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Enantioselective determination of ornidazole in human plasma by liquid chromatography–tandem mass spectrometry on a Chiral-AGP column
Journal of Pharmaceutical and Biomedical Analysis 86 (2013) 182–188
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Enantioselective determination of ornidazole in human plasma by liquid chromatography–tandem mass spectrometry on a Chiral-AGP column Jiangbo Du a , Zhiyu Ma a , Yifan Zhang a , Ting Wang b , Xiaoyan Chen a , Dafang Zhong a,∗ a b
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai 201203, PR China The First Affiliated Hospital of Lanzhou University, 1 West DongGang Road, Lanzhou 730000, PR China
a r t i c l e
i n f o
Article history: Received 28 June 2013 Received in revised form 25 July 2013 Accepted 26 July 2013 Available online 12 August 2013 Keywords: Ornidazole Enantiomers Enantioselective liquid chromatography–tandem mass spectrometry Stereoselective pharmacokinetics
a b s t r a c t A rapid, sensitive, and enantioselective method was developed and validated for determination of ornidazole enantiomers in human plasma by liquid chromatography–tandem mass spectrometry. Ornidazole enantiomers were extracted from 100 l of plasma using ethyl acetate. Baseline chiral separation (Rs = 2.0) was obtained within 7.5 min on a Chiral-AGP column (150 mm × 4.0 mm, 5 m) using an isocratic mobile phase of 10 mM ammonium acetate/acetic acid (100/0.01, v/v). Stable isotopically labeled R(+)-d5 -ornidazole and S-(−)-d5 -ornidazole were synthesized as internal standards. Acquisition of mass spectrometric data was performed in multiple reaction monitoring mode via positive electrospray ionization, using the transitions of m/z 220 → 128 for ornidazole enantiomers, and m/z 225 → 128 for d5 ornidazole enantiomers. The method was linear in the concentration range of 0.030–10.0 g/ml for each enantiomer. The lower limit of quantification for each enantiomer was 0.030 g/ml. The relative standard deviation values of intra- and inter-day precision were 1.8–6.2% and 1.5–10.2% for R-(+)-ornidazole and S-(−)-ornidazole, respectively. The relative error values of accuracy ranged from −4.5% to 1.2% for R-(+)ornidazole and from −5.4% to −0.8% for S-(−)-ornidazole. The validated method was successfully applied to a stereoselective pharmacokinetic study of ornidazole after oral administration of 1000 mg racemic ornidazole. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Ornidazole, (R,S)-1-chloro-3-(2-methyl-5-nitro-1H-imidazol1-yl)propan-2-ol, is a 5-nitroimidazole derivative that has been used as racemate for the prophylaxis and treatment of susceptible protozoal and anaerobic bacterial infections since 1972 [1]. The antimicrobial mechanism of ornidazole involves the formation of a hydroxylamine intermediate in the microbe, thereby damaging
Abbreviations: APCI, atmospheric pressure chemical ionization; AUC0–t , area under the concentration–time curve to the last measurable concentration; AUC0–∞ , area under plasma concentration–time curve to infinity; CL/F, total body clearance; Cmax , maximum plasma concentration; ESI, electrospray ionization; FDA, Food and Drug Administration; IS, internal standard; LC–MS/MS, liquid chromatography–tandem mass spectrometry; LLOQ, lower limit of quantification; ME, matrix effect; MRM, multiple reaction monitoring; MRT, mean residence time; QC, quality control; RE, relative error; Rs , resolution; RSD, relative standard deviation; SIM, selective ion monitoring; S/N, signal to noise ratio; T1/2 , elimination half-life; tmax , time to maximum plasma concentration; ULOQ, upper limit of quantification; Vd , distribution volume; W, peak width. ∗ Corresponding author at: Tel.: +86 21 50800738; fax: +86 21 50800738. E-mail address: [email protected] (D. Zhong). 0731-7085/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2013.07.048
microbial DNA, disrupting transcription, and ultimately causing cell death [2–4]. Despite the comparable activity to metronidazole, ornidazole is generally preferred in certain therapies of clinical practice due to its longer half-life (14.4 h versus 8.4 h for metronidazole) and it has reduced dosage frequency and duration of therapy in many relevant clinical infections [5–7]. Recent studies have revealed that R-(+)-ornidazole elicits much stronger depressant and anticonvulsant effects on the central nervous system than the S-(−)-enantiomer in mice [8]. S-(−)-Ornidazole has already been developed and approved for marketing as a new antimicrobial agent in China since 2009. Few chiral methods have been reported for the enantioselective determination of ornidazole in biological samples. Liu et al. [9,10] have reported two enantioselective HPLC-UV methods to determine ornidazole enantiomers in dog plasma as well as in human plasma and urine using a Chiralcel OB-H column operated in normal-phase mode. However, these methods require long chromatographic run time (≥28 min) and are thus inadequate for high-throughput analysis. In addition, both methods show poor sensitivity with LLOQ ranging from 0.160 g/ml to 0.320 g/ml using a biological sample volume of 200–400 l. Nowadays, chiral liquid chromatography–tandem mass spectrometry (LC–MS/MS)
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has gained its popularity in enantioselective quantification of chiral drugs and/or their metabolites in biological matrices, due to its high specificity and sensitivity [11–19]. The normal-phase HPLC systems reported in previous studies (involving the use of high percentage of hexane as mobile phase) are generally considered incompatible with electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) techniques because of potential explosion hazards and poor ionization issue [20]. To date, no enantioselective LC–MS/MS method has been reported to determine ornidazole enantiomers in human plasma. The present study aimed to (1) develop and validate a rapid, sensitive, and enantioselective LC–MS/MS method for determination of ornidazole enantiomers in human plasma and (2) investigate the stereoselective pharmacokinetics of ornidazole after oral administration of 1000 mg racemic ornidazole to six healthy Chinese volunteers. 2. Experimental 2.1. Chemicals and reagents Racemic ornidazole (98.0% purity) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). S-(−)-Ornidazole (98.0% purity) was purchased from Amresco Chemical (Solon, OH, USA). R-(+)-ornidazole (100% purity) and racemic d5 -ornidazole (100% purity) were synthesized and purified in our laboratory using a previously described method with minor modifications [21]. Reference standards of M10 (sulfate of ornidazole, 100% purity), M16-1 (glucuronide of S-(−)-ornidazole, 100% purity), and M16-2 (glucuronide of R-(+)-ornidazole, 100% purity) were isolated and purified from human urine using a previously reported method [22]. HPLC-grade methanol was purchased from Sigma–Aldrich (St. Louis, MO, USA). HPLC-grade ammonium acetate and acetic acid were purchased from Tedia (Fairfield, OH, USA). Analytical-grade ethyl acetate was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Deionized water was purified by a Millipore Milli-Q gradient water purification system (Molsheim, France). 2.2. Instrumentation An Agilent 1200 liquid chromatography system comprising a G1322A vacuum degasser, a G1312B binary pump, a G1316B column oven, and a G1367D autosampler (Agilent, Waldbronn, Germany) was used for solvent and sample delivery. Mass spectrometric detection was performed on an Agilent 6460 triple quadrupole instrument (Agilent, Waldbronn, Germany) equipped with an ESI source. Data processing was performed using Agilent MassHunter software (version B.03.02, Agilent). 2.3. LC–MS/MS conditions The enantiomers were separated on a Chiral-AGP column (150 mm × 4.0 mm, 5 m) with a Chiral-AGP guard column (10 mm × 3.0 mm) (ChromTech, Haegersten, Sweden) at 20 ◦ C. The mobile phase consisted of 10 mM ammonium acetate/acetic acid (100:0.01, v/v) and was delivered at a flow rate of 1.0 ml/min in the first 3 min, then shifted to 0.5 ml/min in 0.1 min, and held constant until the end of the run time. R-(+)-d5 -ornidazole and S-(−)-d5 -ornidazole were used as internal standards (ISs) for R-(+)ornidazole and S-(−)-ornidazole, respectively. The mass spectrometer was operated with an ESI source in the positive ion mode. The instrument was operated at capillary voltage and charging voltage of +3500 V and +500 V, respectively. Nitrogen was used as a nebulizer gas of 45 psi, a carrier gas of 5 l/min at
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350 ◦ C, and a sheath gas of 12 l/min at 380 ◦ C. The optimized multiple reaction monitoring (MRM) fragmentation transitions used were as follows: m/z 220 → 128 with a fragmenter voltage of 115 V and a collision energy (CE) of 11 V for ornidazole enantiomers; and m/z 225 → 128 with a fragmenter voltage of 115 V and a CE of 11 V for ISs d5 -ornidazole enantiomers. The dwell time for each transition was 80 ms. 2.4. Preparation of calibration standards and quality control (QC) samples Stock solutions of racemic ornidazole were prepared in methanol/water (50:50, v/v) at a concentration of 500 g/ml for each enantiomer. Calibration standards at concentrations of 0.030, 0.100, 0.300, 1.00, 3.00, and 10.0 g/ml for each enantiomer were prepared through a serial dilution of racemic ornidazole stock solution with blank plasma. The LLOQ samples (0.030 g/ml for each enantiomer) and QC samples (0.090, 0.500, and 8.00 g/ml for each enantiomer) were independently prepared in blank plasma with a separate set of stock solution. The ISs working solution (1.00 g/ml R-(+)-d5 -ornidazole and 1.00 g/ml S-(−)-d5 ornidazole) was prepared by diluting the racemic d5 -ornidazole stock solution (1.00 mg/ml) with methanol/water (50:50, v/v). All the solutions were kept refrigerated (4 ◦ C) and brought to room temperature before use. The calibration standards, LLOQ samples and QC samples were dispatched in 500 l aliquots and stored in plastic tubes at −80 ◦ C until analysis. 2.5. Sample preparation The frozen plasma samples were thawed at room temperature and vortexed thoroughly. A 50-l aliquot of ISs solution (1.00 g/ml R-(+)-d5 -ornidazole and 1.00 g/ml S-(−)-ornidazole) and 200 l of water were added to 100 l of plasma sample. The sample was extracted with 3 ml of ethyl acetate by vortexing for 5 min. The organic and aqueous phases were then separated by centrifugation at 2000 × g for 5 min. The upper organic phase was transferred to another tube and evaporated to dryness at 40 ◦ C under a stream of nitrogen in a TurboVap evaporator (Zymark, Hopkinton, MA, USA). The residue was reconstituted in 150 l of mobile phase, and an aliquot of 5 l was subsequently injected into the LC–MS/MS system for analysis. 2.6. Method validation The method was validated for selectivity, linearity, precision and accuracy, matrix effect (ME), recovery, and stability according to the US FDA guidelines [23]. Selectivity was evaluated by analyzing six sources of human blank plasma and 12 spiked plasma samples at the LLOQ level to test interference at the retention times of the analytes and the ISs. The peak areas of the endogenous compounds co-eluted with the analytes should be less than 20% of the peak area of the LLOQ standard and less than 5% of the peak area of the ISs. Linearity was assessed by plotting the peak area ratios of the analyte to the ISs against the analyte concentrations in human plasma using a linearly weighed (1/x2 ) least squares regression method in duplicate on three consecutive validation days. A correlation coefficient (r2 ) greater than 0.99 was required for linearity assay. The deviations of the calculated concentrations should be within ±15% of the nominal concentrations, except the LLOQ with an allowed deviation of ±20%. Precision and accuracy were determined by assessing six replicates of the QC samples at three levels (0.090, 0.500, and 8.00 g/ml for each enantiomer) on three consecutive validation days. Precision was expressed as the relative standard deviation (RSD),
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whereas accuracy was reported as the relative error (RE). The intraand inter-day precision values were required not to exceed 15% and the accuracy to be within ±15%. LLOQ was established by analyzing six blank plasma samples spiked with 0.030 g/ml of both enantiomers with acceptable precision (RSD ≤ 20%) and accuracy (RE within 20%). To assess the accuracy and precision of the method to determine ornidazole enantiomers ratios, ornidazole enantiomers were combined to obtain two mixtures of known R-(+)/S-(−) ratios at 2/1 and 1/2, containing R-(+)-ornidazole and S-(−)-ornidazole at a total plasma concentration of 0.600 g/ml, and were analyzed in six replicates on three consecutive days. Blank plasma from six lots was extracted and then spiked with analytes and ISs to evaluate the ME of each enantiomer and ISs. The corresponding peak area ratios of the analytes to ISs in the spiked plasma post-extraction samples (A) were then compared with those of the water-substituted samples (B) at equivalent concentrations. The ratio (A/B × 100%) is defined as the IS-normalized matrix factor (MF). The RSD of MFs should be less than 15%. The recovery of each enantiomer was determined by comparing the peak area ratios of the analytes to ISs in the regularly pre-treated QC samples at three concentration levels (six samples each) with those of spiked post-extraction samples. Similarly, the recovery of ISs was determined at 0.500 g/ml of each enantiomer. The stabilities of each enantiomer in human plasma were evaluated by analyzing triplicates of the plasma samples at two concentration levels (0.090 and 8.00 g/ml for each enantiomer), which were exposed to the following conditions: (1) short-term stability at room temperature for 6 h; (2) long-term stability at −80 ◦ C for 31 days; (3) autosampler tray stability at ambient temperature for 24 h; and (4) freeze-thaw stability after three freeze-thaw cycles at −80 ◦ C. The analytes were considered stable when the accuracy bias was within ±15% of the nominal concentrations. To evaluate the enantiomeric stability of R-(+)-ornidazole or S-(−)-ornidazole in human plasma, each enantiomer at the upper limit of quantification (ULOQ, 10.0 g/ml) level was incubated in triplicate for 12 h at 37 ◦ C, then extracted and analyzed as described. 2.7. Stereoselective pharmacokinetic study The validated method was used to investigate the plasma profiles of ornidazole enantiomers following an oral dose of 1000 mg ornidazole tablets (Jiudian Pharmaceutical Co., Ltd., Hunan, China) to six healthy Chinese volunteers (male; age range, 22–26 years; body mass index range, 20.1–23.9 kg/m2 ). The clinical study was approved by the Ethics Committee of the First Affiliated Hospital of Lanzhou University (Lanzhou, China). All volunteers provided written informed consent to participate in the study according to the principles of the Declaration of Helsinki and Good Clinical Practice. Blood samples (4 ml) were collected into sodium heparincontaining tubes at 0 (pre-dose), 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 36, 48, 72, and 96 h after oral administration of 1000 mg ornidazole tablets. The blood samples were centrifuged at 2000 × g for 10 min to separate the plasma fractions, and the plasma samples were stored at −80 ◦ C prior to analysis. The pharmacokinetic parameters of ornidazole enantiomers were calculated by non-compartmental analysis using the WinNonlin 5.3 software (Pharsight, St. Louis, MO, USA).
under ESI source was about 50-fold higher than that under APCI. Therefore, the ESI(+) mode was employed for ornidazole quantification in this study. In the Q1 full scan mode, protonated molecules at m/z 220 and m/z 225 were observed for ornidazole and d5 ornidazole, respectively. Fig. 1 shows the product ion spectra of [M+H]+ ions from ornidazole and d5 -ornidazole, as well as their proposed fragmentation patterns. Their most abundant and stable fragment ions at m/z 128 were selected in the MRM transitions. With these transitions, the signal-to-noise ratio (S/N) increased by 7-fold compared with respective selective ion monitoring (SIM). The ESI parameters were optimized to maximize the MS response, including fragmenter voltage, CE, gas flow-rate and temperature. The highest MS responses of ornidazole and d5 -ornidazole were achieved when the fragmenter voltages and CEs were set at 115 V and 11 V, respectively. The flow-rate and temperature of gases (nebulizer gas, carrier gas, and sheath gas) had little effect on the MS response. To resolve ornidazole enantiomers, different types of chiral stationary phases were evaluated, including Chiralpak ASRH (150 mm × 4.6 mm, 5 m), Chirobiotic T (250 mm × 4.6 mm, 5 m), Chirobiotic V (150 mm × 4.6 mm, 5 m), Ultron ES-OVM (150 mm × 4.6 mm, 5 m), and Chiral-AGP (150 mm × 4.0 mm, 5 m). No resolution was observed on the Chirobiotic T column, although polar ionic and reversed-phased modes were tested. When different percentages of acetonitrile, methanol, and acid/base modifiers were used, only partial resolution of ornidazole enantiomers (Rs ≤ 1.3) was achieved on the Chiralpak AS-RH and Chirobiotic V columns. Protein-based chiral columns (Ultron ES-OVM and Chiral-AGP) provided better resolution of ornidazole enantiomers than the other chiral columns. Baseline separation (Rs ≥ 1.5) was achieved on both columns within 20 min using a mobile phase methanol/10 mM ammonium acetate (2/98, v/v). Since the column efficiency of Ultron ES-OVM for ornidazole enantiomers was relatively low (W > 1.7 min), the Chiral-AGP column was chosen for further optimization. When 0.01% acetic acid was added into the aqueous phase, the resolution of ornidazole enantiomers increased from 1.5 to 1.7, and also acidification of the mobile phase facilitated the detection of ornidazole in the positive mode. However, further optimization of increasing methanol proportion (> 2%) or using other organic modifiers (acetonitrile or isopropanol) resulted in poor resolution. Then the proportion of methanol was further decreased from 2% to 0%. As a result, the enantiomers were highly resolved (Rs = 2.6). A flow gradient was used to shorten the total run time (from 12 min to 7.5 min), and the final Rs was 2.0 (calculated at ULOQ level). Generally, atmospheric pressure ionization techniques (ESI and APCI) require high proportions of organic modifiers to achieve a good spray, which is associated with MS sensitivity. Given that the response of ornidazole under the ESI(+) mode was adequate, the use of the final mobile phase (10 mM ammonium acetate containing 0.01% acetic acid) did not compromise too much sensitivity, and a desirable S/N (> 15) was obtained at the LLOQ of each enantiomer. Furthermore, the deuterated ISs were used in this study to compensate for the matrix effects due to their similar chromatographic behaviors and ionization properties to ornidazole.
3. Results and discussion
3.2. Sample preparation
3.1. Mass spectrometric and enantioselective chromatographic conditions
Considering that the separation of ornidazole enantiomers was performed on a Chiral-AGP with full aqueous phase, the coeluent contaminants could cause significant matrix effect and decrease the reproducibility of the method especially with an ESI ionization source. Protein precipitation is a nonselective purification
Preliminary experiments showed that ornidazole could only be ionized in the positive ionization mode and the signal intensity
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Fig. 1. LC–MS/MS total ion chromatogram of blank plasma sample spiked with R-(+)-ornidazole (1.00 g/ml), S-(−)-ornidazole (1.00 g/ml), R-(+)-d5 -ornidazole (1.00 g/ml), and S-(−)-d5 -ornidazole (1.00 g/ml) (A), as well as product ion spectra of [M+H]+ ions of ornidazole (B) and d5 -ornidazole (C). Their proposed fragmentation patterns are displayed as insets. The chiral center is marked with an asterisk.
method that may introduce high amounts of endogenous components; hence, liquid–liquid extraction was firstly considered for its relatively clean extracts. Given that ornidazole is highly soluble in ethyl acetate at pH 7 (∼296 mg/ml) [1], ethyl acetate was chosen as the extraction reagent without acidifying or alkalizing the plasma sample. Ornidazole is extensively metabolized to some Phase II metabolites in humans, including glucuronides M161/M16-2 and sulfate M10 [22,24]. Conjugated metabolites might be unstable and convert to the parent drug during sample preparation. Moreover, conjugated metabolites that were not chromatographically separated with the parent drug could dissociate in ionization source, thereby increasing the signal of the parent drug and causing false over-estimation. To evaluate the effects of these metabolites on ornidazole detection, blank plasma samples spiked with M10 (0.500 g/ml), M16-1 (0.500 g/ml), and M16-2 (0.500 g/ml) were extracted with ethyl acetate under the same condition above. No interference was observed at the retention times of ornidazole enantiomers.
3.3. Method validation 3.3.1. Assay selectivity Selectivity of the method was assessed by comparing the chromatograms of blank human plasma from six sources with the corresponding spiked plasma at the LLOQ concentration. Fig. 2 shows the typical chromatograms of a blank plasma sample, a blank plasma sample spiked with ornidazole enantiomers at LLOQ level and ISs (1.00 g/ml R-(+)-d5 -ornidazole and 1.00 g/ml S-(−)d5 -ornidazole) and a plasma sample obtained at 1.5 h after oral administration of 1000 mg racemic ornidazole to a volunteer. No significant endogenous interference co-eluting with analytes and ISs was observed in the blank human plasma.
3.3.2. Linearity of calibration curve and LLOQ Linear regression curves were obtained over the concentration ranges of 30.0–10 000 for R-(+)-ornidazole and S-(−)-ornidazole, respectively. The following typical equations of the calibration curve were used: (r 2 = 0.9978)
R-(+)-ornidazole :
y = 2.11x − 0.00442
S-(−)-ornidazole :
y = 2.11x − 0.00670 (r 2 = 0.9972)
where y is the peak area ratio of analytes to ISs and x is the concentration of analytes. The LLOQ of both ornidazole enantiomers was 0.030 g/ml. The RSD values of precision at the LLOQ of the two enantiomers were between 3.3% and 6.8%, and RE values of accuracy were between 0.4% and 0.8%. With the present LLOQ, the plasma concentrations of R-(+)-ornidazole and S-(−)-ornidazole could be determined up to 96 h after oral administration of 1000 mg ornidazole tablets, which were sensitive enough to allow for the investigation of stereoselective pharmacokinetics of ornidazole. Based on the S/N of 3, the limit of detection values (LOD) of this method were estimated as 0.006 g/ml for both enantiomers. 3.3.3. Precision and accuracy Intra- and inter-day precision and accuracy values for the QC samples are summarized in Table 1. In this assay, the RSD values of intra- and inter-day precision for R-(+)-ornidazole were less than 6.2%, whereas the RE values of accuracy ranged from −4.5% to 1.2%. For S-(−)-ornidazole, the RSD values of intra- and inter-day precision were less than 10.2% and RE values of accuracy was between −5.4% and −0.8%. The precision and accuracy of ornidazole enantiomeric ratios are presented in Table 2. The RSD values of intra- and inter-day
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Fig. 2. Representative enantioselective MRM chromatograms for R-(+)-ornidazole (I), S-(−)-ornidazole (II), R-(+)-d5 -ornidazole (IS; III), and S-(−)-d5 -ornidazole (IS; IV) in human plasma. (A) Blank plasma sample. (B) Blank plasma sample spiked with R-(+)-ornidazole (0.030 g/ml), S-(−)-ornidazole (0.030 g/ml), R-(+)-d5 -ornidazole (1.00 g/ml), and S-(−)-d5 -ornidazole (1.00 g/ml). (C) Plasma sample at 1.5 h after oral administration of 1000 mg racemic ornidazole to a volunteer.
Table 1 Precision and accuracy data for determination of R-(+)-ornidazole and S-(−)-ornidazole in human plasma (three days, six replicates per day). Analyte
Nominal conc. (g/ml)
Mean ± SD (g/ml)
Intra-day RSD (%)
RE (%)
R-(+)-Ornidazole
0.030 0.090 0.500 8.00
0.030 0.091 0.492 7.64
± ± ± ±
0.001 0.003 0.029 0.28
6.3 2.5 1.8 6.2
3.3 3.4 4.1 3.2
0.8 1.2 −1.7 −4.5
S-(−)-Ornidazole
0.030 0.090 0.500 8.00
0.030 0.088 0.496 7.57
± ± ± ±
0.001 0.003 0.021 0.50
6.8 1.5 5.7 10.2
4.5 3.8 3.7 5.9
0.4 −2.3 −0.8 −5.4
precision were below 4.3% and 7.1%, respectively. The RE values of accuracy ranged from −3.4% to 1.0%.
3.3.4. ME and recovery The MFs from six lots of blank plasma ranged from 97.3% to 104% for all compounds. Inter-subject variability of the ISs-normalized MFs, as measured by the RSD, was lower than 3.4%. Thus, ion suppression or enhancement from plasma matrix was negligible under the current conditions. The mean extraction recoveries of the two enantiomers and ISs were between 96.5% and 102%.
Inter-day RSD (%)
3.3.5. Stability Ornidazole enantiomers were stable in plasma after placement at room temperature for 6 h, undergoing three freeze-thaw cycles and storage at −80 ◦ C for 31 days. The processed samples were stable up to 24 h at the autosampler tray. The results demonstrated good stability of ornidazole enantiomers throughout the experiment. The peak of the other enantiomer was not observed after incubation of plasma samples containing one enantiomer at 37 ◦ C for 12 h. As a result, no chiral inversion occurred between R-(+)ornidazole and S-(−)-ornidazole during storage, processing, and analysis.
Table 2 Precision and accuracy data for determination of R-(+)-ornidazole and S-(−)-ornidazole in human plasma (three days, six replicates per day). R-(+)/S-(−) ratios
Detected ratios (mean ± SD)
Inter-day RSD (%)
Intra-day RSD (%)
RE (%)
0.50 2.00
0.50 ± 0.02 1.93 ± 0.08
7.1 3.1
4.0 4.3
1.0 −3.4
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within 7.5 min. Stable isotopically labeled d5 -ornidazole enantiomers were used as ISs to compensate for the matrix effects. The LLOQ was 0.030 g/ml for ornidazole enantiomers using only 100 l of human plasma. This method was successfully applied to characterize the pharmacokinetic profiles of ornidazole enantiomers after a single dose of 1000 mg racemic ornidazole to six healthy Chinese volunteers. The pharmacokinetic results indicated that ornidazole enantiomers showed similar tmax and Cmax , while S-ornidazole displayed higher metabolic rate than R-enantiomer in humans.
References Fig. 3. Mean plasma concentration–time profiles of R-(+)-ornidazole and S-(−)ornidazole after oral administration of 1000 mg racemic ornidazole to six healthy Chinese volunteers.
Table 3 Pharmacokinetic parameters (mean ± SD) of R-(+)-ornidazole and S-(−)-ornidazole after oral administration of 1000 mg racemic ornidazole to six healthy Chinese volunteers. Parameters
R-(+)-ornidazole
AUC0–t ((g h)/ml) AUC0–∞ ((g h)/ml) tmax (h) Cmax (g/ml) T1/2 (h) MRT (h) CL/F (l/h) Vd (l)
184.6 193.2 1.2 7.9 17.5 25.6 2.6 66.1
± ± ± ± ± ± ± ±
33.1 29.2 0.4 1.3 1.6 2.2 0.4 8.1
S-(−)-ornidazole 128.7 130.2 1.4 7.9 11.5 17.2 3.9 64.2
± ± ± ± ± ± ± ±
19.8 18.5 0.9 1.6 0.7 1.5 0.6 7.1
AUC0–t : area under plasma concentration–time curve to the last measurable concentration; AUC0–∞ : area under plasma concentration–time curve to infinity; tmax : time to maximum plasma concentration; Cmax : maximum plasma concentration; T1/2 : elimination half-life; MRT: mean residence time; CL/F: total clearance; Vd : distribution volume.
3.4. Stereoselective pharmacokinetic study The present enantioselective LC–MS/MS method provided the LLOQ down to 0.030 g/ml for each enantiomer, which met the requirements to evaluate stereoselective pharmacokinetics of ornidazole. With the present LLOQ, the ornidazole enantiomers concentration could be determined in plasma samples up to 96 h postdose. After oral administration of 1000 mg racemic ornidazole to six healthy Chinese volunteers, the profiles of the mean plasma concentration of ornidazole enantiomers versus time are shown in Fig. 3. The plasma concentrations of R-(+)-ornidazole were higher than those of S-(−)-enantiomer in the elimination phase. The pharmacokinetic parameters are summarized in Table 3. The tmax and Cmax were comparable between two enantiomers. The AUC0−∞ of R-(+)-ornidazole was 1.48 times higher than that of S-(−)-enantiomer, and the R-(+)/S-(−) ratio of the total body clearance (CL/F) was 0.68. According to our previous study [22], the stereoselective glucuronidation of ornidazole enantiomers could be one of the major determinants of its enantioselective pharmacokinetics in humans. 4. Conclusion An enantioselective LC–MS/MS method was firstly developed and validated for quantification of ornidazole enantiomers in human plasma. Liquid chromatographic conditions were performed on a Chiral-AGP column operated in the MS-friendly reverse-phase mode. Baseline separation (Rs = 2.0) was achieved
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Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Enantioselective determination of ornidazole in human plasma by liquid chromatography–tandem mass spectrometry on a Chiral-AGP column Jiangbo Du a , Zhiyu Ma a , Yifan Zhang a , Ting Wang b , Xiaoyan Chen a , Dafang Zhong a,∗ a b
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai 201203, PR China The First Affiliated Hospital of Lanzhou University, 1 West DongGang Road, Lanzhou 730000, PR China
a r t i c l e
i n f o
Article history: Received 28 June 2013 Received in revised form 25 July 2013 Accepted 26 July 2013 Available online 12 August 2013 Keywords: Ornidazole Enantiomers Enantioselective liquid chromatography–tandem mass spectrometry Stereoselective pharmacokinetics
a b s t r a c t A rapid, sensitive, and enantioselective method was developed and validated for determination of ornidazole enantiomers in human plasma by liquid chromatography–tandem mass spectrometry. Ornidazole enantiomers were extracted from 100 l of plasma using ethyl acetate. Baseline chiral separation (Rs = 2.0) was obtained within 7.5 min on a Chiral-AGP column (150 mm × 4.0 mm, 5 m) using an isocratic mobile phase of 10 mM ammonium acetate/acetic acid (100/0.01, v/v). Stable isotopically labeled R(+)-d5 -ornidazole and S-(−)-d5 -ornidazole were synthesized as internal standards. Acquisition of mass spectrometric data was performed in multiple reaction monitoring mode via positive electrospray ionization, using the transitions of m/z 220 → 128 for ornidazole enantiomers, and m/z 225 → 128 for d5 ornidazole enantiomers. The method was linear in the concentration range of 0.030–10.0 g/ml for each enantiomer. The lower limit of quantification for each enantiomer was 0.030 g/ml. The relative standard deviation values of intra- and inter-day precision were 1.8–6.2% and 1.5–10.2% for R-(+)-ornidazole and S-(−)-ornidazole, respectively. The relative error values of accuracy ranged from −4.5% to 1.2% for R-(+)ornidazole and from −5.4% to −0.8% for S-(−)-ornidazole. The validated method was successfully applied to a stereoselective pharmacokinetic study of ornidazole after oral administration of 1000 mg racemic ornidazole. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Ornidazole, (R,S)-1-chloro-3-(2-methyl-5-nitro-1H-imidazol1-yl)propan-2-ol, is a 5-nitroimidazole derivative that has been used as racemate for the prophylaxis and treatment of susceptible protozoal and anaerobic bacterial infections since 1972 [1]. The antimicrobial mechanism of ornidazole involves the formation of a hydroxylamine intermediate in the microbe, thereby damaging
Abbreviations: APCI, atmospheric pressure chemical ionization; AUC0–t , area under the concentration–time curve to the last measurable concentration; AUC0–∞ , area under plasma concentration–time curve to infinity; CL/F, total body clearance; Cmax , maximum plasma concentration; ESI, electrospray ionization; FDA, Food and Drug Administration; IS, internal standard; LC–MS/MS, liquid chromatography–tandem mass spectrometry; LLOQ, lower limit of quantification; ME, matrix effect; MRM, multiple reaction monitoring; MRT, mean residence time; QC, quality control; RE, relative error; Rs , resolution; RSD, relative standard deviation; SIM, selective ion monitoring; S/N, signal to noise ratio; T1/2 , elimination half-life; tmax , time to maximum plasma concentration; ULOQ, upper limit of quantification; Vd , distribution volume; W, peak width. ∗ Corresponding author at: Tel.: +86 21 50800738; fax: +86 21 50800738. E-mail address: [email protected] (D. Zhong). 0731-7085/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2013.07.048
microbial DNA, disrupting transcription, and ultimately causing cell death [2–4]. Despite the comparable activity to metronidazole, ornidazole is generally preferred in certain therapies of clinical practice due to its longer half-life (14.4 h versus 8.4 h for metronidazole) and it has reduced dosage frequency and duration of therapy in many relevant clinical infections [5–7]. Recent studies have revealed that R-(+)-ornidazole elicits much stronger depressant and anticonvulsant effects on the central nervous system than the S-(−)-enantiomer in mice [8]. S-(−)-Ornidazole has already been developed and approved for marketing as a new antimicrobial agent in China since 2009. Few chiral methods have been reported for the enantioselective determination of ornidazole in biological samples. Liu et al. [9,10] have reported two enantioselective HPLC-UV methods to determine ornidazole enantiomers in dog plasma as well as in human plasma and urine using a Chiralcel OB-H column operated in normal-phase mode. However, these methods require long chromatographic run time (≥28 min) and are thus inadequate for high-throughput analysis. In addition, both methods show poor sensitivity with LLOQ ranging from 0.160 g/ml to 0.320 g/ml using a biological sample volume of 200–400 l. Nowadays, chiral liquid chromatography–tandem mass spectrometry (LC–MS/MS)
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has gained its popularity in enantioselective quantification of chiral drugs and/or their metabolites in biological matrices, due to its high specificity and sensitivity [11–19]. The normal-phase HPLC systems reported in previous studies (involving the use of high percentage of hexane as mobile phase) are generally considered incompatible with electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) techniques because of potential explosion hazards and poor ionization issue [20]. To date, no enantioselective LC–MS/MS method has been reported to determine ornidazole enantiomers in human plasma. The present study aimed to (1) develop and validate a rapid, sensitive, and enantioselective LC–MS/MS method for determination of ornidazole enantiomers in human plasma and (2) investigate the stereoselective pharmacokinetics of ornidazole after oral administration of 1000 mg racemic ornidazole to six healthy Chinese volunteers. 2. Experimental 2.1. Chemicals and reagents Racemic ornidazole (98.0% purity) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). S-(−)-Ornidazole (98.0% purity) was purchased from Amresco Chemical (Solon, OH, USA). R-(+)-ornidazole (100% purity) and racemic d5 -ornidazole (100% purity) were synthesized and purified in our laboratory using a previously described method with minor modifications [21]. Reference standards of M10 (sulfate of ornidazole, 100% purity), M16-1 (glucuronide of S-(−)-ornidazole, 100% purity), and M16-2 (glucuronide of R-(+)-ornidazole, 100% purity) were isolated and purified from human urine using a previously reported method [22]. HPLC-grade methanol was purchased from Sigma–Aldrich (St. Louis, MO, USA). HPLC-grade ammonium acetate and acetic acid were purchased from Tedia (Fairfield, OH, USA). Analytical-grade ethyl acetate was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Deionized water was purified by a Millipore Milli-Q gradient water purification system (Molsheim, France). 2.2. Instrumentation An Agilent 1200 liquid chromatography system comprising a G1322A vacuum degasser, a G1312B binary pump, a G1316B column oven, and a G1367D autosampler (Agilent, Waldbronn, Germany) was used for solvent and sample delivery. Mass spectrometric detection was performed on an Agilent 6460 triple quadrupole instrument (Agilent, Waldbronn, Germany) equipped with an ESI source. Data processing was performed using Agilent MassHunter software (version B.03.02, Agilent). 2.3. LC–MS/MS conditions The enantiomers were separated on a Chiral-AGP column (150 mm × 4.0 mm, 5 m) with a Chiral-AGP guard column (10 mm × 3.0 mm) (ChromTech, Haegersten, Sweden) at 20 ◦ C. The mobile phase consisted of 10 mM ammonium acetate/acetic acid (100:0.01, v/v) and was delivered at a flow rate of 1.0 ml/min in the first 3 min, then shifted to 0.5 ml/min in 0.1 min, and held constant until the end of the run time. R-(+)-d5 -ornidazole and S-(−)-d5 -ornidazole were used as internal standards (ISs) for R-(+)ornidazole and S-(−)-ornidazole, respectively. The mass spectrometer was operated with an ESI source in the positive ion mode. The instrument was operated at capillary voltage and charging voltage of +3500 V and +500 V, respectively. Nitrogen was used as a nebulizer gas of 45 psi, a carrier gas of 5 l/min at
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350 ◦ C, and a sheath gas of 12 l/min at 380 ◦ C. The optimized multiple reaction monitoring (MRM) fragmentation transitions used were as follows: m/z 220 → 128 with a fragmenter voltage of 115 V and a collision energy (CE) of 11 V for ornidazole enantiomers; and m/z 225 → 128 with a fragmenter voltage of 115 V and a CE of 11 V for ISs d5 -ornidazole enantiomers. The dwell time for each transition was 80 ms. 2.4. Preparation of calibration standards and quality control (QC) samples Stock solutions of racemic ornidazole were prepared in methanol/water (50:50, v/v) at a concentration of 500 g/ml for each enantiomer. Calibration standards at concentrations of 0.030, 0.100, 0.300, 1.00, 3.00, and 10.0 g/ml for each enantiomer were prepared through a serial dilution of racemic ornidazole stock solution with blank plasma. The LLOQ samples (0.030 g/ml for each enantiomer) and QC samples (0.090, 0.500, and 8.00 g/ml for each enantiomer) were independently prepared in blank plasma with a separate set of stock solution. The ISs working solution (1.00 g/ml R-(+)-d5 -ornidazole and 1.00 g/ml S-(−)-d5 ornidazole) was prepared by diluting the racemic d5 -ornidazole stock solution (1.00 mg/ml) with methanol/water (50:50, v/v). All the solutions were kept refrigerated (4 ◦ C) and brought to room temperature before use. The calibration standards, LLOQ samples and QC samples were dispatched in 500 l aliquots and stored in plastic tubes at −80 ◦ C until analysis. 2.5. Sample preparation The frozen plasma samples were thawed at room temperature and vortexed thoroughly. A 50-l aliquot of ISs solution (1.00 g/ml R-(+)-d5 -ornidazole and 1.00 g/ml S-(−)-ornidazole) and 200 l of water were added to 100 l of plasma sample. The sample was extracted with 3 ml of ethyl acetate by vortexing for 5 min. The organic and aqueous phases were then separated by centrifugation at 2000 × g for 5 min. The upper organic phase was transferred to another tube and evaporated to dryness at 40 ◦ C under a stream of nitrogen in a TurboVap evaporator (Zymark, Hopkinton, MA, USA). The residue was reconstituted in 150 l of mobile phase, and an aliquot of 5 l was subsequently injected into the LC–MS/MS system for analysis. 2.6. Method validation The method was validated for selectivity, linearity, precision and accuracy, matrix effect (ME), recovery, and stability according to the US FDA guidelines [23]. Selectivity was evaluated by analyzing six sources of human blank plasma and 12 spiked plasma samples at the LLOQ level to test interference at the retention times of the analytes and the ISs. The peak areas of the endogenous compounds co-eluted with the analytes should be less than 20% of the peak area of the LLOQ standard and less than 5% of the peak area of the ISs. Linearity was assessed by plotting the peak area ratios of the analyte to the ISs against the analyte concentrations in human plasma using a linearly weighed (1/x2 ) least squares regression method in duplicate on three consecutive validation days. A correlation coefficient (r2 ) greater than 0.99 was required for linearity assay. The deviations of the calculated concentrations should be within ±15% of the nominal concentrations, except the LLOQ with an allowed deviation of ±20%. Precision and accuracy were determined by assessing six replicates of the QC samples at three levels (0.090, 0.500, and 8.00 g/ml for each enantiomer) on three consecutive validation days. Precision was expressed as the relative standard deviation (RSD),
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whereas accuracy was reported as the relative error (RE). The intraand inter-day precision values were required not to exceed 15% and the accuracy to be within ±15%. LLOQ was established by analyzing six blank plasma samples spiked with 0.030 g/ml of both enantiomers with acceptable precision (RSD ≤ 20%) and accuracy (RE within 20%). To assess the accuracy and precision of the method to determine ornidazole enantiomers ratios, ornidazole enantiomers were combined to obtain two mixtures of known R-(+)/S-(−) ratios at 2/1 and 1/2, containing R-(+)-ornidazole and S-(−)-ornidazole at a total plasma concentration of 0.600 g/ml, and were analyzed in six replicates on three consecutive days. Blank plasma from six lots was extracted and then spiked with analytes and ISs to evaluate the ME of each enantiomer and ISs. The corresponding peak area ratios of the analytes to ISs in the spiked plasma post-extraction samples (A) were then compared with those of the water-substituted samples (B) at equivalent concentrations. The ratio (A/B × 100%) is defined as the IS-normalized matrix factor (MF). The RSD of MFs should be less than 15%. The recovery of each enantiomer was determined by comparing the peak area ratios of the analytes to ISs in the regularly pre-treated QC samples at three concentration levels (six samples each) with those of spiked post-extraction samples. Similarly, the recovery of ISs was determined at 0.500 g/ml of each enantiomer. The stabilities of each enantiomer in human plasma were evaluated by analyzing triplicates of the plasma samples at two concentration levels (0.090 and 8.00 g/ml for each enantiomer), which were exposed to the following conditions: (1) short-term stability at room temperature for 6 h; (2) long-term stability at −80 ◦ C for 31 days; (3) autosampler tray stability at ambient temperature for 24 h; and (4) freeze-thaw stability after three freeze-thaw cycles at −80 ◦ C. The analytes were considered stable when the accuracy bias was within ±15% of the nominal concentrations. To evaluate the enantiomeric stability of R-(+)-ornidazole or S-(−)-ornidazole in human plasma, each enantiomer at the upper limit of quantification (ULOQ, 10.0 g/ml) level was incubated in triplicate for 12 h at 37 ◦ C, then extracted and analyzed as described. 2.7. Stereoselective pharmacokinetic study The validated method was used to investigate the plasma profiles of ornidazole enantiomers following an oral dose of 1000 mg ornidazole tablets (Jiudian Pharmaceutical Co., Ltd., Hunan, China) to six healthy Chinese volunteers (male; age range, 22–26 years; body mass index range, 20.1–23.9 kg/m2 ). The clinical study was approved by the Ethics Committee of the First Affiliated Hospital of Lanzhou University (Lanzhou, China). All volunteers provided written informed consent to participate in the study according to the principles of the Declaration of Helsinki and Good Clinical Practice. Blood samples (4 ml) were collected into sodium heparincontaining tubes at 0 (pre-dose), 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 36, 48, 72, and 96 h after oral administration of 1000 mg ornidazole tablets. The blood samples were centrifuged at 2000 × g for 10 min to separate the plasma fractions, and the plasma samples were stored at −80 ◦ C prior to analysis. The pharmacokinetic parameters of ornidazole enantiomers were calculated by non-compartmental analysis using the WinNonlin 5.3 software (Pharsight, St. Louis, MO, USA).
under ESI source was about 50-fold higher than that under APCI. Therefore, the ESI(+) mode was employed for ornidazole quantification in this study. In the Q1 full scan mode, protonated molecules at m/z 220 and m/z 225 were observed for ornidazole and d5 ornidazole, respectively. Fig. 1 shows the product ion spectra of [M+H]+ ions from ornidazole and d5 -ornidazole, as well as their proposed fragmentation patterns. Their most abundant and stable fragment ions at m/z 128 were selected in the MRM transitions. With these transitions, the signal-to-noise ratio (S/N) increased by 7-fold compared with respective selective ion monitoring (SIM). The ESI parameters were optimized to maximize the MS response, including fragmenter voltage, CE, gas flow-rate and temperature. The highest MS responses of ornidazole and d5 -ornidazole were achieved when the fragmenter voltages and CEs were set at 115 V and 11 V, respectively. The flow-rate and temperature of gases (nebulizer gas, carrier gas, and sheath gas) had little effect on the MS response. To resolve ornidazole enantiomers, different types of chiral stationary phases were evaluated, including Chiralpak ASRH (150 mm × 4.6 mm, 5 m), Chirobiotic T (250 mm × 4.6 mm, 5 m), Chirobiotic V (150 mm × 4.6 mm, 5 m), Ultron ES-OVM (150 mm × 4.6 mm, 5 m), and Chiral-AGP (150 mm × 4.0 mm, 5 m). No resolution was observed on the Chirobiotic T column, although polar ionic and reversed-phased modes were tested. When different percentages of acetonitrile, methanol, and acid/base modifiers were used, only partial resolution of ornidazole enantiomers (Rs ≤ 1.3) was achieved on the Chiralpak AS-RH and Chirobiotic V columns. Protein-based chiral columns (Ultron ES-OVM and Chiral-AGP) provided better resolution of ornidazole enantiomers than the other chiral columns. Baseline separation (Rs ≥ 1.5) was achieved on both columns within 20 min using a mobile phase methanol/10 mM ammonium acetate (2/98, v/v). Since the column efficiency of Ultron ES-OVM for ornidazole enantiomers was relatively low (W > 1.7 min), the Chiral-AGP column was chosen for further optimization. When 0.01% acetic acid was added into the aqueous phase, the resolution of ornidazole enantiomers increased from 1.5 to 1.7, and also acidification of the mobile phase facilitated the detection of ornidazole in the positive mode. However, further optimization of increasing methanol proportion (> 2%) or using other organic modifiers (acetonitrile or isopropanol) resulted in poor resolution. Then the proportion of methanol was further decreased from 2% to 0%. As a result, the enantiomers were highly resolved (Rs = 2.6). A flow gradient was used to shorten the total run time (from 12 min to 7.5 min), and the final Rs was 2.0 (calculated at ULOQ level). Generally, atmospheric pressure ionization techniques (ESI and APCI) require high proportions of organic modifiers to achieve a good spray, which is associated with MS sensitivity. Given that the response of ornidazole under the ESI(+) mode was adequate, the use of the final mobile phase (10 mM ammonium acetate containing 0.01% acetic acid) did not compromise too much sensitivity, and a desirable S/N (> 15) was obtained at the LLOQ of each enantiomer. Furthermore, the deuterated ISs were used in this study to compensate for the matrix effects due to their similar chromatographic behaviors and ionization properties to ornidazole.
3. Results and discussion
3.2. Sample preparation
3.1. Mass spectrometric and enantioselective chromatographic conditions
Considering that the separation of ornidazole enantiomers was performed on a Chiral-AGP with full aqueous phase, the coeluent contaminants could cause significant matrix effect and decrease the reproducibility of the method especially with an ESI ionization source. Protein precipitation is a nonselective purification
Preliminary experiments showed that ornidazole could only be ionized in the positive ionization mode and the signal intensity
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Fig. 1. LC–MS/MS total ion chromatogram of blank plasma sample spiked with R-(+)-ornidazole (1.00 g/ml), S-(−)-ornidazole (1.00 g/ml), R-(+)-d5 -ornidazole (1.00 g/ml), and S-(−)-d5 -ornidazole (1.00 g/ml) (A), as well as product ion spectra of [M+H]+ ions of ornidazole (B) and d5 -ornidazole (C). Their proposed fragmentation patterns are displayed as insets. The chiral center is marked with an asterisk.
method that may introduce high amounts of endogenous components; hence, liquid–liquid extraction was firstly considered for its relatively clean extracts. Given that ornidazole is highly soluble in ethyl acetate at pH 7 (∼296 mg/ml) [1], ethyl acetate was chosen as the extraction reagent without acidifying or alkalizing the plasma sample. Ornidazole is extensively metabolized to some Phase II metabolites in humans, including glucuronides M161/M16-2 and sulfate M10 [22,24]. Conjugated metabolites might be unstable and convert to the parent drug during sample preparation. Moreover, conjugated metabolites that were not chromatographically separated with the parent drug could dissociate in ionization source, thereby increasing the signal of the parent drug and causing false over-estimation. To evaluate the effects of these metabolites on ornidazole detection, blank plasma samples spiked with M10 (0.500 g/ml), M16-1 (0.500 g/ml), and M16-2 (0.500 g/ml) were extracted with ethyl acetate under the same condition above. No interference was observed at the retention times of ornidazole enantiomers.
3.3. Method validation 3.3.1. Assay selectivity Selectivity of the method was assessed by comparing the chromatograms of blank human plasma from six sources with the corresponding spiked plasma at the LLOQ concentration. Fig. 2 shows the typical chromatograms of a blank plasma sample, a blank plasma sample spiked with ornidazole enantiomers at LLOQ level and ISs (1.00 g/ml R-(+)-d5 -ornidazole and 1.00 g/ml S-(−)d5 -ornidazole) and a plasma sample obtained at 1.5 h after oral administration of 1000 mg racemic ornidazole to a volunteer. No significant endogenous interference co-eluting with analytes and ISs was observed in the blank human plasma.
3.3.2. Linearity of calibration curve and LLOQ Linear regression curves were obtained over the concentration ranges of 30.0–10 000 for R-(+)-ornidazole and S-(−)-ornidazole, respectively. The following typical equations of the calibration curve were used: (r 2 = 0.9978)
R-(+)-ornidazole :
y = 2.11x − 0.00442
S-(−)-ornidazole :
y = 2.11x − 0.00670 (r 2 = 0.9972)
where y is the peak area ratio of analytes to ISs and x is the concentration of analytes. The LLOQ of both ornidazole enantiomers was 0.030 g/ml. The RSD values of precision at the LLOQ of the two enantiomers were between 3.3% and 6.8%, and RE values of accuracy were between 0.4% and 0.8%. With the present LLOQ, the plasma concentrations of R-(+)-ornidazole and S-(−)-ornidazole could be determined up to 96 h after oral administration of 1000 mg ornidazole tablets, which were sensitive enough to allow for the investigation of stereoselective pharmacokinetics of ornidazole. Based on the S/N of 3, the limit of detection values (LOD) of this method were estimated as 0.006 g/ml for both enantiomers. 3.3.3. Precision and accuracy Intra- and inter-day precision and accuracy values for the QC samples are summarized in Table 1. In this assay, the RSD values of intra- and inter-day precision for R-(+)-ornidazole were less than 6.2%, whereas the RE values of accuracy ranged from −4.5% to 1.2%. For S-(−)-ornidazole, the RSD values of intra- and inter-day precision were less than 10.2% and RE values of accuracy was between −5.4% and −0.8%. The precision and accuracy of ornidazole enantiomeric ratios are presented in Table 2. The RSD values of intra- and inter-day
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Fig. 2. Representative enantioselective MRM chromatograms for R-(+)-ornidazole (I), S-(−)-ornidazole (II), R-(+)-d5 -ornidazole (IS; III), and S-(−)-d5 -ornidazole (IS; IV) in human plasma. (A) Blank plasma sample. (B) Blank plasma sample spiked with R-(+)-ornidazole (0.030 g/ml), S-(−)-ornidazole (0.030 g/ml), R-(+)-d5 -ornidazole (1.00 g/ml), and S-(−)-d5 -ornidazole (1.00 g/ml). (C) Plasma sample at 1.5 h after oral administration of 1000 mg racemic ornidazole to a volunteer.
Table 1 Precision and accuracy data for determination of R-(+)-ornidazole and S-(−)-ornidazole in human plasma (three days, six replicates per day). Analyte
Nominal conc. (g/ml)
Mean ± SD (g/ml)
Intra-day RSD (%)
RE (%)
R-(+)-Ornidazole
0.030 0.090 0.500 8.00
0.030 0.091 0.492 7.64
± ± ± ±
0.001 0.003 0.029 0.28
6.3 2.5 1.8 6.2
3.3 3.4 4.1 3.2
0.8 1.2 −1.7 −4.5
S-(−)-Ornidazole
0.030 0.090 0.500 8.00
0.030 0.088 0.496 7.57
± ± ± ±
0.001 0.003 0.021 0.50
6.8 1.5 5.7 10.2
4.5 3.8 3.7 5.9
0.4 −2.3 −0.8 −5.4
precision were below 4.3% and 7.1%, respectively. The RE values of accuracy ranged from −3.4% to 1.0%.
3.3.4. ME and recovery The MFs from six lots of blank plasma ranged from 97.3% to 104% for all compounds. Inter-subject variability of the ISs-normalized MFs, as measured by the RSD, was lower than 3.4%. Thus, ion suppression or enhancement from plasma matrix was negligible under the current conditions. The mean extraction recoveries of the two enantiomers and ISs were between 96.5% and 102%.
Inter-day RSD (%)
3.3.5. Stability Ornidazole enantiomers were stable in plasma after placement at room temperature for 6 h, undergoing three freeze-thaw cycles and storage at −80 ◦ C for 31 days. The processed samples were stable up to 24 h at the autosampler tray. The results demonstrated good stability of ornidazole enantiomers throughout the experiment. The peak of the other enantiomer was not observed after incubation of plasma samples containing one enantiomer at 37 ◦ C for 12 h. As a result, no chiral inversion occurred between R-(+)ornidazole and S-(−)-ornidazole during storage, processing, and analysis.
Table 2 Precision and accuracy data for determination of R-(+)-ornidazole and S-(−)-ornidazole in human plasma (three days, six replicates per day). R-(+)/S-(−) ratios
Detected ratios (mean ± SD)
Inter-day RSD (%)
Intra-day RSD (%)
RE (%)
0.50 2.00
0.50 ± 0.02 1.93 ± 0.08
7.1 3.1
4.0 4.3
1.0 −3.4
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within 7.5 min. Stable isotopically labeled d5 -ornidazole enantiomers were used as ISs to compensate for the matrix effects. The LLOQ was 0.030 g/ml for ornidazole enantiomers using only 100 l of human plasma. This method was successfully applied to characterize the pharmacokinetic profiles of ornidazole enantiomers after a single dose of 1000 mg racemic ornidazole to six healthy Chinese volunteers. The pharmacokinetic results indicated that ornidazole enantiomers showed similar tmax and Cmax , while S-ornidazole displayed higher metabolic rate than R-enantiomer in humans.
References Fig. 3. Mean plasma concentration–time profiles of R-(+)-ornidazole and S-(−)ornidazole after oral administration of 1000 mg racemic ornidazole to six healthy Chinese volunteers.
Table 3 Pharmacokinetic parameters (mean ± SD) of R-(+)-ornidazole and S-(−)-ornidazole after oral administration of 1000 mg racemic ornidazole to six healthy Chinese volunteers. Parameters
R-(+)-ornidazole
AUC0–t ((g h)/ml) AUC0–∞ ((g h)/ml) tmax (h) Cmax (g/ml) T1/2 (h) MRT (h) CL/F (l/h) Vd (l)
184.6 193.2 1.2 7.9 17.5 25.6 2.6 66.1
± ± ± ± ± ± ± ±
33.1 29.2 0.4 1.3 1.6 2.2 0.4 8.1
S-(−)-ornidazole 128.7 130.2 1.4 7.9 11.5 17.2 3.9 64.2
± ± ± ± ± ± ± ±
19.8 18.5 0.9 1.6 0.7 1.5 0.6 7.1
AUC0–t : area under plasma concentration–time curve to the last measurable concentration; AUC0–∞ : area under plasma concentration–time curve to infinity; tmax : time to maximum plasma concentration; Cmax : maximum plasma concentration; T1/2 : elimination half-life; MRT: mean residence time; CL/F: total clearance; Vd : distribution volume.
3.4. Stereoselective pharmacokinetic study The present enantioselective LC–MS/MS method provided the LLOQ down to 0.030 g/ml for each enantiomer, which met the requirements to evaluate stereoselective pharmacokinetics of ornidazole. With the present LLOQ, the ornidazole enantiomers concentration could be determined in plasma samples up to 96 h postdose. After oral administration of 1000 mg racemic ornidazole to six healthy Chinese volunteers, the profiles of the mean plasma concentration of ornidazole enantiomers versus time are shown in Fig. 3. The plasma concentrations of R-(+)-ornidazole were higher than those of S-(−)-enantiomer in the elimination phase. The pharmacokinetic parameters are summarized in Table 3. The tmax and Cmax were comparable between two enantiomers. The AUC0−∞ of R-(+)-ornidazole was 1.48 times higher than that of S-(−)-enantiomer, and the R-(+)/S-(−) ratio of the total body clearance (CL/F) was 0.68. According to our previous study [22], the stereoselective glucuronidation of ornidazole enantiomers could be one of the major determinants of its enantioselective pharmacokinetics in humans. 4. Conclusion An enantioselective LC–MS/MS method was firstly developed and validated for quantification of ornidazole enantiomers in human plasma. Liquid chromatographic conditions were performed on a Chiral-AGP column operated in the MS-friendly reverse-phase mode. Baseline separation (Rs = 2.0) was achieved
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