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Simultaneous stereoisomeric separation of loxoprofen sodium and its alcohol metabolites. Application to a stereoselective pharmacokinetic study
⁎Corresponding authors. ppengfeizhao@163.com (Pengfei Zhao), wangzhaokun@bzmc.edu.cn (Zhaokun Wang)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Abstract
Loxoprofen sodium (LOX) is a propionic acid derivative non-steroidal anti-inflammatory drug (NSAID), existing in the form of four stereoisomers. The main metabolites of LOX in vivo were trans- and cis-alcohol, each consisting of four stereoisomers. The objectives of the present study were to examine the selective pharmacokinetic behavior of LOX stereoisomers and stereoselective formation of its alcohol metabolites in rats based on a chiral liquid chromatographytandem mass spectrometry (LC-MS/MS) method by using an FLM Chiral NQ-RH column, which was reported for the first time. The significant difference in pharmacokinetic parameters of four stereoisomers indicated that stereoselective behavior has occurred in rats after oral administration of LOX. (1′S,2S)-LOX showed the highest concentration among the four stereoisomers in both plasma and urine samples. Trans- and cis-alcohol metabolites of LOX were also detected in plasma and urine, and trans-alcohol was found to be primary and the stereoisomeric fractions (SFs) of its four stereoisomers at different times were calculated. Examination of the stereoisomeric composition indicated a stereo preference for (2S)-configuration with respect to trans-alcohol formation. The overall results of the present study revealed the enantioselective pharmacokinetic properties of LOX stereoisomers in rats, which provided a means to advance understanding of the complex pharmacokinetic of LOX.
Keywords
HPLC-MS/MS
Loxoprofen sodium
Alcohol metabolite
Rat plasma
Rat urine
Stereoselective behavior
1 Introduction
Loxoprofen sodium (LOX) [(±)-2-[4-(2-oxocyclopentylmethyl)phenyl]propionate dihydrate)] is an arylpropionic acid anti-inflammatory drug, which has excellent analgesic and antipyretic activities with fewer NSAID-related adverse events (Mu et al., 2016; Nourwali et al., 2019; Zhang et al., 2020; Endo et al., 2021; Zhou et al., 2022). LOX was firstly developed and approved for marketing as a new NSAID in Japan since 1986 (Ding et al., 2004; Greig and Garnock-Jones, 2016; Daiichi Sankyo Co., Ltd., 2015). LOX is indicated for pain and inflammation related to musculoskeletal and joint disorders (Fan et al., 2019; Sawamura et al., 2014). It is also the common treatments for chronic and transient conditions, such as toothache, headache, menstrual cramps, common cold, etc (Moore et al., 2015; Greig and Garnock-Jones, 2016; Wan et al., 2019). The anti-inflammatory and analgesic effects of LOX were 4 to 6 times as high as those of indomethacin, ketoprofen and naproxen (Tanaka et al., 1983; Ding et al., 2004). Tmax of LOX after oral administration was significantly shorter compared with other profens (derivatives of 2-arylpropionic acids), which results in a more rapid onset of action (Jamali and Brocks, 1990; Davies and Anderson, 1997; Tan et al., 2002; Helmy, 2013). Attributed to these advantages, LOX has been one of the most popular NSAIDs in Japan, and also well-known in Eastern Asia, the Middle East, Latin America, and Africa (Paudel et al., 2019).
With two asymmetrical carbon atoms in its molecular structure, LOX is applied as racemate, existing in four stereoisomeric forms. Actully, LOX is a prodrug. After absorption into the gastrointestinal tract, it is converted to the corresponding cis-alcohol and trans-alcohol metabolites (each containing four isomers) by reduction of the ketone carbonyl (cyclopentanone) (Fujiki et al., 2019; Riendeau et al., 2004; Sawamura et al., 2015). The chemical structures of LOX, trans- and cis- alcohol were shown in Fig. 1. The trans-alcohol metabolite was the main active ingredient (Choo et al., 2001; Sawamura et al., 2015), and the active isomer with the (2S,1′R,2′S)-configuration produces the inhibitory effect on cyclooxygenase activity (Fujiki et al., 2019; Riendeau et al., 2004). As reported for some other profens (Ikuta et al., 2017; Lorier et al., 2016; Suzuki et al., 2014) (e.g., ibuprofen, ketoprofen and naproxen), the stereoselective inversion of (2R)- to (2S)-configuration in α-substituted propionic acid moiety similarly occurred after oral administration of racemic LOX (Nagashima et al., 1984). However, LOX contained more than one chiral centre, which made its stereoselective inversion process more complicated compared to other profens.
The chemical structures of LOX, trans- and cis- alcohol metabolites.
The metabolism and pharmacokinetics process of LOX have been widely reported without considering the difference of its individual stereoisomer since its introduction into therapeutics (Nagashima et al., 1984; Takasaki and Tanaka, 1992; Nagashima et al., 1985). Some researchers also focused on the stereoselective biotransformation behavior of its stereoisomers. Regrettably, due to the difficulty in enantioseparation of four stereoisomers of LOX, nearly all the corresponding studies were only concerned with its configuration inversion dominated by the chiral center in the α-substituted propionic acid moiety (Nagashima et al., 1984; Takasaki and Tanaka, 1992). High performance liquid chromatography (HPLC) was the primary method for simultaneous determination of LOX and its metabolites stereoisomers in biological fluids. The LC method using chiral derivatizing reagent for determination of (2R)/(2S)-LOX and its two monohydroxy metabolites in biological samples has been reported with ordinary stationary phase, based on the μPorasil column and fluorescence detection (Nagashima et al., 1984; Nagashima et al., 1985). Antibody-mediated extraction followed by chiral HPLC has been developed for the stereoselective determination of the active metabolite ((2S,1′R,2′S)-trans-alcohol) containing three chiral centers in the plasma of human and rat subjects having received LOX (Takasaki and Tanaka, 1992). The existing methods mentioned above involve either tedious derivations or complex preprocessing.
In recent years, applying chiral stationary phase (CSP) into HPLC has become a well-established technique for separation of LOX stereoisomers due to its convenience, sensitivity and reproducibility (Li et al., 2018; Rebizi et al., 2018). A normal phase (NP)-LC method with CD detector has been described for separation and (R)/(S)-configuration identification of four LOX stereoisomers, involving the employment of CSP. But the normal phase system is not suitable for LC tandem mass spectrometry which limits its further application in pharmacokinetic study (Li et al., 2012). Our previous work found that reversed phase (RP)-LC method with cellulose-based column (Chiralcel OJ-RH) as CSP showed excellent selectivity toward four stereoisomers of LOX (Wang et al., 2018). However, the validation for quantitative analysis in biological matrices has not been carried out.
In the present work, a stereospecific, also fully validated HPLC-MS/MS method has been developed for the quantification of LOX stereoisomers in plasma and urine, which also showed good stereoselective resolution to the stereoisomers of its trans- and cis-alcohol metabolites. Subsequently, applying the established method, the pharmacokinetic study of LOX four stereoisomers was conducted to reveal the different pharmacokinetic profiles of each stereoisomer in vivo, which also involved the analysis of stereoselective formation of its alcohol-metabolites in the metabolism process.
2 Materials and methods
2.1 Chemicals and reagents
Loxoprofen sodium (LOX) dihydrate was obtained from TCI Chemical Industrial Development Co., Ltd. (Shanghai, China). Racemic trans- and cis-alcohol were purchased from TRC (Toronto, Canada). (S)-naproxen was supplied by Saen Chemical Technology Co., Ltd. (Shanghai, China). Methanol and acetonitrile of HPLC grade were supplied by Concord Technology Co., Ltd. (Tianjin, China). All other reagents were analytical grade. Ethyl acetate was purchased from Tianjin Huihang Chemical Technology Co., Ltd. (Tianjin, China). Sodium hydroxide was from Tianjin Fine Chemical Co., Ltd. (Tianjin, China). Hydrochloric acid was supplied by Yantai Sanhe Chemical Reagent Co., Ltd. (Yantai, China). Formic acid (≥88.0%) came from Tianjin Kermel Chemical Reagent Co., Ltd. (Tianjin, China). Zinc sulfate heptahydrate was obtained from Shanghai Hushi Laboratory Equipment Co., Ltd. (Shanghai, China). Ultrapure water was obtained from Jilin Wahaha Food Co., Ltd. (Jilin, China).
2.2 Animals
Six Male Sprague-Dawley rats weighing between 210 and 220 g, specific-pathogen-free grade, supplied by Jinan Pengyue Experimental Animal Breeding Co., Ltd. (Jinan, China) were used for the pharmacokinetic study. They were housed under controlled laboratory conditions (a 12 h dark-light cycle at ambient temperature (about 25 °C) with a relative humidity of 55–60%).
2.3 Chiral LC-MS/MS conditions
Chiral LC-MS/MS analysis was carried on a SCIEX Triple Quad™ 4500 LC-MS/MS System (AB Sciex, USA). All the operations, acquisition and analysis of data were controlled by Analyst 1.6.2 software (AB Sciex, USA). Simultaneous stereoisomeric separation of LOX and its alcohol metabolites was performed on an FLM Chiral NQ(2)-RH column (250 × 4.6 mm i.d., 5 μm), which was obtained from Guangzhou FLM Scientific Instrument Co., Ltd. (Guangzhou, China). The separation was carried out isocratically using solvent A (HPLC-grade ACN) and solvent B (0.1% formic acid in ultrapure water) at a ratio of 50:50 (v/v) and the flow rate was kept at 0.6 mL/min for 45 min. The column was kept at 20 °C and the temperature in the sample manager was kept at 4 °C. The injection volume was 5 μL. The mass detection was performed in the multiple reaction monitoring transitions in electrospray negative ion mode. Other optimized MS parameters were as follows: ionspray voltage 3.5 of kV; source temperature of 350 °C; ion source gas at pressure of 55 psi. Nitrogen was used as nebulizing and desolvation gas. Argon was used as collision gas. These settings were utilized for all subsequent studies.
2.4 Identification of LOX stereoisomers
Each effluent from the FLM chiral NQ(2)-RH column was collected and then injected into a Chiralcel OJ-RH column by referring to the same separation conditions reported in the previous literature (Ding et al., 2004). The absolute structures of the eluted stereoisomers of LOX on FLM chiral NQ(2)-RH can be known by comparing the elution time of this enantiopure compound.
2.5 Identification of trans-alcohol metabolite stereoisomers
Trans-alcohol metabolite stereoisomers were first isolated using a semi-preparative HPLC. Briefly, trans-alcohol metabolite was injected on an FLM Chiral ND(2) semi-preparative column (250 × 10 mm i.d., 5 μm) flushed with hexane and EtOH (containing 0.25% formic acid) at a proportion of 70:30. The flow-rate was 3.0 mL/min and the detection was at 210 nm. Each eluent from the semi-preparative column was collected and obtained by evaporating the solvent. In order to confirm the identity of these fractions, electronic circular dichroism (ECD) spectra of LOX four stereoisomers were recorded on a Chirascan qCD spectrograph (Applied Photophysics, UK). Spectra were recorded between 200 and 400 nm with a response of 0.5 s. Experiments were carried out using a 0.5 mm path length quartz cuvette. Each spectrum represents an average of 3 consecutive scans subtracted from the background. ECD spectra of the four stereoisomers were obtained by analysis of the ECD software system. The time-dependent density functional theory (DFT) method was carried out to further establish the absolute configuration of (R)/(S)-stereoisomers (Krykunov et al., 2006).
2.6 Preparation of stock solutions, calibration standards and quality control (QC) samples
Stock solutions of racemic LOX and (S)-naproxen (IS) were prepared by dissolving a proper amount of the standards in methanol to a final stereoisomeric concentration of 750 and 1000 μg/mL, respectively. A series of standard working solutions of racemic LOX were prepared by diluting the stock solution with methanol. Calibration standard samples were prepared by spiking these working solutions (10 μL) into drug-free rat plasma and urine. Target stereoisomeric concentrations used for quantification in plasma were 12, 60, 600, 3000, 6000, 30,000 and 60,000 ng/mL. Target stereoisomeric concentrations were set at 12, 30, 60, 300, 3000, 9000 and 45,000 ng/mL for quantification in urine.
The quality control (QC) samples were prepared by the same procedure to achieve the samples containing each stereoisomer at 24, 2400, 48,000 ng/mL for methodological validation in plasma and 24, 240 and 36,000 ng/mL in urine. The internal standard working solutions added in plasma and urine (10 μL) were prepared by diluting the stock (S)-naproxen solution with methanol to a total concentration of 30 and 3 μg/mL, respectively. All the solutions were stored at 4 °C and brought to room temperature before use.
2.7 Sample preparation
Each 100 μL of plasma was mixed with 10 μL of IS ((S)-naproxen, 30 μg/mL in methanol). 10 μL of 10% zinc sulfate solution and 300 μL of acetonitrile were added with mixing (vortexing for 2 min at each step). The resulting solution was centrifuged at 14,000 rpm for 10 min to isolate the precipitate and supernatant. The supernatant was pipetted into a clean vial and evaporated to dryness under a stream of nitrogen at 40 °C. After evaporation of the solvents, the residue was mixed with 100 μL of methanol, vortexed for 3 min and centrifuged at 14,000 rpm for 10 min. 5 μL of the supernatant was subsequently injected into the LC-MS/MS system for analysis.
Urine samples (200 μL) were spiked with 10 μL of IS (3 μg/mL in methanol) and was made alkaline with 200 μL of 1 M NaOH. The purpose of alkaline treatment of urine samples was to convert the excreted glucuronide conjugates into the corresponding free-acids by hydrolysis. After standing for 1 h, the hydrolysed mixture was acidified to pH 1.0–1.5 by the addition of 300 μL of 1 M hydrochloric acid and then extracted with 1 mL of ethyl acetate by shaking for 5 min. The organic phase supernatant was collected into another vial and evaporated to dryness at 40 °C with the aid of a gentle stream of nitrogen. Thereafter, the referred method was followed as originally described.
2.8 Method validation
Specificity of the method was assessed by analyzing the blank plasma and urine from six individual rats to observe the possible disturbances at retention times of LOX and IS.
The calibration curves were plotted as peak area ratios of the analyte relative to IS (y) versus concentrations of the calibration standards (x) with a weighed factor (1/x2) and the linearity was estimated at three different analytical batches. A correlation coefficient (r) >0.99 was required for linearity assay. The LLOQ was regularly defined as the lowest limit of quantification on the calibration curve with a signal-to-noise ratio of at least 10.
The precision and accuracy were performed by analyzing per concentration level at four different levels (LLOQ, low, medium and high QC samples) in six replicates on three consecutive validation days. The intra- and inter-day precision were determined by calculating the coefficient of variation. While accuracy was estimated based on the mean percentage error of measured and actual concentration.
The recovery of each stereoisomer was determined by directly comparing the analyte responses (peak areas) of six regularly pre-treated QC samples at low, medium and high quality control levels (A) with those of post-extracted blank samples spiked with the analytes (B). The ratio (A/B × 100)% was used to evaluate the recovery. Recovery of IS was determined in the same samples simultaneously.
For each LOX stereoisomer and IS, the matrix effect (ME) should be evaluated by comparing the corresponding peak area in the presence of matrix (B) (measured by analyzing six different blank biosamples spiked with standards after extraction) with that of pure standard solutions at equivalent concentrations at three levels (C). The ratio (B/C × 100)% was used to evaluate the matrix effect.
The stability was evaluated by subjecting six aliquots of QC samples at low and high concentration levels which were exposed to the following conditions: (1) short-term stability at room temperature for 8 h; (2) freeze–thaw stability after two freeze–thaw cycles at − 80 °C.
2.9 Method application
2.9.1 Drug administration and sampling
The animals were fasted for 12 h with free access to water prior to oral administration of racemic LOX at the dose of 50 mg/kg. 250 μL of orbital blood samples were collected in tubes containing heparin sodium as the anticoagulant at 15, 30, 50, 75, 120, 240 and 360 min post-dosing. Urine samples were collected at 1, 2, 4, 6, 8, 12, 24, 48 h after administration. The volume of each fraction was measured. Blood and urine samples were immediately centrifuged at 4 °C with 3500 rpm for 15 min and 14,000 rpm for 10 min respectively and then the supernatant was kept frozen at −80 °C until analysis.
2.9.2 Pharmacokinetic data analysis
The plasma concentrations of the LOX stereoisomers and the stereoisomer levels in urine at different times were calculated from the daily calibration curve. Analysis of the pharmacokinetic data and the cumulative amount excreted into urine were performed using the pharmacokinetic program DAS 2.0 (Chinese Pharmacological Society). The differences in the pharmacokinetic parameters were evaluated by SPSS 16.0 using independent Samples t-test (Statistical Package for the Social Science). Differences were considered to be significant at *P < 0.05, **P < 0.01. All values were reported as the mean ± standard deviation.
3 Results and discussion
3.1 Optimization of mass spectrometry conditions
Herein, the LC conditions were referenced to previously reported chiral separation method (Cao et al., 2021). The standard solutions of LOX, its alcohol metabolites and IS were directly injected into the mass spectrometer to obtain optimum ionization efficiency and sensitivity. These analytes could be ionized in the negative ionization mode because all compounds contain carboxyl groups. Therefore, the ESI(−) mode was employed in this study. The ESI parameters were optimized to maximize the MS response, including DP, CE, ionspray voltage and ion source temperature. The ion transitions at m/z 245.00 → 83.00 for LOX and m/z 229.00 → 185.00 for IS were selected in following quantitative analysis. In the same way, the transition of m/z for MRM analysis of metabolites were identified also. Optimized MS/MS conditions of analytes were presented in Table 1. Under the conditions above, the separation results were shown in Fig. 2.
Analytes
Dwell time (ms)
MRM transitions (m/z)
Declustering potential (eV)
Collision energy (eV)
LOX
100
245.00 > 83.00
−35.00
−14.00
Trans-alcohol
100
247.10 > 201.10
−40.00
−11.40
Cis-alcohol
100
247.10 > 217.10
−56.80
−11.80
Naproxen
100
229.00 > 185.00
−38.00
−10.00
Stereoisomeric separation chromatograms of LOX, trans- and cis-alcohol (each consisting of four stereoisomers).
3.2 Stereochemical configuration of LOX
In order to confirm the absolute structures of the stereoisomers of LOX, analysis of each enantiopure isomer and comparison with the enantioseparation chromatogram of rac-LOX were performed. The elution orders of LOX stereoisomers were then determined as follows: (1′S,2R)-LOX, (1′R,2R)-LOX, (1′R,2S)-LOX and (1′S,2S)-LOX.
3.3 Stereochemical configuration of trans-alcohol metabolite
Fig. 3 shows the CD spectra of the stereoisomers of trans-alcohol. As a consequence, the overall pattern of the experimental CD spectrum of S3 and S4 at 226 nm were consistent with the theoretical CD data for (2S)-isomer. Whereas, the CD spectrum of S1 and S2 at the same wavelengths displayed reverse cotton effects, which corresponded to the calculated CD spectrum of (2R)-isomer.
CD spectra of the stereoisomers of trans-alcohol.
3.4 Optimization of sample preparation
In the previous studies (Nagashima et al., 1985; Choo et al., 2001), LOX and its alcohol metabolites in the plasma and urine were extracted by protein precipitation and liquid–liquid extraction method, respectively. In the present study, we tried to optimize these methods to obtain satisfactory recovery. Finally, acetonitrile was used for protein precipitation and ethyl acetate as solvent was used for liquid–liquid extraction. The details of the extraction were depicted in the “Sample preparation” part.
3.5 Method validation
3.5.1 Specificity
The typical chromatograms of blank rat plasma (urine), spiked plasma (urine) sample at the LLOQ level and a real plasma (urine) sample obtained after oral administration of 50 mg/kg racemic LOX to rats were shown in Fig. S1 (Fig. S2). Obviously, no matrix interference was observed at the retention times of the analyte and IS, reflecting the acceptable specificity of the proposed method.
3.5.2 Linearity and LLOQ
Data of calibration curves and correlation coefficients in plasma and urine were shown in Table S1 and Table S2, respectively. The LLOQ of each stereoisomer in rat plasma and urine was 12 ng/mL.
3.5.3 Precision and accuracy
The results of relative error (RE) and intra- and inter-day precision were presented in Table S3 and Table S4, which indicated that the present method has good precision and accuracy.
3.5.4 Extraction recovery and matrix effect
The mean range of pretreatment recoveries were 69.32–92.79% in plasma and 92.56–107.82% in urine. Mean recovery values of IS were 103.08% in plasma and 98.10% in urine.
The matrix effects in plasma were all between 116.65 and 146.76% for the stereoisomers of LOX and the matrix effect of IS was 132.17%. The matrix effects in urine for LOX stereoisomers were in the range of 96.27–124.76% and for IS was 101.40%, respectively. The results of extraction recovery and matrix effect were presented in Table S5 and Table S6.
3.5.5 Stability
The stability data of QC samples were summarized in Table S7 and Table S8.
3.6 Stereoselective pharmacokinetic study
The validated method was successfully applied to measure the concentration of each stereoisomer of LOX and the stereoisomeric fractions of trans-alcohol metabolite in urine and plasma after a single oral administration of 50 mg/kg racemic LOX to male SD rats. Plasma concentration–time curves and the cumulative urinary curves of each stereoisomer were illustrated in Fig. 4 and Fig. 5, respectively. The corresponding pharmacokinetic parameters in plasma and urine were listed in Table 2.
The plasma concentration–time curves of each stereoisomer of LOX.
The cumulative urinary curves of each stereoisomer of LOX.
Parameters
Stereoisomers
1′S,2R
1′R,2R
1′R,2S
1′S,2S
Plasma
Cmax (ng/mL)
28648.04 ± 3276.69
12329.35 ± 963.325
21678.43 ± 4521.96
31993.10 ± 5568.91
Tmax (h)
0.375 ± 0.14
0.29 ± 0.10
0.35 ± 0.14
0.38 ± 0.14
t1/2 (h)
0.69 ± 0.23
0.75 ± 0.24
3.81 ± 1.63
1.61 ± 0.44
AUC0-6h (ng·h/mL)
25369.02 ± 4034.85
7938.99 ± 1117.47
38342.88 ± 10606.08
62955.94 ± 11041.37
AUC0-∞ (ng·h/mL)
25461.58 ± 3985.02
7972.00 ± 1116.41
58598.99 ± 21707.29
68846.77 ± 13439.22
CL/F (L/h/kg)
0.50 ± 0.07
1.60 ± 0.24
0.25 ± 0.12
0.19 ± 0.04
Urine
Ae0-48h (ng)
2674.40 ± 779.16
2602.92 ± 772.99
21364.70 ± 9786.89
22334.60 ± 10280.30
Apparently selective pharmacokinetic parameters resulted from the chiral center in asymmetric 2-carbon in the α-phenylpropionic acid moiety. It was observed that both (2R)-isomers (1′S,2R and 1′R,2R) showed a faster elimination and a shorter t1/2 than (2S)-forms (1′R,2S and 1′S,2S) (P < 0.05 or P < 0.01). The AUC0-6h and AUC0-∞ of (2S)-isomers were about 3.04 and 3.81 times higher than that of their antipodes, respectively. In addition, the absolute configuration of chiral center in 1′-position of LOX structure also had an impact on the stereoselective pharmacokinetics. In the two (2R)-isomers, (1′S)-forms showed the relatively higher Cmax, AUC0-6h and AUC0-∞ but lower CL/F than (1′R)-forms (P < 0.01). This tendency was also observed in the two (2S)-isomers. Though, for (2S)-form, AUC0-∞ and CL/F were not significantly different between 1′R,2S and 1′S,2S (P > 0.05), their Cmax and AUC0-6h were significantly different (P < 0.01). The results suggested the stereoselectivity also occurred in chiral centre of 1′-position, which has not been reported previously.
Overall, the order of AUC values for the individual isomers of LOX was as follows: 1′S,2S > 1′R,2S > 1′S,2R > 1′R,2R. The CL/F just followed the reverse order (1′S,2S < 1′R,2S < 1′S,2R < 1′R,2R).
The cumulative amounts of each stereoisomer excreted in urine, containing both free and conjugated forms, accounted for approximately 0.45% of the administered dose of LOX during the 48 h collection period. The results were much lower than that reported in the literature, where the total values of rac-LOX recovery amounted to 25.0% of the analytes in urine of human (Nagashima et al., 1985; Choo et al., 2001). This difference is possibly attributed to the greater activity of hepatic metabolizing enzyme of rat. Moreover, cumulative amounts excreted in urine of each stereoisomer were 0.205% for 1′S,2S, 0.196% for 1′R,2S, 0.025% for 1′S,2R and 0.024% for 1′R,2R of the administered dose, which showed the marked stereoselective disposition behavior.
To some extent, the noticeably different levels of four LOX stereoisomers in both plasma and urine may be mainly attributed to the irreversible optical inversion of (2R)- to (2S)-LOX, which has been confirmed in the previous papers (Nagashima et al., 1984). In addition, the stereoselective behavior also found between (1′S)- and (1′R)-LOX. The reason caused this stereoselectivity maybe originated from the stereoselective interactions between the stereoisomer and enzymes, or some other unknown mechanisms.
3.7 Analysis of trans- and cis- alcohol stereoisomers in rat plasma and urine
Representative chromatographic profiles of trans-alcohol and cis-alcohol at different points were shown in Fig. 6. For trans-alcohol, its four stereoisomers could be detected without the interference from cis-alcohol at their retention time. The time-dependent changes in the SFs of trans-alcohol were recorded in Fig. 7, which depicted that trans-alcohol existed predominantly as the S3-form (having the (2S)-configuration). SF results of 2S ≫ 2R showed that stereoselective behavior also existed in trans-alcohol in both plasma and urine. Unfortunately, it has not yet been possible to exactly determine the absolute configuration of each stereoisomer of trans-alcohol due to that the configurations of chiral center in 1′- and 2′-position were unclear, but the corresponding work is in progress that will allow their identification in our further study.
Representative chromatographic profiles of trans-alcohol and cis-alcohol at different points.
Stereoisomeric fractions (SFs) of trans-alcohol in plasma and urine.
Separation of cis-alcohol stereoisomers was also conducted in our research. Although cis-alcohol was interfered by trans-alcohol in real samples yet, detection signals belonging to cis-alcohol can be identified according to the retention time and elution order (Fig. 2). Based on the present findings (Fig. 6), the concentration of cis-alcohol observed in plasma and urine is much lower than that of trans-alcohol. As shown in Fig. 6, the S1, S2 and S4 of cis-alcohol can be identified in a different time period, but the S3 was undetectable. Besides, the concentration of S4 is higher than other stereoisomers of cis-alcohol, indicating that stereoselectivity also occurred in the formation of cis-alcohol.
Overall, through this study, we have evaluated the specific behaviour of four stereoisomers of LOX occurred in rat. The reduction process of the ketone carbonyl in vivo showed a stereo preference for (2S)-configuration. What’s more, it was also found that the chiral centre of 1′-position in LOX had some impact on the stereoselective pharmacokinetic behavior, which was first reported. Results from the present study provided some new confidence in the configuration inversion from (2R)- to (2S)-form, and a means to advance understanding of the complex pharmacokinetic of LOX stereoisomers.
4 Conclusion
To conclude, simultaneous separation of four stereoisomers of LOX and its alcohol metabolites (trans- and cis- alcohol) in plasma and urine using a chiral LC-MS/MS method has been achieved in one analytical run, in a short time (up to 45 min) and was reported for the first time. (1′S,2S)-LOX showed the highest concentration among the four stereoisomers of LOX in both plasma and urine samples. SF results showed that stereoselective behavior also existed in the stereoisomers of its trans-alcohol metabolite in both plasma and urine, which was the first evidence focusing on the stereoselectivity of each stereoisomer of LOX as well as its metabolite in vivo.
Ethical approval
All the animal experiments were carried out according to the Guideline for Binzhou Medical University Animal Experimentation with permission by institutional Animal Ethics Committee.
Acknowledgements
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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Appendix A
Supplementary material
Supplementary material to this article can be found online at https://doi.org/10.1016/j.arabjc.2023.104942.
Appendix A
Supplementary material
The following are the Supplementary material to this article:Supplementary data 1
Supplementary data 1