Research Article
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[email protected] Simultaneous determination of ornidazole and its main metabolites in human plasma by LC–MS/MS: application to a pharmacokinetic study Background: Ornidazole is a 5-nitroimidazole antimicrobial agent used for almost 40 years. A novel LC–MS/MS assay was developed and validated for the simultaneous determination of ornidazole and its main metabolites (M3, M6, M16–1, and M16–2) in human plasma. Results: After extraction from 100 μl of plasma by protein precipitation with acetonitrile, all the analytes were separated on a Capcell PAK MG C18 column (100 × 4.6 mm, 5 μm) within 5.0 min and detected by ESI-MS/MS in the positive mode. The validation results met the acceptance criteria as per the US FDA and EMA guidelines. Conclusion: The validated method was successfully applied to a pharmacokinetic study after oral administration of 1000 mg ornidazole to six healthy Chinese volunteers.
Ornidazole [(R,S)-1-chloro-3-(2-methyl5-nitro-1H-imidazol-1-yl)propan-2-ol] is a 5-nitroimidazole derivative that has been used for the prophylaxis and treatment of susceptible protozoal or anaerobic bacterial infections since 1970s [1] . The antimicrobial activity of ornidazole is dependent on the formation of a hydroxylamine intermediate in the microbe, which damages microbial DNA, inhibits its repair, disrupts transcription, and eventually causes cell death [2–4] . Although it shows similar activity to metronidazole, ornidazole requires less frequent administration and shorter treatment duration for many clinically relevant infections because of its longer half-life (14.4 vs 8.4 h for metronidazole) [5–7] . After intravenous infusion, ornidazole is extensively metabolized in humans. The major metabolites have been identified as two diastereoisomeric glucuronides, a dihydrodiol metabolite, and a sulfate conjugate in human urine [8] . In our preliminary study, ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF MS) was used to characterize ornidazole metabolites in human plasma after oral administration. Based on the MS peak area ratios (%) of
10.4155/BIO.14.117 © 2014 Future Science Ltd
the metabolite to the parent drug, the main metabolites of ornidazole in human plasma included 2′,3′-epoxy ornidazole (M3, 9.5%), 2′,3′-dihydrodiol ornidazole (M6, 3.4%), and two diastereoisomeric glucuronides of ornidazole (M16–1, 1.1%; M16–2, 0.2%). The circulating metabolites may contribute to the pharmacology or toxicity of the drugs [9] . M3 and M6 were reported to be as potent as the parent drug in vitro, while they showed different toxicologic profiles compared with ornidazole (LD50 value in mice: M6 > ornidazole > M3) [10,11] . Therefore, the formation of M3 would elevate the toxicity of ornidazole, whereas conversion of M3 to M6, probably by epoxide hydrolase [12–14] , was considered as a detoxification process in vivo. Determination of M3 and M6 would help us better understand the pharmacology and toxicology of ornidazole in humans. Glucuronide conjugates are generally (but not all) not expected to be pharmacologically active or present toxicologic risks. However, glucurondation is the primary metabolic pathway of ornidazole and the main cause of its enantioselective pharmacokinetics in humans [8,15] , thus knowing the exposure of the glucuronide metabolites would provide further insight into the enantioselective
Bioanalysis (2014) 6(18), 2343–2356
Jiangbo Du1, Zhiyu Ma1, Yifan Zhang1, Ting Wang2, Xiaoyan Chen1 & Dafang Zhong*,1 1 State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai 201203, PR China 2 The First Affiliated Hospital of Lanzhou University, 1 West Donggang Road, Lanzhou 730000, PR China *Author for correspondence: Tel.: +86 21 5080 0738 Fax: +86 21 5080 0738
[email protected] part of
ISSN 1757-6180
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Research Article Du, Ma, Zhang, Wang, Chen & Zhong
Key terms Diastereoisomeric glucuronides: Glucuronide conjugates that have different configurations at one or more of the drug-related stereocenters. Circulating metabolites: Metabolites present in the circulation. Circulating metabolites have potential impact on the safety and efficacy of the drugs. Enantioselective pharmacokinetics: Drug enantiomers possess different pharmacokinetic properties in vivo. Biological fluids: Include blood, plasma, serum, urine, bile, saliva, and different kinds of tissue fluids.
disposition of ornidazole enantiomers. To this end, it is essential to establish a reliable method for the determination of ornidazole and these main metabolites in human plasma. Although numerous analytical methods have been developed to detect ornidazole in biological fluids [16–25] , few studies have sought to determine its metabolites. Heizmann et al. [26] reported an HPLC-UV method to quantify ornidazole and two of its metabolites (6-hydroxy ornidazole and 2´,3´-dihydrodiol ornidazole) in biological fluids. However, their method required a large volume of plasma (250 μl) and laborious clean-up procedures (liquid–liquid extraction). And obviously, the LLOQ of 50.0 ng/ml for both metabolites (>1/20 of respective Cmax) could not guarantee comprehensive evaluation of their pharmaco kinetic parameters. Furthermore, these two metabolites are minor metabolites of ornidazole in plasma, and their circulating concentrations turn out to be very low [26] . To date, no studies have clarified the exposure of the main circulating metabolites of ornidazole in humans. In this study, we developed and validated a simple and rapid LC–MS/MS method for the simultaneous determination of ornidazole and its main metabolites in human plasma. This method was successfully applied to a pharmacokinetic study following oral administration of 1000 mg ornidazole to six healthy Chinese volunteers. Experimental
with minor modifications, in which d5-epichlorohydrin was used as the starting material instead of epichlorohydrin [10,11] . HPLC-grade methanol, acetonitrile, and formic acid were obtained from Sigma-Aldrich (MO, USA). HPLC-grade ammonium acetate was supplied by Tedia (OH, USA). Deionized water was purified using a Millipore Milli-Q Gradient Water Purification System (Molsheim, France). Heparinized blank human plasma was obtained from the First Affiliated Hospital of Lanzhou University (Lanzhou, China). Chromatographic conditions
An Agilent 1200 LC 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. The analytes were separated on a Capcell PAK MG C18 column (100 × 4.6 mm, 5 μm; Shiseido, Tokyo, Japan) equipped with a Security-Guard C18 column (4.0 × 3.0 mm, 5 μm; Phenomenex, CA, USA) at ambient temperature. The mobile phase consisted of methanol/5 mM ammonium acetate/formic acid (45:55:0.055, v/v/v), and was delivered using a flow velocity gradient at 1.0 min/ml for 0–1.2 min and at 0.6 ml/min for 1.2–5.0 min. A diverter valve was used after the analytical column (0–1.2 min, to waste; 1.2–5.0 min, to source) to avoid contaminating the ion source by nonvolatile salts and endogenous components of the plasma. MS conditions
MS detection was performed on an Agilent 6460 Triple Quad LC/MS instrument (Agilent, Waldbronn, Germany) with an electrospray ionization (ESI) source in the positive multiple reaction monitoring (MRM) mode. Data were processed using Agilent MassHunter workstation (version B.03.02, Agilent). The instrument was operated at capillary voltage and nozzle voltage of +3500 and +500 V, respectively. Nitrogen was used as the nebulizer gas (45 psi), the carrier gas (5 l/min at 350°C), and the sheath gas (11 l/min at 380°C). A dwell time of 80 ms was set for each transition. The optimized MS parameters are listed in Table 1.
Chemicals & reagents
Ornidazole (98.0% purity) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Reference standards of M3 (99.2% purity), M6 (99.6% purity), M16–1 (100% purity), and M16–2 (100% purity) were isolated and purified from human urine, as previously described [8] . d5-Ornidazole (100% purity), d5-M3 (100% purity), and d5-M6 (100% purity) were synthesized and purified in our laboratory as internal standards (ISs) using a previously described method
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Preparation of calibration standards, quality control samples & IS solutions
Standard and quality control (QC) stock solutions (∼1 mg/ml) of ornidazole, M3, M6, M16–1, and M16–2 were separately prepared by dissolving accurately weighed reference substances in acetonitrile. Combined standard working solutions of ornidazole/M3/M6/M16–1/M16–2 were diluted with acetonitrile from standard stock solutions to yield the following concentrations:
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Determination of ornidazole & its main metabolites in human plasma
Research Article
Table 1. MS parameters for ornidazole, M3, M6, M16–1, M16–2, d5 -ornidazole (internal standard for ornidazole, M16–1, and M16–2), d5 -M3 (internal standard for M3), and d5 -M6 (internal standard for M6). Compound
Precursor ion [M + H] + (m/z)
Product ion (m/z)
Fragmentor voltage (V) Collision energy (eV)
Ornidazole
220
128
115
11
d5 -Ornidazole
225
128
115
11
M3
184
128
90
8
d5 -M3
189
129
90
8
M6
202
128
90
8
d5 -M6
207
128
90
8
M16–1
396
220
160
12
396
128
130
30
M16–2
396
220
160
12
396
128
130
30
100/10.0/5.00/3.00/3.00, 300/30.0/10.0/9.00/9.00, 1000/100/20.0/30.0/30.0, 3000/300/50.0/90.0/90.0, 10000/1000/100/300/300, and 30000/3000/200/ 900/900 ng/ml, respectively. The calibration standards were prepared by spiking 100 μl of standard working solutions to 100 μl of blank human plasma. The QC working solutions were prepared by serial dilution with acetonitrile from QC stock solutions. For the LLOQ and QC samples (low QC, medium QC, and high QC), 50 μl of the appropriate diluted QC working solutions were spiked into 950 μl of blank plasma to yield final concentrations of 100/10.0/5.00/3.00/3.00, 300/30.0/12.0/9.00/9.00, 3000/300/30.0/90.0/90.0, and 24000/2700/160/720/720 for ornidazole/M3/ M6/M16–1/M16–2, respectively. An IS working solution (2000/1000/200 ng/ml for d5-ornidazole/d5-M3/ d5-M6, respectively) was prepared by mixing the three IS stock solutions and diluting the mixture with acetonitrile. The LLOQ and QC samples were distributed for single use and then stored at -80°C until analysis. Stock and working solutions were stored at 4°C. Sample preparation
After thawed and vortexed thoroughly, a 50 μl aliquot of the IS working solution and 250 μl of acetonitrile were added to 100 μl of plasma sample. The mixture was vortexed for 1 min and centrifuged at 11000 × g for 5 min. All the supernatant was transferred to a 10-ml glass tube and evaporated to dryness at 40°C under nitrogen in a TurboVap evaporator (Zymark, MA, USA). The dry residue was reconstituted in 75 μl of the mobile phase, and then 5 μl was injected into the LC–MS/MS system for analysis. Method validation
The assay was validated as per the guidelines of the US FDA and EMA [27,28] .
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To evaluate the selectivity of the method, six different lots of blank plasma and 12 spiked LLOQ samples were analyzed. The co-eluted interference at the retention times of each analyte and IS was required to be less than 20% of the peak area of the LLOQ standard and less than 5% of the peak area of the IS, respectively. Linearity was evaluated by linear least squares regression of the peak area ratios of analyte to IS versus the analyte concentrations in duplicate on 3 validation days. The linearity assay was considered acceptable when the correlation coefficient (r2) was greater than 0.99 and each back-calculated concentration was within ±15% of the nominal concentration (±20% at the LLOQ). The LLOQ, defined as the lowest calibration standard [27,28] , was analyzed in six replicates on 3 validation days. The intra- and inter-assay precision of less than 20% and the accuracy of ±20% were required for the LLOQ. The limit of detection (LOD) was estimated as the analyte concentration that provided a signal-to-noise ratio (S/N) of 3. Precision and accuracy were assessed using three concentrations of QC samples (300/30.0/12.0/9.00/9.00, 3000/300/30.0/90.0/90.0, and 24000/2700/160/ 720/720 ng/ml for ornidazole/M3/M6/M16–1/ M16–2, respectively) in six replicates on 3 validation days. The precision and accuracy of the assay were expressed as the relative standard deviation (RSD) and the relative error (RE), respectively. The intra- and inter-assay precision should not exceed 15%, and the accuracy was required to be within ±15%. Dilution integrity experiment was performed to evaluate the precision and accuracy of the method after sample dilution. QC samples at 120000/13500/800/3600/3600 ng/ml for ornidazole/M3/M6/M16–1/M16–2 (diluted QC samples) were diluted 5-fold with blank plasma in six replicates prior to analysis. The dilution procedure was
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Research Article Du, Ma, Zhang, Wang, Chen & Zhong considered valid if the precision (RSD) was less than 15% and the accuracy (RE) was within ±15% of the nominal value. The matrix effect was determined at two concentrations (300/30.0/12.0/9.00/9.00 and 24000/2700/160/720/720 ng/ml for ornidazole/ M3/M6/M16–1/M16–2, respectively) by comparing corresponding peak area ratios of analyte to IS in the spiked-after-extraction samples (A, n = 6) with those of water-substituted samples (B, n = 3). The IS-normalized matrix factor (MF) was calculated as the ratio (A/B × 100%). The RSD of the MFs was required to be less than 15%. The recoveries of ornidazole, M3, M6, M16–1, and M16–2 after extraction were determined at three QC levels by comparing the peak area ratios of analyte to IS in the regularly prepared QC samples (n = 6) with those of spiked-after-extraction samples (n = 3). For regularly prepared QC samples, the analytes and ISs were spiked before extraction, while the analytes and ISs were back-spiked after extraction for spikedafter-extraction samples. The recoveries of the ISs were estimated using the QC samples at the medium concentration as references. To evaluate the stability of ornidazole and its four metabolites in human plasma, we analyzed three replicates of spiked plasma samples exposed to different time and temperature conditions at two QC levels (300/30.0/12.0/9.00/9.00 and 24000/2700/160/720/720 ng/ml for ornidazole/ M3/M6/M16–1/M16–2, respectively). Short-term stability was assessed after placing spiked plasma samples at room temperature for 6 h. Post-preparative stability was evaluated after placing the reconstituted samples in autosampler vials at room temperature for 24 h. Freeze–thaw cycle stability was assessed after three complete freeze–thaw cycles (-80°C to room temperature) on consecutive days. Long-term stability was determined after storage of the standard spiked plasma samples at -80°C for 61 days. The analytes were considered stable in plasma if the determined concentrations were within ±15% of the nominal values. For sample collection and handling stability [29,30] , fresh human whole blood (sodium heparin) was spiked with two concentrations of all analytes (300/30.0/12.0/9.00/9.00 and 24000/2700/160/720/720 ng/ml for ornidazole/ M3/M6/M16–1/M16–2, respectively). The spiked whole blood samples were immediately split into two Key term Incurred sample reanalysis: Reanalysis of a portion of the incurred samples to determine whether the initial analytical results are reproducible.
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aliquots (I and II). One aliquot (I) was processed and analyzed immediately, while the other aliquot (II) was exposed at room temperature for 2 h before preparation and analysis. Stability of the analytes in blood was proven if the mean peak area ratios of analyte to IS in the test samples (II) were between ±15% of those in the reference samples (I). To evaluate whether sample preparation process would cleave glucuronide metabolites of ornidazole, QC samples only spiked with M16–1/M16–2 (1000/1000 ng/ml) were processed and analyzed as described. The stock solution stability of all the analytes and ISs was assessed for short-term stability after 6 h of storage at room temperature and for long-term stability after 12 days at 4°C. The solutions were considered stable if the peak area difference between the stored solution and a freshly prepared solution was within 10%. Interference test
To evaluate if the presence of high concentrations of ornidazole would influence the analysis of its metabolites (M3, M6, M16–1, and M16–2) in human plasma [31] , we analyzed the LLOQ samples containing individual metabolites and high concentrations of ornidazole with different concentration ratios (1:10 for M3, 1:1000 for M6, 1:100 for M16–1, and 1:1000 for M16–2) in six replicates. The precision of less than 20% and the accuracy of ±20% were required for the interference test. Application to a pharmacokinetic study
The validated method was used to investigate the pharmacokinetics of ornidazole and its main metabolites following a single oral dose of 1000 mg ornidazole tablets (Jiudian Pharmaceutical Co., Ltd, Hunan, China) to six healthy Chinese volunteers (male; age range: 22–26 years; BMI range: 20.1–23.9 kg/m2) under fasting conditions. The clinical study was approved by the Ethics Committee of the First Affiliated Hospital of Lanzhou University (Lanzhou, China). All volunteers provided their 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, and centrifuged at 2000 × g for 10 min. The plasma samples were split into duplicates immediately and stored at -80°C prior to analysis. The pharmacokinetic parameters of ornidazole and its four metabolites were calculated by noncompartmental analysis using the WinNonlin 5.3 software (Pharsight, MO, USA).
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Research Article
Determination of ornidazole & its main metabolites in human plasma
Incurred sample reanalysis
The reproducibility of the analytical method was evaluated with incurred sample reanalysis (ISR). Samples close to the maximum concentration and near the end of the elimination phase were selected for ISR. The ISR data were compared with the original data, and the differences should be within ±20% of the mean for at least 67% of the repeats [32–34] . Results & discussion The primary objective of this study was to develop a sensitive and rapid method for the simultaneous determination of ornidazole and its main metabolites (M3, M6, M16–1, and M16–2) in human plasma. Since 5-nitroimidazole structure is a good chromophore (λmax = 318 nm), HPLC-UV becomes the first choice to determine 5-nitroimidazole derivatives in biological matrices [20–23,26] . However, HPLC-UV methods always require
a long run time to achieve optimal resolution and avoid co-eluted interference substances. In addition, standard HPLC assays generally provide limited sensitivity and are inadequate for quantification of drugs and/or metabolites at low concentrations. In our pilot study, the concentrations of ornidazole metabolites were estimated to be in ng/ml level, thus an HPLC-UV assay was not expected to provide practical sensitivity for their quantification. In the recent decades, LC–MS/MS has became a dominant technique in the field of bioanalysis, due to its inherent high selectivity, sensitivity, and throughput. To this end, we developed and validated an LC–MS/MS method for the quantification of ornidazole and its main metabolites in human plasma. MS conditions
Preliminary studies demonstrated that ornidazole, M3, and M6 could only be ionized in the positive mode
A
B +ESI product ion (220.0 -> **) ornidazole
x104 2.0 1.6
N
CH3
Counts
Counts
OH O2N
m/z 128
N 0.8 82.0
1.4
D
1.2
D
O2N
Cl
D OH N
1.0
CH3
N
m/z 128
0.8 224.9
0.4 0.2
92.9 0
81.9 97.8
61.8
0 60
90
120
150
180
210
240
Mass-to-charge (m/z)
C
60
90
120
128.1
129.0
N
CH3 N
1.6
m/z 128
183.9
81.9
D
2.5 Counts
O2N
2.0
O2N
1.0
0.8 110.8
0.4
240
D
D
O
D
H/D Exchange N
CH3 m/z 129
N
2.0 1.5
1.2
210
D
3.0
2.4
180
+ESI product ion (189.0 -> **) d5-M3
x103 3.5
O
150
Mass-to-charge (m/z)
D
+ESI product ion (184.0 -> **) M3
2.8
Counts
D
D
0.6
219.9
0.4
x103
128.0
1.6
Cl
1.2
+ESI product ion (225.0 -> **) d5-ornidazole
x104
128.0
188.9
83.0 61.9 98.9 110.8
0.5 0
0 60
90
120
150
180
Mass-to-charge (m/z)
210
240
60
90
120
150
180
210
240
Mass-to-charge (m/z)
Figure 1. Product ion mass spectra and tentative fragmentation patterns. (A) Ornidazole, (B) d5 -ornidazole, (C) M3 and (D) d5 -M3. ** represents all product ions of the parent ion under the optimized MS parameters. ESI: Electrospray ionization.
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Research Article Du, Ma, Zhang, Wang, Chen & Zhong
A
B x103
+ESI product ion (202.0 -> **) M6 127.9
1.8
OH
1.6
1.2
CH3 N
1.0 0.8 0.6
m/z 128 202.1
81.9
D D O2N
1.25
0.2
0.25
0
0 90
120
150
180
OH N
CH3 m/z 128 206.9
81.9
210
240
189.1 61.9 90
60
120
Mass-to-charge (m/z)
150
180
210
240
Mass-to-charge (m/z) D
C +ESI product ion (396.0 -> **) M16-1 219.9
1.2
Cl HOOC O O
1.0 128.0 0.8
O2N
81.9
N
CH3 N
m/z 128
173.8
0.6
x103 1.2 OH OH OH
220.0
m/z 220 395.9
0.6
128.1 O2N 81.9
174.1
m/z 128
N
CH3 N
OH OH OH
m/z 220 396.1
0.4
0.2
Cl HOOC O O
0.8
184.1
0.4
+ESI product ion (396.0 -> **) M16-2
1.0
Counts
x10
3
Counts
OH
D
N
1.00
0.50
0.4
60
D
0.75
183.9
61.8
D
1.50 Counts
Counts
O2N
N
127.9
1.75
OH
1.4
+ESI product ion (207.0 -> **) d5-M6
x103 2.0
184.1
0.2 0
0 100
150
200
250
300
350
400
450
Mass-to-charge (m/z)
100
150
200
250
300
350
400
450
Mass-to-charge (m/z)
Figure 2. Product ion mass spectra and tentative fragmentation patterns. (A) M6, (B) d5 -M6, (C) M16–1 and (D) M16–2. ** represents all product ions of the parent ion under the optimized MS parameters. ESI: Electrospray ionization.
by virtue of the presence of imidazole ring [15] , while the two glucuronides (M16–1 and M16–2) exhibited comparable signal intensities in the positive and negative mode. It was also found that ESI provided much higher MS responses for all analytes than atmospheric pressure chemical ionization, especially for M16–1 and M16–2, which were subjected to severe ion-source dissociation in the atmospheric pressure chemical ionization source. Therefore, the ESI-positive mode was used in the present study. In the Q1 full scan mode, protonated molecules at m/z 220, 225, 184, 189, 202, 207, 396, and 396 were observed for ornidazole, d5-ornidazole, M3, d5-M3, M6, d5-M6, M16–1, and M16–2, respectively. Considering the specificity and stability of the product ions, the MRM transitions chosen for quantitative analysis were m/z 220 → 128 for ornidazole, m/z 225 → 128 for
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Bioanalysis (2014) 6(18)
d5-ornidazole, m/z 184 → 128 for M3, m/z 189 → 129 for d5-M3, m/z 202 → 128 for M6, m/z 207 → 128 for d5-M6, respectively. Since one MRM transition has already provided practical sensitivity and selectivity for those analytes, no more transitions were incorporated during their quantification in this study. However, M16–1 and M16–2 had low plasma concentrations and poor MS responses. To maximize their signal intensities, the summation of two MRM transitions was used in the detection program: m/z 396 → 220 + 128, resulting in a 1.6-fold sensitivity improvement compared with a single transition (m/z 396 → 220). Figures 1 & 2 present the product ion spectra of [M + H]+ ions from the analytes and the ISs, as well as their tentative fragmentation patterns. Moreover, other ESI parameters including fragmentor voltage and collision energy were also carefully optimized in order to achieve maximum sen-
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Determination of ornidazole & its main metabolites in human plasma
sitivity. The optimal MS parameters for each transition are shown in Table 1. Chromatographic conditions
Due to the high specificity of LC–MS/MS, it is not necessary to resolve the individual analytes to avoid interference. However, in the present study, M16–1 and M16–2 are diastereoisomeric glucuronides of ornidazole, which share the same MRM transitions. Furthermore, they could be subjected to in-source dissociation to convert to ornidazole, thus causing an overestimation of the parent A
drug when baseline resolution was not achieved. Therefore, the chromatographic separation of ornidazole, M16–1, and M16–2 was crucial in this study. To solve this problem, we evaluated several HPLC columns, including Eclispe XDB-C18, Capcell PAK MG C18, Zorbax Eclipse Plus C18, and Eclipse XDB-Phenyl, in terms of sensitivity, peak symmetry, and separation capacity for ornidazole, M16–1, and M16–2. The results showed that the Capcell PAK MG C18 column exhibited satisfactory sensitivity, peak shape, and chromatographic separation of each analyte of interest. Thus, this column
B
x101 +ESI MRM (220.0 -> 128.0) blank 5
I
C
x102 +ESI MRM (220.0 -> 128.0) sd_o I
2
x101 +ESI MRM (225.0 -> 128.0) blank 3.8
II
D
x103 +ESI MRM (220.0 -> 128.0) LLOQ I 5.0
x104 +ESI MRM (225.0 -> 128.0) sd_o 4 II 2
x104 +ESI MRM (225.0 -> 128.0) LLOQ 4
x105 +ESI MRM (220.0 -> 128.0) 03_12 I 2 1
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x101 +ESI MRM (202.0 -> 128.0) blank 6
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x104 +ESI MRM (189.0 -> 129.0) sd_o 2 IV
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1
x101 +ESI MRM (202.0 -> 128.0) sd_o V
x104 +ESI MRM (184.0 -> 128.0) 03_12 III 1
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x104 +ESI MRM (202.0 -> 128.0) 03_12 V 1
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x101 +ESI MRM (207.0 -> 128.0) blank 4.5 VI
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x104 +ESI MRM (207.0 -> 128.0) sd_o 2 VI 1
x101 +ESI MRM (396.0 -> 220.0, 128.0) blank 4
x102 +ESI MRM (184.0 -> 128.0) LLOQ III 4
8
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x101 +ESI MRM (184.0 -> 128.0) sd_o 8 III 6 4
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1 2 3 4 5 Counts vs acquisition time (min)
+ESI MRM (207.0 -> 128.0) LLOQ x104 +ESI MRM (207.0 -> 128.0) 03_12 1.50 VI VI 0.75
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x101 +ESI MRM (396.0 -> 220.0, 128.0) sd_o 4
x104 1.50
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1 2 3 4 5 Counts vs acquisition time (min)
x101 +ESI MRM (396.0 -> 220.0, 128.0) LLOQ VII VIII 4
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1 2 3 4 5 Counts vs acquisition time (min)
VIII
1 4 5 2 3 Counts vs acquisition time (min)
Figure 3. Typical multiple reaction monitoring chromatograms. (I) Ornidazole, (IS, II) d5 -ornidazole, (III) M3, (IS, IV) d5 -M3, (V) M6, (IS, VI) d5 -M6, (VII) M16–1, and (VIII) M16–2 in human plasma. (A) Blank plasma sample. (B) Blank plasma spiked with 1000 ng/ml d5 -ornidazole (IS), 500 ng/ml d5 -M3 (IS), and 100 ng/ml d5 -M6 (IS). (C) Blank plasma spiked with 100 ng/ml ornidazole, 10.0 ng/ml M3, 5.00 ng/ml M6, 3.00 ng/ml M16–1, 3.00 ng/ml M16–2, 1000 ng/ml d5 -ornidazole (IS), 500 ng/ml d5 -M3 (IS), and 100 ng/ml d5 -M6 (IS). (D) A plasma sample obtained at 36 h after oral administration of 1000 mg ornidazole to a volunteer. ESI: Electrospray ionization; MRM: Multiple reaction monitoring.
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Table 2. Precision and accuracy data for analysis of ornidazole, M3, M6, M16–1, and M16–2 in human plasma (3 days with six replicates per day). Analyte
Concentration (ng/ml)
RSD (%)
RE (%)
Spiked
Calculated
Intra-day
Inter-day
Ornidazole
100 (LLOQ)
105
5.1
5.8
5.3
300 (LQC)
300
0.7
2.1
-0.1
3000 (MQC)
3067
0.7
1.6
2.2
24,000 (HQC)
25,170
0.4
0.5
4.9
120,000 (DQC)
111,713
1.3
–
-6.9
M3
10.0 (LLOQ)
10.7
4.1
11.6
7.2
30.0 (LQC)
32.4
4.5
5.6
8.0
300 (MQC)
328
4.4
7.2
9.4
2700 (HQC)
2943
1.2
6.4
9.0
13,500 (DQC)
12,874
0.7
–
-4.6
M6
5.00 (LLOQ)
5.30
4.8
9.3
6.0
12.0 (LQC)
12.9
1.4
2.7
7.3
30.0 (MQC)
32.8
4.8
7.3
9.4
160 (HQC)
171
4.1
7.5
7.0
800 (DQC)
790
4.1
–
-1.3
M16–1
3.00 (LLOQ)
3.26
8.2
15.8
8.6
9.00 (LQC)
9.76
4.5
9.3
8.5
90.0 (MQC)
91.2
4.4
10.9
1.3
720 (HQC)
764
3.7
1.9
6.1
3600 (DQC)
3295
4.8
–
-8.5
M16–2
3.00 (LLOQ)
3.21
6.6
14.9
6.9
9.00 (LQC)
8.96
8.8
6.1
-0.5
90.0 (MQC)
93.0
4.2
8.6
3.3
720 (HQC)
757
2.0
4.2
5.1
3600 (DQC)
3527
2.4
–
-2.0
DQC: Diluted QC samples; HQC: High QC samples; LLOQ: Lower limit of quantification samples; LQC: Low QC samples; MQC: Medium QC samples; RE: Relative error; RSD: Relative standard deviation.
was selected for further optimization. Ammonium acetate was used as a buffer to guarantee the reproducibility and robustness of the method. Moreover, a trace amount of formic acid in the mobile phase improved the MS signal for all analytes without compromising the resolution of ornidazole and its glucuronides. Notably, methanol provided higher intensity and better resolution for M16–1 and M16–2 than acetonitrile. Consequently, the mobile phase was finally optimized as methanol/5 mM ammonium acetate/formic acid (45:55:0.055, v/v/v). A stable isotopically labeled IS is generally the first choice to minimize the influence of matrix effects on an LC–MS/MS method, particularly when an ESI ion source is used [35] . Deuterated ISs of ornidazole, M3, and M6 were used to compensate for the matrix effects in the present study. However, the deuterated ISs for M16–1
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Bioanalysis (2014) 6(18)
and M16–2 were very difficult to prepare, so d5-ornidazole, d5-M3, and d5-M6 were all tried as the IS for these metabolites. Since d5-ornidazole provided the best linearity, it was chosen as the IS for the quantification of M16–1 and M16–2 in the current study. A flow velocity gradient was used to shorten the total run time (from 8.0 to 5.0 min), and ultimately the retention times of ornidazole, d5-ornidazole, M3, d5-M3, M6, d5-M6, M16–1, and M16–2 under the current chromatographic conditions were 3.73, 3.65, 2.61, 2.56, 1.64, 1.61, 2.29, and 2.95 min, respectively (Figure 3). Method validation Selectivity Figure 3 shows typical MRM chromatograms of blank
human plasma (A), blank plasma sample only spiked
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• M6: y = 2.79 × 10 -2x − 7.59 × 10-2 (r2 = 0.9987);
with the ISs (B), blank plasma sample spiked with ornidazole, M3, M6, M16–1, and M16–2 at LLOQ concentrations and the ISs (C), and a plasma sample obtained at 36 h after oral administration of 1000 mg ornidazole to a volunteer (D). As shown in the figure, there was no evidence of significant endogenous interference at the retention times of the analytes and the ISs in blank human plasma.
• M16–1: y = 7.73 × 10 -5x + 1.36 × 10 -5 (r2 = 0.9951); • M16–2: y = 6.92 × 10 -5x + 6.17 × 10 -5 (r2 = 0.9938). where y is the peak area ratio of analyte to IS and x is the plasma concentration of analyte. The LLOQ was established as 100, 10.0, 5.00, 3.00, 3.00 ng/ml for ornidazole, M3, M6, M16–1, and M16–2, respectively. The RSD values of precision at LLOQ ranged from 4.1 to 15.8% for the five analytes, and the RE values of accuracy were between 5.3 and 8.6% (Table 2) . Based on the S/N of 3, the LOD was estimated as 0.400, 0.333, 0.333, 1.00, and 1.00 ng/ml for ornidazole, M3, M6, M16–1, and M16–2, respectively.
Linearity & LLOQ
Calibration curves were best fitted to linear regression analysis with a weighted factor (1/x 2), and were established in the range of 100–30,000 ng/ml for ornidazole, 10.0–3000 ng/ml for M3, 5.00–200 ng/ml for M6, and 3.00–900 ng/ml for M16–1 and M16–2 in plasma. The linearity results complied with the predefined acceptance criteria. The typical equations of the calibration curves were as follows:
Precision & accuracy
• Ornidazole: y = 1.28 × 10 -3 x − 4.71 × 10 -4 (r2 = 0.9992);
The intra- and inter-assay precision and accuracy values for all analytes at three QC levels (LQC, MQC, and HQC) are summarized in Table 2. The RSD values of intra- and inter-assay precision were less than 8.8
• M3: y = 5.67 × 10 -3x + 1.80 × 10 -3 (r2 = 0.9963);
Table 3. Matrix effects and recoveries of ornidazole, M3, M6, d5 -ornidazole (internal standard for ornidazole, M16–1, and M16–2), d5 -M3 (internal standard for M3), and d5 -M6 (internal standard for M6). Analyte
Concentration (ng/ml)
Recovery
Matrix effect
Mean (%)
RSD (%)
Mean (%)
RSD (%)
Ornidazole
300 (LQC)
96.0
0.2
99.2
2.7
3000 (MQC)
99.9
0.8
–
–
24,000 (HQC)
99.4
0.5
98.7
0.4
M3
30.0 (LQC)
97.3
2.8
99.4
1.8
900 (MQC)
101
5.2
–
–
2700 (HQC)
108
0.5
100
1.0
M6
12.0 (LQC)
98.6
1.3
100
0.7
30.0 (MQC)
98.7
5.5
–
–
160 (HQC)
97.4
4.4
99.4
1.1
M16–1
9.00 (LQC)
99.1
2.3
103
13.5
90.0 (MQC)
98.5
4.5
–
–
720 (HQC)
103
3.7
104
1.8
M16–2
9.00 (LQC)
100
9.4
103
10.2
90.0 (MQC)
94.6
5.4
–
–
720 (HQC)
99.2
2.1
101
1.5
d5 -Ornidazole
1000
100
1.2
101
1.1
d5 -M3
500
99.5
1.4
100
1.0
d5 -M6
100
102
0.8
101
1.1
HQC: High QC samples; LQC: Low QC samples; MQC: Medium QC samples; RSD: Relative standard deviation.
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Table 4. Stability data of ornidazole, M3, M6, M16–1, and M16–2 in human plasma and blood exposed to various storage conditions (n = 3). Storage conditions
Concentration
Ornidazole
M3
M6
M16–1
M16–2
RSD (%)
RE (%)
RSD (%)
RE (%)
RSD (%)
RE (%)
RSD (%)
RE (%)
RSD (%)
RE (%)
Short-term
LQC
2.1
4.4
1.4
6.5
0.3
6.8
9.4
-0.4
4.4
2.9
(RT 6 h)
HQC
0.4
4.2
2.4
4.7
0.3
7.2
1.5
1.4
0.9
3.5
Post-preparative
LQC
1.1
4.3
1.3
5.6
0.7
6.9
5.8
-0.7
8.3
-1.3
(RT 24 h)
HQC
0.6
4.0
1.8
6.4
2.8
7.2
2.5
4.3
1.2
4.4
3 freeze–thaw
LQC
3.0
3.9
1.6
9.8
1.0
5.4
5.3
1.9
7.4
0.7
(-80°C to RT)
HQC
0.6
3.6
0.9
7.1
0.7
8.6
2.3
5.5
1.1
5.2
Long-term
LQC
3.2
-1.8
3.4
-1.5
3.2
-4.1
4.7
-4.0
4.3
-0.9
(-80°C 61 days)
HQC
1.0
-4.7
1.0
-3.5
3.3
-3.2
1.5
-4.7
1.2
-1.0
Whole blood
LQC
2.8
2.0
0.4
0.4
1.6
1.6
8.0
-1.9
3.8
-2.5
(RT 2 h)
HQC
1.7
0.7
1.6
0.3
1.1
0.6
2.7
1.3
3.1
1.6
HQC: High QC samples; LQC: Low QC samples; RE: Relative error; RSD: Relative standard deviation; RT: Room temperature.
and 10.9% for all analytes, respectively. The RE values of mean accuracy ranged from -0.5 to 9.4%. The RSD values of precision of the diluted QC samples (Table 2) for all analytes were less than 4.8%, and the RE values of accuracy ranged from -8.5% to -1.3%, indicating that a fivefold dilution of human plasma samples containing the analytes above the upper limit of quantification is acceptable.
Recovery & matrix effect
As shown in Table 3, the simple protein precipitation procedure used in the current study achieved good and consistent recovery (∼100%) of ornidazole and its four metabolites in human plasma. The MFs from six different lots of blank plasma ranged from 98.7 to 104% for all compounds. Intersubject variability of the IS-normalized MFs, as
Table 5. Stability data of ornidazole, M3, M6, M16–1, M16–2, and the internal standards in stock solutions under various storage conditions (n = 3). Storage conditions
Analyte
Freshly prepared
After storage
Stock solutions at room temperature for 6 h
Ornidazole
1.5
2.3
1.2
M3
6.7
8.4
-0.8
M6
2.6
1.8
2.2
M16–1
2.7
3.1
1.6
M16–2
2.6
2.7
1.3
d5 -Ornidazole
2.7
3.8
0.5
d5 -M3
8.2
8.9
-1.1
d5 -M6
1.8
0.7
1.7
Ornidazole
0.7
0.8
-2.4
M3
0.2
0.3
-2.4
M6
1.4
0.2
-2.3
M16–1
3.3
0.2
-4.7
M16–2
3.0
1.0
-3.6
d5 -Ornidazole
1.0
0.8
-3.0
d5 -M3
0.8
0.6
-2.2
d5 -M6
2.7
0.6
-1.6
Stock solutions at 4°C for 12 days
RSD (%)
RE (%)
RE: Relative error; RSD: Relative standard deviation.
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Bioanalysis (2014) 6(18)
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Determination of ornidazole & its main metabolites in human plasma
Research Article
Table 6. Precision and accuracy data for analysis of M3, M6, M16–1, and M16–2 in human plasma in the presence of high concentrations of ornidazole with different ratios (n = 6). Analyte
Concentration (ng/ml)
RSD (%)
RE (%)
10.4
2.2
3.9
5.00
5.24
2.6
4.8
3.00
3.19
5.2
6.5
3.00
2.93
4.9
-2.2
Spiked
Calculated
M3 (1:10)
10.0
M6 (1:1000) M16–1 (1:100) M16–2 (1:1000)
RE: Relative error; RSD: Relative standard deviation.
measured by the RSD, was lower than 13.5%. The results showed that under the current LC–MS/MS conditions, the matrix effects of ornidazole, M3, M4, M6, M16–1, and M16–2 were negligible. Stability
The stability tests in the present study were designed to accommodate all the anticipated conditions encountered when dealing with practical clinical samples. The peak of ornidazole was not observed after preparation of the QC samples only spiked with M16–1/ M16–2, indicating that M16–1 and M16–2 exhibited good stability during sample processing. As shown
in Table 4, the results demonstrated good stability of ornidazole and its four metabolites during sample collection, storage, extraction process throughout the current study. A summary of stock solution stability is presented in Table 5. The stock solutions of ornidazole, M3, M6, M16–1, M16–2, and the ISs were stable for 6 h at room temperature and under refrigeration at 4°C for up to 12 days. Interference test
Determination of individual metabolites in human plasma was performed in the presence of high
100,000 Ornidaozle M3 M6
10,000 Plasma concentration (ng/ml)
M16–1 M16–2 1000
100
10
1
0
12
24
36
48
60
72
84
96
Time (h) Figure 4. Typical plasma concentration–time profiles of ornidazole, M3, M6, M16–1, and M16–2 after oral administration of 1000 mg ornidazole to six healthy Chinese volunteers. Error bars represent the standard deviation of the mean concentration (n = 6).
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Table 7. Pharmacokinetic parameters of ornidazole and its main metabolites in plasma after oral administration of 1000 mg ornidazole (mean ± SD; n = 6). Parameters
Ornidazole
M3
M6
M16–1
M16–2
AUC0 –t (ng·h/ml)
323424 ± 52067
52253 ± 29647
6833 ± 1382
22616 ± 4640
4538 ± 1314
AUC0 – ∞ (ng·h/ml)
332948 ± 46529
53513 ± 29725
7206 ± 1020
22885 ± 4482
4791 ± 1216
tmax (h)
1.21 ± 0.68
2.38 ± 2.85
16.0 ± 6.2
3.75 ± 1.94
2.42 ± 1.91
Cmax (ng/ml)
16185 ± 2613
3098 ± 1159
157 ± 17
1008 ± 188
179 ± 80
t1/2 (h)
14.7 ± 0.8
15.1 ± 2.1
15.9 ± 1.9
10.4 ± 0.5
17.6 ± 2.6
AUC0 –t: Area under the plasma concentration–time curve to the last measurable concentration; AUC0 – ∞ : Area under the plasma concentration–time curve to infinity; Cmax: Maximum plasma concentration; t1/2: Elimination half-life; tmax: Time to the maximum plasma concentration.
concentrations of ornidazole. The concentration ratios of metabolites and the parent drug were designed to cover all the anticipated concentration difference during clinical samples analysis. As shown in Table 6, the results indicated that the presence of high concentrations of ornidazole did not interfere with the quantification of its metabolites. Application to a pharmacokinetic study
The validated LC–MS/MS method was successfully applied to the determination of ornidazole, M3, M6, M16–1, and M16–2 in human plasma after oral administration of 1000 mg ornidazole tablets to six healthy Chinese volunteers. The diluted QC validated in this study covered the highest concentration of all clinical samples, and the designed concentration ratios of metabolites and ornidazole in the interference test also covered the concentration difference in all clinical samples. The plasma concentration–time curves of ornidazole and its four metabolites are shown in Figure 4. The pharmacokinetic parameters obtained from all six Chinese volunteer are listed in Table 7. Based on the molar AUC0–96h, the most abundant circulating metabolite was M3, followed by M16–1, M6, and M16–2, which accounted for 19.4, 3.9, 2.3, and 0.8% of the exposure of the parent drug, respectively. Additionally, the exposure of M16–1 was approximately fivefold higher than M16–2, indicating that the glucuronidation of ornidazole is stereoselective in humans, which is consistent with the in vitro study [8] . Incurred sample reanalysis
As shown in Figure 4 & Table 7, ornidazole and its metabolites exhibited different pharmacokinetic profiles (e.g., tmax, t1/2) in humans. Therefore in this study, we chose the samples, which met the criteria of being close to the maximum concentration or near the end of the elimination phase (but ≥3 × LLOQ) for as much analytes as possible, to do the ISR test. For those samples that needed dilution, the same dilution process as the initial assay was preformed to obtained the
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Bioanalysis (2014) 6(18)
ISR results. Due to the small size of the study, only 12 samples were selected for ISR. A total of 100, 91.7, 91.7, 83.3, and 83.3% of the test samples met the predefined acceptance criteria for ornidazole, M3, M6, M16–1, and M16–2, respectively, indicating that the test results were reproducible. Conclusion To the best of our knowledge, this is the first LC−MS/MS method for the simultaneous determination of ornidazole and its main circulating metabolites (M3, M6, M16–1, and M16–2) in human plasma. A chromatographic run time of 5.0 min was achieved using a flow velocity gradient. The concentrations of ornidazole and its metabolites were determined using a simple and rapid protein precipitation with a small amount of plasma (100 μl). Potential interference between ornidazole and metabolites was fully assessed in this study. This method was successfully applied to characterize the pharmacokinetic profiles of ornidazole and its four metabolites after oral administration of 1000 mg ornidazole to six healthy Chinese volunteers. The reproducibility of the pharmacokinetic results was demonstrated through ISR test. For the first time, we demonstrated that M3 is the most abundant metabolite of ornidazole in the human circulation after oral administration. Future perspective A comprehensive understanding of drug metabolic profiles in vivo is essential for bioanalytical scientists to develop a reliable quantitative method. Due to the limitation of research techniques, the metabolic profile of ornidazole in humans was not systematically investigated until recent years. Since conjunction pathways (i.e., glucuronidation, sulfation) played an important role in the elimination of ornidazole (also other 5-nitroimidazole antimicrobial agents), cautions must be taken to investigate the possible interference of those labile metabolites with the parent drug, especially when using a LC−MS/MS method. Though a
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Determination of ornidazole & its main metabolites in human plasma
few LC−MS/MS methods have been published for the quantification of ornidazole and other 5-nitroimidazole antimicrobials in biological samples, the evaluations of potential metabolite back-conversion or interference lacked adequate addressing, which might raise the concerns of over-estimation of the parent drug. In this study, we determined ornidazole, as well as its active metabolites (M3, M6), and two glucuronides in human plasma by LC−MS/MS. The potential interference issues were fully assessed with typical QC samples and avoided by adequate chromatographic separation. The method reproducibility was demonstrated by ISR test. Furthermore, the described method has several benefits, including fast throughput (5.0 min), small sample size requirement (100-μl plasma), ease of sample preparation (protein participation), and high sensitivity. We anticipate that this new method will be a useful tool for studying the disposition and potential drug–drug interactions of ornidazole in humans. In addition, we believe that this method can be extended for quantification of other 5-nitroimidazole antibiotics and in other matrices with little modifications.
Research Article
Key term Drug–drug interaction: A situation in which a drug affects the activity or pharmacokinetics of another drug when both are administered together.
Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
Ethical conduct of research The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.
Executive summary Method development & validation • A simple and rapid LC−MS/MS method was proposed for the simultaneous determination of ornidazole and its main metabolites (M3, M6, M16–1, and M16–2) in human plasma. • All the analytes were extracted from 100 μl plasma by protein precipitation, separated on a Capcell PAK MG C18 column (100 × 4.6 mm, 5 μm) within 5.0 min, and detected by ESI-MS/MS in the positive mode. • The proposed method was validated as per the US FDA and EMA guidelines, and the potential interference issues between ornidazole and metabolites were fully assessed in this study.
Method application • The validated method was successfully applied to a pharmacokinetic study after oral administration of 1000 mg ornidazole to six healthy Chinese volunteers. • The reproducibility of the study data has been demonstrated through incurred sample reanalysis test. • For the first time, we have shown that M3 is the most abundant metabolite of ornidazole in the human circulation after oral administration.
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Excellent review on how to eliminate or reduce matrix effect in an LC–MS/MS assay.
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Bioanalysis (2014) 6(18)
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