries (above 90% for these three extraction solvent systems). However,
357 when using trimethylpentane as the extraction solvent, its vapor could form explosive
358 mixture, which was in danger of combustion and explosion in case of heat source and
359 open fire. Thus, trimethylpentane-IPA was abandoned. When using n-hex-DEE (80:20,
360 v/v) as the extraction solvent, the recovery of FBP in plasma was lower than that of
361 using n-hex-EtAc (60:40, v/v), and DEE is also costlier than ethyl acetate.
362 Consequently, n-hex-EtAc (60:40, v/v) was selected as the final extraction solvent for
363 LLE process. The overall extraction recovery results of FBP (mean ± SD) are
364 summarized in Table 1.
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366
367
Table 1
The recovery results for FBP after using various sample preparation procedures.
Recovery (%, mean ± SD)
Recovery (%, mean ± SD)
Liquid-liquid
Solid-phase
extraction (LLE)
extraction (SPE)
R-(−)-FBP
91.4 ± 8.5
88.5 ± 7.1
66.9 ± 5.9
S-(+)-FBP
92.3 ± 8.7
89.3 ± 7.8
68.1 ± 5.4
R-(−)-FBP
92.8 ± 4.4
74.1 ± 4.0
50.2 ± 6.7
S-(+)-FBP
94.3 ± 3.5
75.8 ± 3.2
53.2 ±7.3
N-hex-EtAc
(60:40, v/v)
N-hex-DEE
(80:20, v/v)
N-hex-IPA (95:5,
v/v)
Sep-pak C18
Oasis HLB
Sep-pak NH2
368
369
During the development of the SPE extraction procedure several different
370 cartridges were also examined including the Sep-pak C18, Oasis HLB and Sep-pak
371 NH2. They were checked in the same experimental conditions: the biological sample
372 (100 µL of plasma) with 10 µL aliquot of the IS working solution before SPE; 5% (v)
373 MeOH in water as the washing agent (1mL) and MeOH used as an eluting solvent
374 (1mL). After preliminary optimization, C18 columns offered slightly more effective
375 FBP extraction than HLB and NH2. Additionally, other modifications of the sample
376 matrix, before SPE with C18 cartridges, were tested for checking whether the addition
377 of organic solvent or decreasing the pH of the sample can improve the extraction
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378 efficiency of FBP from plasma. Thus, 0.5 mL of the mixture of MeOH-water (10:90,
379 v/v), 0.01% (v) formic acid, 0.05% (v) formic acid and 0.1% (v) formic acid,
380 respectively, was added to the plasma samples, and next, the SPE procedure was carried
381 out in the same experimental conditions as described above. As it is shown in Table 1,
382 the highest recoveries of FBP from plasma was acquired after the acidification of
383 biological matrix with 0.1% (v) formic acid. Summarizing, the SPE methodologies with
384 C18 cartridges offered better extraction of FBP from plasma samples than the tested
385 LLE procedures. The detailed description of the developed SPE method with C18
386 cartridges chosen as the most optimal for the preparation of rat plasma was presented
387 in the section “Sample Preparation”. It should be noted that the developed SPE
388 procedure based on C18 cartridges allowed higher recoveries of FBP was the first report
389 on the extraction of FBP from plasma and the results were comparable to the previous
390 data [12,19,21] reported in the literature. In addition to the high extraction recovery and
391 stable recovery results, the samples treated by the SPE process were also considered to
392 be less interfered by endogenous substances, especially for the expensive chiral column,
393 which can prolong its service life and reduce the analysis cost.
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395 3.3 Method validation
396 3.3.1 Specificity
397
398
Assay selectivity is demonstrated by the absence of interfering peaks at the
399 retention times of R-(−)-FBP and S-(+)-FBP and IS in extracted blank plasma samples.
400 Fig. 3 showed the typical chromatograms of a blank plasma sample, a blank plasma
401 sample added to racemic FBP at the LLOQ level (10 ng mL-1 per enantiomer), and an
402 actual rat plasma sample obtained at 0.25h after oral administration of 10 mg kg-1 FBP
403 racemic. The retention times for R-(−)-FBP and S-(+)-FBP were about 6.5 min and 9.0
404 min respectively. These chromatograms demonstrate the absence of any
405 chromatographic and/or mass spectrometric interference.
406
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408
409
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411
Fig. 3. Typical chromatograms of FBP enantiomers and R-(−)-IBF (IS) for (a) blank
plasma sample, (b) extracted rat plasma at LLOQ level, (c) extracted rat plasma
obtained at 0.25 h after oral administration of 10 mg kg-1 racemic FBP.
412 3.3.2 Linearity and LLOQ
413
414
Calibration curves for both enantiomers were validated in the concentration range
415 of 0.01–20.0 µg mL-1 in rat plasma. The degree of linear correlation was calculated as
416 the coefficient R2 >0.996 for standard curves based on three replicates of an 8-point
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417 calibration curve. With the stated assay conditions, this method offered an LLOQ of
418 10.0 ng mL-1 for each enantiomer with an accuracy and precision of 5.4 and 8.9% for
419 R-(−)-FBP, and 4.7 and 8.3% for S-(+)-FBP, respectively. The proposed method had
420 much higher sensitivity than the limits of quantification 100 ng mL-1[12] or 50 ng mL-
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[19,21] reported in literatures and the results were sufficient for the research on
422 pharmacokinetics in rat following oral administration of FBP.
423
424 3.3.3 Accuracy and precision
425
426
The outcomes of precision and accuracy for QC samples at three concentration
427 levels (LQC, MQC and HQC) analyzed in six replicates on three consecutive days are
428 presented in Table 2. The RSD values of intra- and inter-day precision for R-(−)-FBP
429 were <6.3%, whereas the RE values of accuracy varied from 4.8 to 9.4%. For S-(+)-
430 FBP, the RSD values of intra- and inter-day precision were within 7.3% while the RE
431 values of accuracy ranged from 2.6 to 3.9 %. These data fulfilled the desired criteria,
432 demonstrating that this method was feasible and reproducible for the measurement of
433 FBP enantiomers in rat plasma.
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435
436
Table 2
Intra- and inter-day accuracy and precision of R-(−)-FBP and S-(+)-FBP in plasma samples.
Analyte
Nominal
concentration
(ng mL-1)
Intra-day(n=6)
Calculated
Inter-day(n=18)
Calculated
RE
RSD
(%)
RE
RSD
(%)
concentration
(ng mL-1)
(%)
concentration
(ng mL-1)
(%)
R-(–)-FBP
S-(+)-FBP
20.0
21.2 ± 0.98
5.8
9.4
5.5
2.9
3.9
3.2
4.6
4.4
5.0
3.1
6.9
5.2
21.2 ± 1.34
5.8
9.3
4.8
3.1
3.8
2.6
6.3
5.1
5.4
5.8
7.3
5.3
1000.0
16000.0
20.0
1094.8 ± 48.1
16886.0 ± 837.8
20.6 ± 0.64
1092.7 ± 55.5
16760.9 ±907.4
20.6 ± 1.19
1000.0
16000.0
1039.0 ± 72.1
16518.2 ± 865.5
1037.9 ± 76.2
16421.1 ±864.2
437
438 3.3.4 Extraction recovery and matrix effect
439
440
The recoveries of R-(−)-FBP and S-(+)-FBP changed from 88.9 to 101.2% and
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441 from 89.9 to 100.5% at three distinct QC concentrations in rat plasma with all RSD
5
6
442 values within 6.1%. The IS-normalized MFs from six lots of blank plasma were
7
8
443 between 90.5 and 106.8% for the two enantiomers. These results confirm the validity
444 of the extraction procedure for sample preparation before chromatography to obtain
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445 reproducible and reliable quantitative results for the analyte, without interference of
446 matrix components.
447
448 3.3.5 Stability
449
450
The results of the stability studies are presented in Table 3. No significant
451 degradation of analytes or IS was observed in all the conditions as described in Section
452 2.5.5. As per the stability conditions described above, the accuracy in plasma ranged
453 from -7.3 % to 7.7 % and -7.7 % to 7.8% for R-(−)-FBP and S-(+)-FBP respectively.
454
455
456
Table 3
Stability results of R-(−)-FBP and S-(+)-FBP under various storage conditions (n = 6).
Nominal
concentration
(ng mL-1)
Sample conditions
Room temperature
for 12 h
Three freeze–thaw
cycles
−80 ◦C for 30 days
On the autosampler
tray 4 ◦C for 24 h
RE (%) RSD (%)
RE (%) RSD (%)
RE (%)
RSD (%)
RE (%)
RSD (%)
R-(−)-FBP
20.0
5.2
6.3
4.5
1.9
7.7
6.3
4.8
3.3
3.8
3.7
4.3
2.1
5.5
4.1
16000.0
S-(+)-FBP
20.0
-2.9
-7.3
4.9
6.1
5.2
2.5
7.8
6.7
3.7
2.2
3.2
3.0
3.6
2.2
4.5
3.9
16000.0
457
-2.8
-7.7
458 3.3.6 Carry over effect
459
460
No corresponding peak at the retention time of analytes and IS was found in blank
461 samples injected immediately following the analysis of the high concentration standard
462 sample, which indicated that the carry over effect in this proposed approach was absent.
463
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464 3.3.7 Dilution effect
4
5
6
7
8
465
466
It was shown that the accuracy values of 1/10 dilution specimens were 5.3% for R-
467 (−)-FBP and 4.8% for S-(+)-FBP, while the precision results were 6.2% for R-(−)-FBP
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468 and 5.1% for S-(+)-FBP. All of the results were within the acceptance criteria.
469
470 3.4 Application to a pharmacokinetic study
471
472
The applicability of the method described in this study was demonstrated through
473 the determination of R-(−)-FBP and S-(+)-FBP in rat plasma after oral administration
474 of 10 mg kg-1 of racemic FBP. The mean plasma concentration-time profiles of FBP
475 enantiomers quantified until 24 h are presented in Fig. 4, and the corresponding
476 pharmacokinetic parameters are summarized in Table 4.
477
478
As can be seen in Fig. 4, the concentrations of the two enantiomers both rapidly
479 increased following oral absorption, and reached the maximum plasma concentration
480 after 1.5h. The plasma concentration of S-(+)-FBP was consistently higher than that of
481 R-(−)-FBP after oral administration of racemic FBP, which was in accordance with the
482 literature data [21]. By comparing the drug concentration-time curves of the two
483 enantiomers more precisely, we can find that the mean Cmax value of S-(+)-FBP was 1.4
484 times greater than that of R-(−)-FBP, and the area under the plasma concentration versus
485 time curve (AUC0–∞) was 1.5-fold higher than that of R-(−)-FBP, which repeatedly
486 corroborated that S-(+)-FBP presented the enhanced pharmacodynamic activity
487 compared with R-(−)-FBP. Significant differences could be observed between
488 enantiomers with relatively higher plasma concentration (P < 0.05) and AUC for R-(−)-
489 FBP than that obtained for S-(−)-FBP. This significantly stereoselectivity data analyses
490 were first analyzed and observed in our study. Moreover, the plasma concentration of
491 S-(+)-FBP after oral administration had always been much higher than that of R-(−)-
492 FBP, which might be beneficial for S-(+)-FBP to perform the anti-inflammatory action
493 in vivo. Besides, this was also consistent with the previous literatures data of the
494 analogue drug, ibuprofen [28] in which the S-isomer form of ibuprofen was almost
495 entirely responsible for the anti-inflammatory action.
496
To some extent, conversion between the two enantiomers in vivo may contribute
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497 to the result. However, according to the results in previously reported literature [19],
498 there was no indication to prove the chiral inversion in the whole process. Therefore,
499 according to the previous research, all these pharmacokinetic phenomena and results of
500 R-(−)-FBP and S-(+)-FBP of the noticeable difference in plasma level could be mainly
501 due to the stereoselectivity of protein binding [20]. Briefly, this study warranted a sound
502 conclusion that the pharmacokinetics of FBP enantiomers after oral administration of
503 racemic FBP was stereoselective.
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Fig. 4. Mean plasma concentration–time curve of R-(−)- and S- (+)-FBP after oral
administration of 10 mg kg-1 racemic FBP to SD rats (n = 6).
Table 4
Pharmacokinetic parameters of R-(−)-FBP and S-(+)-FBP after oral administration of 10 mg kg-1
racemic FBP to SD rats (n = 6).
Racemic FBP
Parameters
R-(−)-FBP
S-(+)-FBP
Cmax (µg mL-1)
Tmax (h)
11.78 ± 1.11
1.50 ± 0.00
3.39 ± 0.51
16.99 ± 1.82
1.50 ± 0.00
4.31 ± 0.74
t1/2 (h)
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AUC0–t (µg h mL-1)
AUC0−∞ (µg h mL-1)
57.56 ± 4.28
57.89 ± 4.36
86.01 ± 4.84
89.12 ± 2.91
512
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513 4. Conclusion
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515
A new, specific, accurate and precise LC-MS/MS method for the quantification of
516 FBP enantiomers in rat plasma was developed and validated. The presented LC-MS/MS
517 method, based on rapid and effective SPE with C18 cartridges, which reduced the
518 sample volume and the time required for sample preparation and enantioseparation, can
519 be considered as an interesting analytical tool in comparison to previously reported
520 HPLC methods. This new LC-MS/MS method has been proved to be used for the
521 application for quantifying the rat plasma concentration of each enantiomer in real
522 samples successfully. The pharmacokinetic outcomes revealed that except for the same
523 Tmax value of two enantiomers of FBP, S-(+)-FBP also displayed prominently higher
524 Cmax and AUC values than R-(−)-enantiomer. There was no chiral inversion between R-
525 (−)- and S-(+)-FBP during the whole experimental process. Thereby, a high level of
526 stereoselectivity of FBP pharmacokinetics in rat plasma was preliminarily proved.
527 According to the previous literature, this is probably due to the different protein binding
528 rate.
529
530 Declaration of Competing Interest
531
532
533
None.
534 Acknowledgments
535
536
This work was supported by National Natural Science Foundation of China (No.
537 81703467).
538
539
540 Reference
541
542 [1] C.N. Huang, Y. Li, J.J. Yang, J.Y. Peng, J. Tan, Y. Fan, L.X. Wang, J.P. Chen,
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543 Hyperbranched mixed-mode anion-exchange polymeric sorbent for highly selective
544 extraction of nine acidic non-steroidal anti-inflammatory drugs from human urine,
545 Talanta.190 (2018)15–22. https://doi.org/10.1016/j.talanta. 2018.07.033
546 [2] N.H. Hashim, S.J. Khan, Enantioselective analysis of ibuprofen, ketoprofen and
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548 4746–4754. https://doi.org/ 10.1016/j.chroma.2011.05.046
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