- Email: [email protected]
A chiral high-performance liquid chromatography–tandem mass spectrometry method for the stereospecific determination of morinidazole in human plasma
Accepted Manuscript Title: A chiral high-performance liquid chromatography–tandem mass spectrometry method for the stereospecific determination of morinidazole in human plasma Author: Kan Zhong Zhiwei Gao Qin Li Dafang Zhong Xiaoyan Chen PII: DOI: Reference:
S1570-0232(14)00293-1 http://dx.doi.org/doi:10.1016/j.jchromb.2014.04.049 CHROMB 18918
To appear in:
Journal of Chromatography B
Received date: Revised date: Accepted date:
8-2-2014 22-4-2014 28-4-2014
Please cite this article as: K. Zhong, Z. Gao, Q. Li, D. Zhong, X. Chen, A chiral high-performance liquid chromatographyndashtandem mass spectrometry method for the stereospecific determination of morinidazole in human plasma, Journal of Chromatography B (2014), http://dx.doi.org/10.1016/j.jchromb.2014.04.049 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title Page
A chiral high-performance liquid chromatography–tandem mass
ip t
spectrometry method for the stereospecific determination of
us
cr
morinidazole in human plasma
Kan Zhong a, Zhiwei Gao a, Qin Li b, Dafang Zhong a, Xiaoyan Chen a,* Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike
an
a
Road, Shanghai 201203, PR China b
Jiangsu Hansoh Pharmaceutical Co., Ltd, 9 Dongjin Road, Economic & Technical
Ac ce p
te
d
M
Development Zone, Lianyungang, Jiangsu 222069, PR China
*
Corresponding author.
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai 201203, P.R. China Tel/Fax: +86 21 50800738; Email: [email protected]
1
Page 1 of 26
Abstract Morinidazole is a novel 5-nitroimidazole derivative used for the treatment of amoebiasis, trichomoniasis, and anaerobic bacterial infections. Morinidazole possesses a chiral carbon and is clinically administered as a racemate. In the present study, an enantioselective and sensitive liquid chromatography–tandem mass
ip t
spectrometry method of determining morinidazole enantiomers in human plasma was developed and validated to characterize the stereoselective pharmacokinetics. Plasma
cr
samples were processed by liquid–liquid extraction using tert-butyl methyl ether.
Chiral separation was optimized within 8.5 min on a cellulose column using an
us
isocratic mobile phase of methanol/water (80:20, v/v). Detection was using mass spectrometry in multiple reaction monitoring mode, using the transitions of m/z
an
271→144 for morinidazole enantiomers, and m/z 275→148 for d4-morinidazole (internal standard). The calibration curves were linear over 5.00–6000 ng/mL for each
M
enantiomer. The lower limit of quantification for each enantiomer was established at 5.00 ng/mL. Intra- and inter-day precisions were less than 6.4% for each enantiomer
d
in terms of relative standard deviation, and accuracies were between −2.5% and 6.4% in terms of relative error for each enantiomer. No chiral inversion was observed
te
during sample storage, preparation procedure and analysis. Major glucuronide and
Ac ce p
sulfate conjugates were not observed to interfere with the determination of morinidazole enantiomers. The method was applied to study the stereoselective pharmacokinetics of morinidazole in humans. Moderate stereoselectivity was observed in healthy subjects and patients with severe renal impairment.
Keywords: Morinidazole enantiomer ∙ Liquid chromatography–tandem mass
spectrometry ∙ Stereoselective pharmacokinetics ∙ 5-Nitroimidazole antimicrobial drug
2
Page 2 of 26
1. Introduction Metronidazole and tinidazole are 5-nitroimidazole-based agents recommended for the treatment of vaginal trichomoniasis and are the most-prescribed drugs for the
ip t
treatment of giardiasis [1]. However, clinical resistances to these drugs are emerging [2,3]. Thus, the development of new 5-nitroimidazole derivatives as alternative agents
cr
against anaerobic pathogens is needed.
Morinidazole [R,S-1-(2-methyl-5-nitro-1H-imidazol-1-yl)-3-morpholinopropan-
us
2-ol] is a novel 5-nitroimidazole antimicrobial agent that exhibits great antiparasitic activity against trichomoniasis and amoebiasis and produces relatively low toxicity
an
based on in vitro and preclinical studies [4]. Morinidazole is administered as a racemate at a recommended intravenous injection dose of 500 mg. Recent in vitro
M
assays have demonstrated that higher antiparasitic potency is observed in Smorinidazole than in R-morinidazole [5].
Previous studies have shown that after the intravenous administration of 500 mg
d
of racemic morinidazole to healthy human subjects, morinidazole is extensively
te
metabolized before being excreted mainly in the urine [6]. The principal pathways are stereoselective N+-glucuronidation (M8-1/2) and sulfation (M7). The plasma exposure
Ac ce p
of N+-glucuronide from R-morinidazole (M8-2) is sixfold higher than that from S-
morinidazole (M8-1) [6]. Meanwhile, the ratio of S-/R-morinidazole sulfate is approximately 50 according to hepatocyte incubation tests. However, the stereoselective pharmacokinetics of the parent morinidazole remains unclear. Gao et al. [7] reported a liquid chromatography-tandem mass spectrometry (LC-
MS/MS) method for the simultaneous determination of morinidazole and its four major metabolites in human plasma. To the best of our knowledge, no chiral method for the quantification of individual morinidazole enantiomers has been reported. Therefore, an enantioselective method of determining the circulating levels and characterizing the pharmacokinetics of morinidazole enantiomers must be established. In the present study, a novel and sensitive chiral LC–MS/MS method for the quantification of morinidazole enantiomers in human plasma was developed and 3
Page 3 of 26
validated. The method was successfully applied to characterize the pharmacokinetic profiles of morinidazole enantiomers after the intravenous administration of 500 mg of morinidazole racemate to healthy human subjects and patients with severe renal
ip t
impairment. 2. Experimental
cr
2.1 Chemicals and reagents
us
Racemic morinidazole (99.8% purity), R-morinidazole hydrochloride (99.1% purity) and S-morinidazole hydrochloride (98.9% purity) were kindly provided by Jiangsu Hansoh Pharmaceutical Co., Ltd. (Lianyungang, China). The sulfate
an
conjugate of morinidazole (M7, 100% purity), the N+-glucuronide of S-morinidazole (M8-1, 100% purity), and the N+-glucuronide of R-morinidazole (M8-2, 100% purity)
M
were isolated and purified from human urine as previously described [6]. D4-(±)morinidazole (93.8% chemical purity, 100% isotopic purity) as the internal standard
d
(IS) was synthesized in-house according to previously described methods with minor modifications [8,9]. Methanol of HPLC grade was purchased from Sigma (St. Louis,
te
MO, USA). Deionized water was obtained from a Millipore Milli-Q Gradient Water
Ac ce p
Purification System (Molsheim, France). Analytical-grade tert-butyl methyl ether was supplied by Sinopharm Group Chemical Reagent Co. Ltd. (Shanghai, China). 2.2 Instrumentation
A Shimadzu HPLC system consisting of a DGU-20A3 vacuum degasser, a LC-
20AD binary pump, a SIL-20AC autosampler, and a CTO-20A column oven (Shimadzu, Kyoto, Japan) was used. Mass analysis and detection were carried out on an API 4000 triple quadrupole mass spectrometer (Applied Biosystems, Concord, Ontario, Canada) equipped with an electrospray ionization (ESI) source. Data acquisition and processing were performed using Analyst software v1.5.2 (Applied Biosystems). 2.3 LC–MS/MS analytical conditions 4
Page 4 of 26
The chromatographic separation of enantiomers was achieved on Lux Cellulose4 column (250 mm × 4.6 mm; 5 μm particle size, Phenomenex, Torrance, CA, USA). A mixture of methanol/water (80:20, v/v) was used as mobile phase at a flow rate of 1.00 mL/min for the first 4.5 min and then at 0.6 mL/min until the end of the run. The
ip t
total analysis time was 8.5 min. The column temperature was set at 30 °C.
The mass spectrometer was operated in the positive ion mode. The optimized
cr
tuning parameters were as follows: the ion spray voltage, 5500 V; source temperature, 400°C; nebulizer gas (Gas 1), 50 psi; heater gas (Gas 2), 50 psi; curtain gas, 30 psi
us
and collision activated dissociation (CAD) gas, 6 psi. Declustering potential (DP), entrance potential (EP) and collision cell exit potential (CXP) were set at 50, 10 and
an
20 V for each enantiomer and the IS. The optimized multiple reaction monitoring (MRM) fragmentation transitions (Figure 1) were m/z 271 → m/z 144 and m/z 275 →
M
m/z 148 with a collision energy (CE) of 21 eV for morinidazole enantiomers and d4morinidazole enantiomers, respectively. The dwell time for each transition was kept at
d
80 ms.
te
2.4 Preparation of calibration standards and quality control (QC) samples Standard and QC stock solutions of racemic morinidazole were prepared in
Ac ce p
methanol separately to obtain 500 μg/mL for each enantiomer. Standard curve
samples were serially diluted from stock solutions with blank plasma to obtain the concentrations of 5.00, 15.0, 50.0, 150, 500, 2000, and 6000 ng/mL for each enantiomer. The lower limit of quantification (LLOQ) and QC samples were independently prepared in blank plasma at different concentrations (LLOQ, low, medium, and high concentrations at 5.00, 10.0, 500, and 4800 ng/mL for each enantiomer, respectively). The 1.00 mg/mL IS stock solution of racemic d4-
morinidazole was further diluted with 50% aqueous methanol to yield a working solution (100 ng/mL for each enantiomer). All stock solutions were stored at –20°C and working solutions were stored at 4°C and were brought to room temperature before use. Standard-curve samples and QC samples were stored frozen at –20°C until analysis. 5
Page 5 of 26
2.5 Sample preparation To an aliquot of 50 μL of plasma sample, 50 μL of IS working solution (racemic d4-morinidazole, 100 ng/mL for each enantiomer) and 300 μL of water were added.
ip t
The sample was vortex-mixed and extracted with 3 mL of tert-butyl methyl ether by vortexing for 5 min. After centrifuging the samples at 3500 × g for 5 min, the upper organic layers were transferred to another glass tube and evaporated to dryness at 40
cr
°C under a gentle stream of nitrogen in a TurboVap evaporator (Zymark, Hopkinton,
us
MA, USA). The residues were reconstituted in 100 μL of the mobile phase of methanol/water (80: 20, v/v) and an aliquot of 5.0 μL was injected for LC-MS/MS
an
analysis. 2.6 Method validation
M
The assay was validated with respect to selectivity, linearity, precision and accuracy, extract recovery, matrix effects and stabilities in human plasma according to
d
the FDA guidelines on bioanalytical method validation [10]. Selectivity of the method was performed by analyzing six independent sources of
te
blank plasma and the same matrix spiked with analytes at LLOQ level. Samples were
Ac ce p
extracted and analyzed, and the peak areas in blank sample were compared with those of LLOQ samples at the retention times of the enantiomers and their corresponding IS. The acceptance criteria required that the integrated response of co-eluted endogenous matrix components was lower than 20% that of the LLOQ and <5% for the IS. The major metabolites glucuronides (M8-1 and M8-2) and sulfate (M7) at the maximum clinical concentrations were also spiked with blank plasma and the IS to evaluate for potential interference. Calibration standards at eight levels were prepared and analyzed in duplicate for three independent validation days. Calibration curves were obtained by analyzing the peak area ratio of each analyte to the IS (analyte/IS) versus the nominal concentrations in human plasma, using a linearly weighed (1/x2) least squares regression model. A correlation coefficient (r) of 0.995 or greater was accepted for
6
Page 6 of 26
linearity determination. The deviations of back-calculated concentrations should not exceed ±15% of the spiked concentrations, while a deviation of ±20% was permitted for the LLOQ. Intra- and inter-day accuracy and precision were evaluated by replicate analyses
ip t
of six sets of QC samples at low, medium and high levels. Intra-assay data were
determined by comparing data from within run (n = 6) and inter-assay data were
cr
assessed for three consecutive days (n = 18). The values of accuracy and precision
were expressed as relative error (RE, %) and relative standard deviation (RSD, %),
us
respectively. Intra- and inter-day precision values were required to be below 15% and accuracy was required to be within ±15%. The LLOQ was defined as the lowest
an
concentration on the calibration curve with precision not exceeding 20% and accuracy within ±20%. To evaluate the accuracy and precision of different morinidazole
M
enantiomeric ratios, morinidazole enantiomers were combined and diluted with blank plasma to obtain three mixtures of known S/R ratios at 2:1, 4:1, and 8:1 containing R-
d
morinidazole and S-morinidazole at 500 ng/mL total concentration. Samples were
separate days.
te
analyzed using an enantioselective LC-MS/MS system in six replicates for three
Ac ce p
The extraction efficiency of each enantiomer was determined by comparing the mean peak areas of the analyte (A) spiked before extraction (six samples each) with those (B) spiked after extraction. The ratio (A/B × 100) was defined as the extraction efficacy. The extraction recovery of morinidazole enantiomers was carried out at three QC levels. IS recovery was determined using the same method. The matrix effects of each enantiomer and IS were evaluated by preparing two
sets of samples at low and high QC levels, namely, the peak area of plasma extracts from six different lots spiked with analytes after extraction (A) and the peak area of standards in neat solutions at the same concentration (B). The results were expressed as the matrix factor (MF; A/B × 100%). The coefficient of variation of the ISnormalized matrix factors should not be greater than 15%. An MF value of 100% suggested that the signals of plasma samples and solutions were similar and that matrix effect was considered negligible. A value greater than 100% indicated 7
Page 7 of 26
ionization enhancement, whereas a value less than 100% indicated ionization suppression. The stabilities of each enantiomer in human plasma were evaluated in triplicate by analyzing replicates (n = 3) of low- and high-concentrations plasma samples
ip t
exposed to different storage conditions. The stability of extracts in the autosampler
was assessed at 4 °C for 36 h. The short-term bench top stability of morinidazole
cr
enantiomers in plasma over 8 h was determined at ambient temperature (25 °C). The long-term stability was evaluated after storage of the standard spiked plasma samples
us
at –20 °C for 56 days. We also assessed the stabilities of plasma samples after three
within ± 15% of the nominal concentration.
an
freeze–thaw cycles. The samples were considered stable when the assay RE was
To evaluate the enantiomeric stability of R-morinidazole or S-morinidazole in
M
human plasma, spiked plasma samples were prepared in triplicate at 500 ng/mL for each enantiomer and incubated at 37 °C for 12 h. The samples were then extracted
d
and analyzed as previously described. Chiral inversion was evaluated by detecting the
te
appearance of the other enantiomer in the samples. 2.7 Stereoselective pharmacokinetic studies
Ac ce p
Pharmacokinetic studies were approved by the Ethics Committees and conducted
in accordance with the Declaration of Helsinki and the principles of Good Clinical Practice. Four healthy male Chinese volunteers and four patients with severe renal impairment were included in the study. Written informed consent was obtained from all subjects before the study. Venous blood samples (4 mL) were collected in heparincontaining tubes after a continuous infusion injection for 45 min of 500 mg of racemic morinidazole at 0 (pre-dose), 0.375, 0.75, 1.25, 1.5, 1.75, 2.75, 4.75, 6.75, 8.75, 12.75, 24.75, 36.75, and 48.75 h after the start of infusion. Plasma was obtained after centrifugation of the blood samples and stored at −20°C until analysis. The pharmacokinetic parameters were calculated based on the plasma concentration versus time curves by non-compartmental analysis using the WinNolin program, V5.3 (Pharsight Corporation, Mountain View, CA, USA). All parameters were expressed as 8
Page 8 of 26
the mean ± standard deviation. 3. Results and discussion 3.1 Mass spectrometry and enantioselective chromatography conditions
ip t
Our preliminary study showed that ESI could offer higher intensity for
morinidazole enantiomers than atmospheric pressure chemical ionization (APCI) (>
cr
15-fold). Accordingly, ESI was used for method development in the present work. The
us
MS conditions were in a previously described method of our group, with minor modifications to achieve the highest response under the present chromatography conditions [7]. The stable isotope labeled as d4-(±)-morinidazole was synthesized in
an
house and chosen as the IS because of its similarity to morinidazole in terms of chromatographic behavior, extraction recovery and ionization efficiency.
M
Chiral HPLC separation based on various chiral stationary phases (CSPs) coupled with MS detection are high sensitive and selective, thus, this technique is
d
widely applied in enantioselective pharmacokinetics. The properties of CSPs and the compatibility of the mobile phase with the LC-MS/MS interface should first be
te
considered. In the early stage of method development, several types of CSP columns
Ac ce p
were attempted, including macrocyclic glycopeptide-based (Chirobiotic T and V2), polysaccharide-based (Chiralpak AS-RH and Lux cellulose-4), and protein-based (chiral-AGP and Ultron ES-OVM) columns. No obvious resolution was observed for morinidazole enantiomers on the macrocyclic glycopeptide-based, AS-RH and ESOVM columns, although various organic modifiers (acetonitrile, methanol, and 2propanol) and water with or without typical modifiers and buffers (ammonium formate and ammonium acetate) at different pH values were tested. Fortunately, sufficient separation (RS = 1.7) and suitable retention time (Rt = 5.6 and 6.6 min) were achieved from the AGP column, when the mobile phase was 100% aqueous phase containing 20 mM ammonium formate at a flow rate of 0.6 mL/min. However, poor ionization efficiency was observed under this chromatographic condition, leading to low MS response at LLOQ level (5.00 ng/mL). To enhance the signal intensities in
9
Page 9 of 26
positive ionization mode, further optimization of increasing organic modifiers (methanol, acetonitrile or 2-propanol) proportion to 1% or adding 0.005% acetic acid or formic acid was attempted, but all resulted in poor resolution. Adjustment of the organic-phase proportion enabled the peak resolution to reach complete separation (RS
ip t
= 3.8) with high signal intensity on Lux cellulose-4 column using the mobile phase of
methanol/water (80:20, v/v). However, a retention time drift was observed after
cr
multiple injections. With increased column temperature from 20 °C to 30 °C, the
reproducibility of retention time and the peak symmetry were improved. Further
us
optimization of using 5 mM ammonium acetate as a substitute for water was tried, but MS signal intensity was found to decrease. Although good resolution and sensitivity
an
were obtained from the Lux cellulose-4 column with methanol/water (80:20, v/v) as mobile phase, the analytical time (12.5 min) was slightly long for bioanalysis. To
M
shorten the total run time, a flow gradient was used and the analytical time was reduced to 8.5 min. The elution order on the Lux cellulose-4 column was R-
d
morinidazole (5.8 min) followed by S-morinidazole (7.5 min). The chiral recognition mechanism of polysaccharide phases is generally
te
considered to be due to differences in the combination of interaction forces such as
Ac ce p
hydrogen bonding, hydrophobic interactions, π-π interactions and dipole-dipole interactions between the two enantiomers and CSP [11]. Regarding CSPs based on phenylcarbamate derivatives, hydrogen bonding to the carbamate group is considered to be the strongest attractive interaction [12]. 5-Nitroimidazole chiral drugs usually contain a hydroxyl group at the chiral center, a nitro group and an imidazole group in their structure. The hydroxyl group at the chiral center is pivotal for chiral resolution and interaction with the polar carbamate moiety of CSP through hydrogen bonding with –NH group. The nitro and imidazole groups could also interact with CSP through hydrogen bonding and dipole–dipole interaction that is inherent in the polar carbamate group. In addition, phenyl groups in CSP may have π-π stacking interactions with the imidazole group of enantiomers. In particular, the morinidazole structure consists of a morpholine ring, and may greatly strengthen the hydrogen binding force between the enantiomers and CSP. Therefore, the specific configuration 10
Page 10 of 26
of
the
Lux
cellulose-4
stationary
phase
[cellulose
tris(4-chloro-3-
methylphenylcarbamate)] can be speculated to provide suitable sites for morinidazole enantiomers and make them interact with CSP with different affinities, leading to different retentions. As analogs of morinidazole, our pilot studies showed that
ip t
ornidazole and secnidazole enantiomers could also be baseline resolved on Lux celluse-4 column (data not shown). Thus, the Lux celluse-4 column may have
cr
implications in the stereoselective pharmacokinetics of other 5-nitroimidazole
us
derivatives. 3.2 Sample preparation
an
Considering that liquid–liquid extraction (LLE) could give cleaner extracts and less signal suppression or enhancement especially with an ESI ionization source [13],
M
we performed LLE as our first attempt. After extraction with ethyl acetate, tert-butyl methyl ether or ethyl ether–dichloromethane (2:1, v/v), high response and less matrix interference were obtained with tert-butyl methyl ether. Although a pilot study
d
indicated that ethyl acetate had the highest extract efficiency for morinidazole
te
enantiomers (up to 90%), signal response declined during the repeated injections, which was presumably due to the interference of endogenous matrix extracted.
Ac ce p
Morinidazole is a weakly basic compound; thus, conducting the extraction after plasma pH adjustment was evaluated. However, compared with the response of the sample extracted at pH 7, no significant difference was observed in extraction efficacy.
3.3 Method validation
3.3.1 Assay selectivity. For evaluation of the selectivity of the method, six different batches of blank human plasma and the corresponding spiked plasma at LLOQ level were analyzed for peak inference. Figure 2 shows the typical chromatograms included blank plasma sample, blank plasma sample spiked only with R-d4-morinidazole and S-d4-
11
Page 11 of 26
morinidazole, blank plasma sample with R-morinidazole and S-morinidazole at the LLOQ and IS, and plasma sample obtained 2.75 h after infusion injection of 500 mg of racemic morinidazole to a healthy volunteer. No interfering matrix peak was observed at the retention time of enantiomers of the analyte and IS. The possibilities
ip t
of back-conversion of sulfate conjugate M7 and glucuronide conjugates M8-1 and M8-2 were evaluated at 100, 250, and 2000 ng/mL, respectively. No back-conversion
cr
was observed, suggesting that metabolites did not interfere with the determination of
the parent drug, including the extraction procedures or in the chromatographic and
us
MS mode used. The chromatographs proved that the method was selective for analyzing morinidazole enantiomers.
an
3.3.2 Linearity and LLOQ.
The calibration curves were linear over the concentration range of 5.00–6000
M
ng/mL for each enantiomer in human plasma. The mean (± SD) regression equations from calibration curves on different days were as follows: R-morinidazole: y = (0.0108 0.0002)x + (0.0023 0.0019), r2 = 0.9991 0.0002
d
S-morinidazole: y = (0.0110 0.0002)x + (0.0049 0.0012), r2 = 0.9995 0.0002
Ac ce p
concentration.
te
where y is the peak-area ratio of each enantiomer to IS and x is the nominal plasma
The LLOQ was 5.00 ng/mL for the determination of morinidazole enantiomers
in plasma and the signal-to-noise ratio of LLOQ was >10. The values of intra- and inter-day at LLOQ precision and accuracy are shown in Table 1, and these values were accepted with RSD below 6.4% and RE within 2.8%. With the present LLOQ, the concentrations could be determined up to more than six half-life for R-
morinidazole and S-morinidazole, suggesting that the method was sensitive for investigating the stereoselective pharmacokinetics of morinidazole after an intravenous administration of 500 mg of racemic morinidazole. 3.3.3 Precision and accuracy. Table 1 summarizes the intra- and inter-day precision and accuracy for morinidazole enantiomers from QC samples. The intra-day precision by calculating RSD was 3.4% or less for R-morinidazole and 2.9% or less for S-morinidazole, and 12
Page 12 of 26
the inter-day precision was 4.0% or less for R-morinidazole and 4.1% or less for Smorinidazole. The accuracy in terms of RE ranged from 0.7% to 6.4% for Rmorinidazole and from 0.2% to 4.6% for S-morinidazole at all QC levels. The precision and accuracy for morinidazole enantiomeric ratios are also presented in
ip t
Table 2. The intra-day and inter-day precision were less than 2.2% and 1.2%. The accuracy ranged from 0.3% to 4.5% (RE). Figure 3 presents the typical
cr
chromatograms of R-morinidazole and S-morinidazole from QC samples with different enantiomeric ratios at a total morinidazole plasma concentration of 500
us
ng/mL. 3.3.4 Extraction recovery and matrix effect.
an
Mean extraction recoveries of R-morinidazole were 60.0 ± 4.9%, 56.1 ± 5.2% and 58.5 ± 4.4% at the concentrations of 10.0, 500 and 4800 ng/mL, respectively. The
M
recoveries of S-morinidazole were 63.6 ± 3.4%, 57.8 ± 5.4%, and 58.5 ± 3.9%, respectively. The recoveries of R-d4-morinidzole and S-d4-morinidazole were 55.8 ±
d
7.7% and 56.7 ± 7.8%. The precision (RSD) values of recoveries for the analytes and IS were <10.0%. The recoveries were consistent and reproducible.
te
The matrix factors at low and high concentration levels ranged within 96.9–
Ac ce p
98.9% for R-morinidazole and 101–102% for S-morinidazole, and the deviations from six batches of plasma were less than 3.8%. Obviously, ion suppression or enhancement caused by plasma matrix was negligible for the determination of morinidazole enantiomers under the present conditions. 3.3.5 Stability.
The stabilities of racemic morinidazole and major conjugated metabolites have
been evaluated in a previous study [7]. In the present study, the enantiomers were found to be stable under the following conditions: in plasma at room temperature for 8 h, in autosampler for 36 h post-extraction, in plasma after three freeze−thaw cycles, and in plasma at −20 °C for 56 days. The signal of the other enantiomer was not
detected after incubation of plasma samples containing one enantiomer at 37 °C for 12 h, suggesting that chiral inversion did not occur between R- and S-morinidazole during storage, sample preparation procedure and analysis. 13
Page 13 of 26
3.4 Method application The developed chiral LC–MS/MS method was successfully applied to the determination of plasma concentrations of morinidazole enantiomers in healthy
ip t
human subjects and patients with severe renal impairment after intravenous administration of 500 mg of racemic morinidazole. Figure 4 shows the mean plasma
cr
concentration–time profiles of morinidazole enantiomers. Table 4 lists the major pharmacokinetic parameters of R-morinidazole and S-morinidazole in humans.
us
After an intravenous administration of 500 mg of morinidazole racemate to healthy subjects, significant difference was observed between the pharmacokinetics of
an
the two enantiomers, with S-morinidazole providing slightly higher exposure than the R-enantiomer, i.e., the average AUC0-t ratio of the S-form to R-form was 1.71. The mean S/R plasma concentration ratio values gradually increased to 12 in healthy
M
subjects from the start of infusion time to 36.75 h. Mean elimination half-life (t1/2) of S-morinidazole was 1.6 times as long as R-morinidazole. The total plasma clearance
d
(CL) of R-morinidazole was higher than that of its antipode, and the CL ratio value
te
between two enantiomers was 1.72. Based on these findings, we speculated that the higher exposure of S-morinidazole was due to a lower clearance.
Ac ce p
Meanwhile, we determined the plasma concentrations of the main metabolites of
M7, M8-1, and M8-2 in these four healthy human subjects using the method previously described by Gao et al [7]. If the amount ratio of S-/R-morinidazole sulfate
in hepatocyte incubation was representative of that in plasma, we speculated that the sulfate conjugate M7 was mainly from S-morinidazole. Therefore, the sum of AUC values of S-morinidazole, glucuronide conjugate M8-1, and sulfate M7 was calculated (based on molar concentrations), and this sum was 1.28-fold higher than the sum of R-
morinidazole and its glucuronide conjugate M8-2. The ratio value of S-form to Rform drastically decreased, suggesting that the stereoselective metabolism of morinidazole was the key determinant for the stereoselective pharmacokinetics. Morinidazole and its main metabolites are eliminated primarily through kidney, with the excreted amount accounting for approximately 71% of the dose over the 0– 14
Page 14 of 26
36 h period [6]. Thus, the effect of renal function on the pharmacokinetics of morinidazole enantiomers was evaluated. A slight increase in systematic exposure of each enantiomer in patients with severe renal impairment was observed. The AUC values of R-morinidazole and S-morinidazole were 37% and 29% higher than those in
ip t
healthy subjects, respectively. However, the enantiomeric ratio of AUC (S to R) was not influenced by renal function, with a value of 1.60 in patients with severe renal
cr
impairment.
us
4. Conclusions
An enantioselective LC-MS/MS method determining morinidazole enantiomers
an
in human plasma using a deuterated IS by combining separation on a cellulose chiral stationary phase column was developed and validated for the first time. The LLOQ
M
was 5.00 ng/mL both for R-morinidazole and S-morinidazole using 50 μL of the plasma sample. No interference derived from the morinidazole conjugated metabolites was observed. Under the present conditions, no retention time drift and signal
d
intensity attenuation were found after about 100 injections in one analytical run. The
te
validated method was successfully applied to characterize the pharmacokinetic profiles of morinidazole enantiomers in healthy volunteers and patients with severe
Ac ce p
renal impairment after administration of therapeutic doses of racemic morinidazole, and the results verified the stereoselective pharmacokinetics of morinidazole in humans. These findings can help elucidate the stereoselective pharmacokinetics and the development of the single enantiomer of morinidazole.
15
Page 15 of 26
References [1] J.D. Sobel, P. Nyirjesy, W. Brown, Clin Infect Dis, 33 (2001) 1341-1346.
ip t
[2] J.A. Upcroft, L.A. Dunn, J.M. Wright, K. Benakli, P. Upcroft, P. Vanelle, Antimicrob Agents Chemother, 50 (2006) 344-347.
4210.
us
[4] A. Lv, China Patent CN 1981764A, (2007) 636-643.
cr
[3] J.R. Schwebke, F.J. Barrientes, Antimicrob Agents Chemother, 50 (2006) 4209-
[5] C. Zhang, X. Tao, Z. Teng, China Patent CN 1850086A, (2006).
(2012) 556-567.
an
[6] R. Gao, L. Li, C. Xie, X. Diao, D. Zhong, X. Chen, Drug Metab Dispos, 40
[7] R. Gao, D. Zhong, K. Liu, Y. Xia, R. Shi, H. Li, X. Chen, J Chromatogr B Analyt
M
Technol Biomed Life Sci, 908 (2012) 52-58.
[8] J. Liu, X. Tang, S.L. Harbeson, C.E. Masse, US Patent No. 0,160,247 (2010).
d
[9] S. Khaksar, A. Heydari, M. Tajbakhsh, H.R. Bijanzadeh, Journal of fluorine
te
chemistry, 131 (2010) 106-110.
[10] USFDA, Guidance for Industry: Bioanalytical Method Validation, (2001)
Ac ce p
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/ Guidances/ucm070107.pdf.
[11] P. Wang, S. Jiang, D. Liu, P. Wang, Z. Zhou, J Biochem Biophys Methods, 62 (2005) 219-230.
[12] D.T. Witte, J.P. Franke, F.J. Bruggeman, D. Dijkstra, R.A. De Zeeuw, Chirality, 4 (1992) 389-394.
[13] M. Jemal, Z. Ouyang, Y.Q. Xia, Biomed Chromatogr, 24 (2010) 2-19.
16
Page 16 of 26
Legends Fig. 1 Product ion spectra of [M + H]+ of morinidazole (A) and d4-morinidazole (B)
ip t
Fig. 2 Typical enantioselective MRM chromatograms of R-morinidazole (I), Smorinidazole (II), R-d4-morinidazole (III), and S-d4-morinidazole (IV) in human
plasma: (A) blank plasma sample; (B) plasma spiked with R-d4-morinidazole (100
cr
ng/mL) and S-d4-morinidazole (100 ng/mL); (C) plasma spiked with R-morinidazole
(5.00 ng/mL), S-morinidazole (5.00 ng/mL), R-d4-morinidazole (100 ng/mL) and S-
us
d4-morinidazole (100 ng/mL); and (D) plasma sample 2.75 h after a continuous
an
infusion injection of 500 mg of racemic morinidazole to a healthy volunteer
Fig. 3 Typical chromatograms of R-morinidazole and S-morinidazole from different enantiomeric ratios (total plasma concentration = 500 ng/mL): (A) S/R = 2:1; (B) S/R
M
= 4:1; and (C) S/R = 8:1. Peaks I, II, III and IV refer to R-morinidazole, S-
d
morinidazole, R-d4-morinidazole, and S-d4-morinidazole, respectively
te
Fig. 4 Plasma concentration versus time profiles (A) and semi-log scale plots (B) of R-morinidazole and S-morinidazole after a continuous infusion injection of 500 mg
Ac ce p
of racemic morinidazole to healthy volunteers and patients with severe renal impairment
18
Page 17 of 26
*Highlights (for review)
Research highlights: Baseline separation of morinidazole enantiomers was achieved on a cellulose chiral stationary phase column using a high proportion of organic phase. D4-morinidazole was synthesized as the internal standard to compensate for matrix effects and variables during sample preparation. The analysis time was short by using a flow rate gradient.
ip t
The method shows advantages of high selectivity and reproducibility.
The method was successfully applied to clinical stereoselective pharmacokinetics
Ac
ce pt
ed
M
an
us
cr
study of morinidazole.
Page 18 of 26
Ac
ce
pt
ed
M
an
us
cr
i
Figure 1
Page 19 of 26
Ac
ce
pt
ed
M
an
us
cr
i
Figure 2
Page 20 of 26
Ac
ce
pt
ed
M
an
us
cr
i
Figure 3
Page 21 of 26
Ac ce p
te
d
M
an
us
cr
ip t
Figure 4
Page 22 of 26
Tables
Table 1 Precision and accuracy data of the analysis of R-morinidazole, and S-morinidazole in
Added
Found
Intra-day Inter-day
5.00
5.14
5.1
4.0
10.0
10.6
3.4
4.0
500
503
1.0
2.3
0.7
4800
4835
2.1
2.1
0.7
5.00
4.87
6.4
3.7
-2.5
10.0
10.5
2.9
3.7
4.6
500
501
1.2
2.5
0.2
4800
4893
4.1
1.9
2.5
ip t
RE (%)
2.8 6.4
Ac
ce pt
ed
M
S-morinidazole
RSD (%)
cr
R-morinidazole
Concentration (ng/mL)
an
Analyte
us
human plasma (3 days with 6 replicates per day).
Page 23 of 26
Table 2 Precision and accuracy data of the analysis of morinidazole enantiomeric ratios in human plasma (3 days with 6 replicates per day).
Found 2.01 4.05 8.36
Intra-day 2.2 1.7 1.0
RE (%) Inter-day 1.0 1.2 0.6
0.3 1.2 4.5
Ac
ce pt
ed
M
an
us
cr
Added 2.00 4.00 8.00
RSD (%)
ip t
S/R ratios
Page 24 of 26
ip t cr us
Table 3
RSD (%) 3.0 0.7 3.3 3.6
Long-term (−20◦C for 56 days)
RSD (%) 3.2 4.7 3.3 3.6
RSD (%) 4.3 1.4 2.1 1.0
RSD (%)
RE (%)
9.50 3.50 4.10 1.50
0.6 -5.7 5.1 -2.9
M
RE (%) 11.2 3.4 6.7 1.6
d
S-morinidazole
(ng/mL) 10.0 4800 10.0 4800
3 Freeze–thaw (−20◦C to room temperature)
RE (%) 5.9 -0.8 1.9 1.6
RE (%) 3.9 0.3 1.0 2.9
ep te
R-morinidazole
Concentration
Autosampler for 36 h (4°C)
Ac c
Analyte
Short-term (room temperature for 8 h)
an
Stability of R-morinidazole, and S-morinidazole in human plasma under various storage conditions (n = 3).
Page 25 of 26
ip t cr us
Table 4
an
Mean pharmacokinetic parameters of R-morinidazole, and S-morinidazole after administration of 500 mg of racemic morinidazole by continuous intravenous infusion for 45 min to four healthy subjects and four patients with severe renal impairment.
T1/2 (h)
M
Analyte
AUC0-t (h*ng/mL)
CL (mL/h)
3.9 ± 0.2
0.7 ± 0.2
4975 ± 458
23751 ± 1715
10522 ± 724
S-morinidazole
6.4 ± 0.2
0.7 ± 0.2
5300 ± 556
40590 ± 1394
6128 ± 210
R-morinidazole
5.5 ± 1.7
0.9 ± 0.3
4503 ± 801
32671 ±8326
8140 ± 2763
S-morinidazole
7.8 ± 1.6
1.0 ± 0.3
4930 ± 856
52243 ± 7142
4782 ± 702
Ac c
ep te
Severe renal impairment
Cmax (ng/mL)
R-morinidazole
d
Healthy subjects
Tmax (h)
Page 26 of 26
S1570-0232(14)00293-1 http://dx.doi.org/doi:10.1016/j.jchromb.2014.04.049 CHROMB 18918
To appear in:
Journal of Chromatography B
Received date: Revised date: Accepted date:
8-2-2014 22-4-2014 28-4-2014
Please cite this article as: K. Zhong, Z. Gao, Q. Li, D. Zhong, X. Chen, A chiral high-performance liquid chromatographyndashtandem mass spectrometry method for the stereospecific determination of morinidazole in human plasma, Journal of Chromatography B (2014), http://dx.doi.org/10.1016/j.jchromb.2014.04.049 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title Page
A chiral high-performance liquid chromatography–tandem mass
ip t
spectrometry method for the stereospecific determination of
us
cr
morinidazole in human plasma
Kan Zhong a, Zhiwei Gao a, Qin Li b, Dafang Zhong a, Xiaoyan Chen a,* Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike
an
a
Road, Shanghai 201203, PR China b
Jiangsu Hansoh Pharmaceutical Co., Ltd, 9 Dongjin Road, Economic & Technical
Ac ce p
te
d
M
Development Zone, Lianyungang, Jiangsu 222069, PR China
*
Corresponding author.
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai 201203, P.R. China Tel/Fax: +86 21 50800738; Email: [email protected]
1
Page 1 of 26
Abstract Morinidazole is a novel 5-nitroimidazole derivative used for the treatment of amoebiasis, trichomoniasis, and anaerobic bacterial infections. Morinidazole possesses a chiral carbon and is clinically administered as a racemate. In the present study, an enantioselective and sensitive liquid chromatography–tandem mass
ip t
spectrometry method of determining morinidazole enantiomers in human plasma was developed and validated to characterize the stereoselective pharmacokinetics. Plasma
cr
samples were processed by liquid–liquid extraction using tert-butyl methyl ether.
Chiral separation was optimized within 8.5 min on a cellulose column using an
us
isocratic mobile phase of methanol/water (80:20, v/v). Detection was using mass spectrometry in multiple reaction monitoring mode, using the transitions of m/z
an
271→144 for morinidazole enantiomers, and m/z 275→148 for d4-morinidazole (internal standard). The calibration curves were linear over 5.00–6000 ng/mL for each
M
enantiomer. The lower limit of quantification for each enantiomer was established at 5.00 ng/mL. Intra- and inter-day precisions were less than 6.4% for each enantiomer
d
in terms of relative standard deviation, and accuracies were between −2.5% and 6.4% in terms of relative error for each enantiomer. No chiral inversion was observed
te
during sample storage, preparation procedure and analysis. Major glucuronide and
Ac ce p
sulfate conjugates were not observed to interfere with the determination of morinidazole enantiomers. The method was applied to study the stereoselective pharmacokinetics of morinidazole in humans. Moderate stereoselectivity was observed in healthy subjects and patients with severe renal impairment.
Keywords: Morinidazole enantiomer ∙ Liquid chromatography–tandem mass
spectrometry ∙ Stereoselective pharmacokinetics ∙ 5-Nitroimidazole antimicrobial drug
2
Page 2 of 26
1. Introduction Metronidazole and tinidazole are 5-nitroimidazole-based agents recommended for the treatment of vaginal trichomoniasis and are the most-prescribed drugs for the
ip t
treatment of giardiasis [1]. However, clinical resistances to these drugs are emerging [2,3]. Thus, the development of new 5-nitroimidazole derivatives as alternative agents
cr
against anaerobic pathogens is needed.
Morinidazole [R,S-1-(2-methyl-5-nitro-1H-imidazol-1-yl)-3-morpholinopropan-
us
2-ol] is a novel 5-nitroimidazole antimicrobial agent that exhibits great antiparasitic activity against trichomoniasis and amoebiasis and produces relatively low toxicity
an
based on in vitro and preclinical studies [4]. Morinidazole is administered as a racemate at a recommended intravenous injection dose of 500 mg. Recent in vitro
M
assays have demonstrated that higher antiparasitic potency is observed in Smorinidazole than in R-morinidazole [5].
Previous studies have shown that after the intravenous administration of 500 mg
d
of racemic morinidazole to healthy human subjects, morinidazole is extensively
te
metabolized before being excreted mainly in the urine [6]. The principal pathways are stereoselective N+-glucuronidation (M8-1/2) and sulfation (M7). The plasma exposure
Ac ce p
of N+-glucuronide from R-morinidazole (M8-2) is sixfold higher than that from S-
morinidazole (M8-1) [6]. Meanwhile, the ratio of S-/R-morinidazole sulfate is approximately 50 according to hepatocyte incubation tests. However, the stereoselective pharmacokinetics of the parent morinidazole remains unclear. Gao et al. [7] reported a liquid chromatography-tandem mass spectrometry (LC-
MS/MS) method for the simultaneous determination of morinidazole and its four major metabolites in human plasma. To the best of our knowledge, no chiral method for the quantification of individual morinidazole enantiomers has been reported. Therefore, an enantioselective method of determining the circulating levels and characterizing the pharmacokinetics of morinidazole enantiomers must be established. In the present study, a novel and sensitive chiral LC–MS/MS method for the quantification of morinidazole enantiomers in human plasma was developed and 3
Page 3 of 26
validated. The method was successfully applied to characterize the pharmacokinetic profiles of morinidazole enantiomers after the intravenous administration of 500 mg of morinidazole racemate to healthy human subjects and patients with severe renal
ip t
impairment. 2. Experimental
cr
2.1 Chemicals and reagents
us
Racemic morinidazole (99.8% purity), R-morinidazole hydrochloride (99.1% purity) and S-morinidazole hydrochloride (98.9% purity) were kindly provided by Jiangsu Hansoh Pharmaceutical Co., Ltd. (Lianyungang, China). The sulfate
an
conjugate of morinidazole (M7, 100% purity), the N+-glucuronide of S-morinidazole (M8-1, 100% purity), and the N+-glucuronide of R-morinidazole (M8-2, 100% purity)
M
were isolated and purified from human urine as previously described [6]. D4-(±)morinidazole (93.8% chemical purity, 100% isotopic purity) as the internal standard
d
(IS) was synthesized in-house according to previously described methods with minor modifications [8,9]. Methanol of HPLC grade was purchased from Sigma (St. Louis,
te
MO, USA). Deionized water was obtained from a Millipore Milli-Q Gradient Water
Ac ce p
Purification System (Molsheim, France). Analytical-grade tert-butyl methyl ether was supplied by Sinopharm Group Chemical Reagent Co. Ltd. (Shanghai, China). 2.2 Instrumentation
A Shimadzu HPLC system consisting of a DGU-20A3 vacuum degasser, a LC-
20AD binary pump, a SIL-20AC autosampler, and a CTO-20A column oven (Shimadzu, Kyoto, Japan) was used. Mass analysis and detection were carried out on an API 4000 triple quadrupole mass spectrometer (Applied Biosystems, Concord, Ontario, Canada) equipped with an electrospray ionization (ESI) source. Data acquisition and processing were performed using Analyst software v1.5.2 (Applied Biosystems). 2.3 LC–MS/MS analytical conditions 4
Page 4 of 26
The chromatographic separation of enantiomers was achieved on Lux Cellulose4 column (250 mm × 4.6 mm; 5 μm particle size, Phenomenex, Torrance, CA, USA). A mixture of methanol/water (80:20, v/v) was used as mobile phase at a flow rate of 1.00 mL/min for the first 4.5 min and then at 0.6 mL/min until the end of the run. The
ip t
total analysis time was 8.5 min. The column temperature was set at 30 °C.
The mass spectrometer was operated in the positive ion mode. The optimized
cr
tuning parameters were as follows: the ion spray voltage, 5500 V; source temperature, 400°C; nebulizer gas (Gas 1), 50 psi; heater gas (Gas 2), 50 psi; curtain gas, 30 psi
us
and collision activated dissociation (CAD) gas, 6 psi. Declustering potential (DP), entrance potential (EP) and collision cell exit potential (CXP) were set at 50, 10 and
an
20 V for each enantiomer and the IS. The optimized multiple reaction monitoring (MRM) fragmentation transitions (Figure 1) were m/z 271 → m/z 144 and m/z 275 →
M
m/z 148 with a collision energy (CE) of 21 eV for morinidazole enantiomers and d4morinidazole enantiomers, respectively. The dwell time for each transition was kept at
d
80 ms.
te
2.4 Preparation of calibration standards and quality control (QC) samples Standard and QC stock solutions of racemic morinidazole were prepared in
Ac ce p
methanol separately to obtain 500 μg/mL for each enantiomer. Standard curve
samples were serially diluted from stock solutions with blank plasma to obtain the concentrations of 5.00, 15.0, 50.0, 150, 500, 2000, and 6000 ng/mL for each enantiomer. The lower limit of quantification (LLOQ) and QC samples were independently prepared in blank plasma at different concentrations (LLOQ, low, medium, and high concentrations at 5.00, 10.0, 500, and 4800 ng/mL for each enantiomer, respectively). The 1.00 mg/mL IS stock solution of racemic d4-
morinidazole was further diluted with 50% aqueous methanol to yield a working solution (100 ng/mL for each enantiomer). All stock solutions were stored at –20°C and working solutions were stored at 4°C and were brought to room temperature before use. Standard-curve samples and QC samples were stored frozen at –20°C until analysis. 5
Page 5 of 26
2.5 Sample preparation To an aliquot of 50 μL of plasma sample, 50 μL of IS working solution (racemic d4-morinidazole, 100 ng/mL for each enantiomer) and 300 μL of water were added.
ip t
The sample was vortex-mixed and extracted with 3 mL of tert-butyl methyl ether by vortexing for 5 min. After centrifuging the samples at 3500 × g for 5 min, the upper organic layers were transferred to another glass tube and evaporated to dryness at 40
cr
°C under a gentle stream of nitrogen in a TurboVap evaporator (Zymark, Hopkinton,
us
MA, USA). The residues were reconstituted in 100 μL of the mobile phase of methanol/water (80: 20, v/v) and an aliquot of 5.0 μL was injected for LC-MS/MS
an
analysis. 2.6 Method validation
M
The assay was validated with respect to selectivity, linearity, precision and accuracy, extract recovery, matrix effects and stabilities in human plasma according to
d
the FDA guidelines on bioanalytical method validation [10]. Selectivity of the method was performed by analyzing six independent sources of
te
blank plasma and the same matrix spiked with analytes at LLOQ level. Samples were
Ac ce p
extracted and analyzed, and the peak areas in blank sample were compared with those of LLOQ samples at the retention times of the enantiomers and their corresponding IS. The acceptance criteria required that the integrated response of co-eluted endogenous matrix components was lower than 20% that of the LLOQ and <5% for the IS. The major metabolites glucuronides (M8-1 and M8-2) and sulfate (M7) at the maximum clinical concentrations were also spiked with blank plasma and the IS to evaluate for potential interference. Calibration standards at eight levels were prepared and analyzed in duplicate for three independent validation days. Calibration curves were obtained by analyzing the peak area ratio of each analyte to the IS (analyte/IS) versus the nominal concentrations in human plasma, using a linearly weighed (1/x2) least squares regression model. A correlation coefficient (r) of 0.995 or greater was accepted for
6
Page 6 of 26
linearity determination. The deviations of back-calculated concentrations should not exceed ±15% of the spiked concentrations, while a deviation of ±20% was permitted for the LLOQ. Intra- and inter-day accuracy and precision were evaluated by replicate analyses
ip t
of six sets of QC samples at low, medium and high levels. Intra-assay data were
determined by comparing data from within run (n = 6) and inter-assay data were
cr
assessed for three consecutive days (n = 18). The values of accuracy and precision
were expressed as relative error (RE, %) and relative standard deviation (RSD, %),
us
respectively. Intra- and inter-day precision values were required to be below 15% and accuracy was required to be within ±15%. The LLOQ was defined as the lowest
an
concentration on the calibration curve with precision not exceeding 20% and accuracy within ±20%. To evaluate the accuracy and precision of different morinidazole
M
enantiomeric ratios, morinidazole enantiomers were combined and diluted with blank plasma to obtain three mixtures of known S/R ratios at 2:1, 4:1, and 8:1 containing R-
d
morinidazole and S-morinidazole at 500 ng/mL total concentration. Samples were
separate days.
te
analyzed using an enantioselective LC-MS/MS system in six replicates for three
Ac ce p
The extraction efficiency of each enantiomer was determined by comparing the mean peak areas of the analyte (A) spiked before extraction (six samples each) with those (B) spiked after extraction. The ratio (A/B × 100) was defined as the extraction efficacy. The extraction recovery of morinidazole enantiomers was carried out at three QC levels. IS recovery was determined using the same method. The matrix effects of each enantiomer and IS were evaluated by preparing two
sets of samples at low and high QC levels, namely, the peak area of plasma extracts from six different lots spiked with analytes after extraction (A) and the peak area of standards in neat solutions at the same concentration (B). The results were expressed as the matrix factor (MF; A/B × 100%). The coefficient of variation of the ISnormalized matrix factors should not be greater than 15%. An MF value of 100% suggested that the signals of plasma samples and solutions were similar and that matrix effect was considered negligible. A value greater than 100% indicated 7
Page 7 of 26
ionization enhancement, whereas a value less than 100% indicated ionization suppression. The stabilities of each enantiomer in human plasma were evaluated in triplicate by analyzing replicates (n = 3) of low- and high-concentrations plasma samples
ip t
exposed to different storage conditions. The stability of extracts in the autosampler
was assessed at 4 °C for 36 h. The short-term bench top stability of morinidazole
cr
enantiomers in plasma over 8 h was determined at ambient temperature (25 °C). The long-term stability was evaluated after storage of the standard spiked plasma samples
us
at –20 °C for 56 days. We also assessed the stabilities of plasma samples after three
within ± 15% of the nominal concentration.
an
freeze–thaw cycles. The samples were considered stable when the assay RE was
To evaluate the enantiomeric stability of R-morinidazole or S-morinidazole in
M
human plasma, spiked plasma samples were prepared in triplicate at 500 ng/mL for each enantiomer and incubated at 37 °C for 12 h. The samples were then extracted
d
and analyzed as previously described. Chiral inversion was evaluated by detecting the
te
appearance of the other enantiomer in the samples. 2.7 Stereoselective pharmacokinetic studies
Ac ce p
Pharmacokinetic studies were approved by the Ethics Committees and conducted
in accordance with the Declaration of Helsinki and the principles of Good Clinical Practice. Four healthy male Chinese volunteers and four patients with severe renal impairment were included in the study. Written informed consent was obtained from all subjects before the study. Venous blood samples (4 mL) were collected in heparincontaining tubes after a continuous infusion injection for 45 min of 500 mg of racemic morinidazole at 0 (pre-dose), 0.375, 0.75, 1.25, 1.5, 1.75, 2.75, 4.75, 6.75, 8.75, 12.75, 24.75, 36.75, and 48.75 h after the start of infusion. Plasma was obtained after centrifugation of the blood samples and stored at −20°C until analysis. The pharmacokinetic parameters were calculated based on the plasma concentration versus time curves by non-compartmental analysis using the WinNolin program, V5.3 (Pharsight Corporation, Mountain View, CA, USA). All parameters were expressed as 8
Page 8 of 26
the mean ± standard deviation. 3. Results and discussion 3.1 Mass spectrometry and enantioselective chromatography conditions
ip t
Our preliminary study showed that ESI could offer higher intensity for
morinidazole enantiomers than atmospheric pressure chemical ionization (APCI) (>
cr
15-fold). Accordingly, ESI was used for method development in the present work. The
us
MS conditions were in a previously described method of our group, with minor modifications to achieve the highest response under the present chromatography conditions [7]. The stable isotope labeled as d4-(±)-morinidazole was synthesized in
an
house and chosen as the IS because of its similarity to morinidazole in terms of chromatographic behavior, extraction recovery and ionization efficiency.
M
Chiral HPLC separation based on various chiral stationary phases (CSPs) coupled with MS detection are high sensitive and selective, thus, this technique is
d
widely applied in enantioselective pharmacokinetics. The properties of CSPs and the compatibility of the mobile phase with the LC-MS/MS interface should first be
te
considered. In the early stage of method development, several types of CSP columns
Ac ce p
were attempted, including macrocyclic glycopeptide-based (Chirobiotic T and V2), polysaccharide-based (Chiralpak AS-RH and Lux cellulose-4), and protein-based (chiral-AGP and Ultron ES-OVM) columns. No obvious resolution was observed for morinidazole enantiomers on the macrocyclic glycopeptide-based, AS-RH and ESOVM columns, although various organic modifiers (acetonitrile, methanol, and 2propanol) and water with or without typical modifiers and buffers (ammonium formate and ammonium acetate) at different pH values were tested. Fortunately, sufficient separation (RS = 1.7) and suitable retention time (Rt = 5.6 and 6.6 min) were achieved from the AGP column, when the mobile phase was 100% aqueous phase containing 20 mM ammonium formate at a flow rate of 0.6 mL/min. However, poor ionization efficiency was observed under this chromatographic condition, leading to low MS response at LLOQ level (5.00 ng/mL). To enhance the signal intensities in
9
Page 9 of 26
positive ionization mode, further optimization of increasing organic modifiers (methanol, acetonitrile or 2-propanol) proportion to 1% or adding 0.005% acetic acid or formic acid was attempted, but all resulted in poor resolution. Adjustment of the organic-phase proportion enabled the peak resolution to reach complete separation (RS
ip t
= 3.8) with high signal intensity on Lux cellulose-4 column using the mobile phase of
methanol/water (80:20, v/v). However, a retention time drift was observed after
cr
multiple injections. With increased column temperature from 20 °C to 30 °C, the
reproducibility of retention time and the peak symmetry were improved. Further
us
optimization of using 5 mM ammonium acetate as a substitute for water was tried, but MS signal intensity was found to decrease. Although good resolution and sensitivity
an
were obtained from the Lux cellulose-4 column with methanol/water (80:20, v/v) as mobile phase, the analytical time (12.5 min) was slightly long for bioanalysis. To
M
shorten the total run time, a flow gradient was used and the analytical time was reduced to 8.5 min. The elution order on the Lux cellulose-4 column was R-
d
morinidazole (5.8 min) followed by S-morinidazole (7.5 min). The chiral recognition mechanism of polysaccharide phases is generally
te
considered to be due to differences in the combination of interaction forces such as
Ac ce p
hydrogen bonding, hydrophobic interactions, π-π interactions and dipole-dipole interactions between the two enantiomers and CSP [11]. Regarding CSPs based on phenylcarbamate derivatives, hydrogen bonding to the carbamate group is considered to be the strongest attractive interaction [12]. 5-Nitroimidazole chiral drugs usually contain a hydroxyl group at the chiral center, a nitro group and an imidazole group in their structure. The hydroxyl group at the chiral center is pivotal for chiral resolution and interaction with the polar carbamate moiety of CSP through hydrogen bonding with –NH group. The nitro and imidazole groups could also interact with CSP through hydrogen bonding and dipole–dipole interaction that is inherent in the polar carbamate group. In addition, phenyl groups in CSP may have π-π stacking interactions with the imidazole group of enantiomers. In particular, the morinidazole structure consists of a morpholine ring, and may greatly strengthen the hydrogen binding force between the enantiomers and CSP. Therefore, the specific configuration 10
Page 10 of 26
of
the
Lux
cellulose-4
stationary
phase
[cellulose
tris(4-chloro-3-
methylphenylcarbamate)] can be speculated to provide suitable sites for morinidazole enantiomers and make them interact with CSP with different affinities, leading to different retentions. As analogs of morinidazole, our pilot studies showed that
ip t
ornidazole and secnidazole enantiomers could also be baseline resolved on Lux celluse-4 column (data not shown). Thus, the Lux celluse-4 column may have
cr
implications in the stereoselective pharmacokinetics of other 5-nitroimidazole
us
derivatives. 3.2 Sample preparation
an
Considering that liquid–liquid extraction (LLE) could give cleaner extracts and less signal suppression or enhancement especially with an ESI ionization source [13],
M
we performed LLE as our first attempt. After extraction with ethyl acetate, tert-butyl methyl ether or ethyl ether–dichloromethane (2:1, v/v), high response and less matrix interference were obtained with tert-butyl methyl ether. Although a pilot study
d
indicated that ethyl acetate had the highest extract efficiency for morinidazole
te
enantiomers (up to 90%), signal response declined during the repeated injections, which was presumably due to the interference of endogenous matrix extracted.
Ac ce p
Morinidazole is a weakly basic compound; thus, conducting the extraction after plasma pH adjustment was evaluated. However, compared with the response of the sample extracted at pH 7, no significant difference was observed in extraction efficacy.
3.3 Method validation
3.3.1 Assay selectivity. For evaluation of the selectivity of the method, six different batches of blank human plasma and the corresponding spiked plasma at LLOQ level were analyzed for peak inference. Figure 2 shows the typical chromatograms included blank plasma sample, blank plasma sample spiked only with R-d4-morinidazole and S-d4-
11
Page 11 of 26
morinidazole, blank plasma sample with R-morinidazole and S-morinidazole at the LLOQ and IS, and plasma sample obtained 2.75 h after infusion injection of 500 mg of racemic morinidazole to a healthy volunteer. No interfering matrix peak was observed at the retention time of enantiomers of the analyte and IS. The possibilities
ip t
of back-conversion of sulfate conjugate M7 and glucuronide conjugates M8-1 and M8-2 were evaluated at 100, 250, and 2000 ng/mL, respectively. No back-conversion
cr
was observed, suggesting that metabolites did not interfere with the determination of
the parent drug, including the extraction procedures or in the chromatographic and
us
MS mode used. The chromatographs proved that the method was selective for analyzing morinidazole enantiomers.
an
3.3.2 Linearity and LLOQ.
The calibration curves were linear over the concentration range of 5.00–6000
M
ng/mL for each enantiomer in human plasma. The mean (± SD) regression equations from calibration curves on different days were as follows: R-morinidazole: y = (0.0108 0.0002)x + (0.0023 0.0019), r2 = 0.9991 0.0002
d
S-morinidazole: y = (0.0110 0.0002)x + (0.0049 0.0012), r2 = 0.9995 0.0002
Ac ce p
concentration.
te
where y is the peak-area ratio of each enantiomer to IS and x is the nominal plasma
The LLOQ was 5.00 ng/mL for the determination of morinidazole enantiomers
in plasma and the signal-to-noise ratio of LLOQ was >10. The values of intra- and inter-day at LLOQ precision and accuracy are shown in Table 1, and these values were accepted with RSD below 6.4% and RE within 2.8%. With the present LLOQ, the concentrations could be determined up to more than six half-life for R-
morinidazole and S-morinidazole, suggesting that the method was sensitive for investigating the stereoselective pharmacokinetics of morinidazole after an intravenous administration of 500 mg of racemic morinidazole. 3.3.3 Precision and accuracy. Table 1 summarizes the intra- and inter-day precision and accuracy for morinidazole enantiomers from QC samples. The intra-day precision by calculating RSD was 3.4% or less for R-morinidazole and 2.9% or less for S-morinidazole, and 12
Page 12 of 26
the inter-day precision was 4.0% or less for R-morinidazole and 4.1% or less for Smorinidazole. The accuracy in terms of RE ranged from 0.7% to 6.4% for Rmorinidazole and from 0.2% to 4.6% for S-morinidazole at all QC levels. The precision and accuracy for morinidazole enantiomeric ratios are also presented in
ip t
Table 2. The intra-day and inter-day precision were less than 2.2% and 1.2%. The accuracy ranged from 0.3% to 4.5% (RE). Figure 3 presents the typical
cr
chromatograms of R-morinidazole and S-morinidazole from QC samples with different enantiomeric ratios at a total morinidazole plasma concentration of 500
us
ng/mL. 3.3.4 Extraction recovery and matrix effect.
an
Mean extraction recoveries of R-morinidazole were 60.0 ± 4.9%, 56.1 ± 5.2% and 58.5 ± 4.4% at the concentrations of 10.0, 500 and 4800 ng/mL, respectively. The
M
recoveries of S-morinidazole were 63.6 ± 3.4%, 57.8 ± 5.4%, and 58.5 ± 3.9%, respectively. The recoveries of R-d4-morinidzole and S-d4-morinidazole were 55.8 ±
d
7.7% and 56.7 ± 7.8%. The precision (RSD) values of recoveries for the analytes and IS were <10.0%. The recoveries were consistent and reproducible.
te
The matrix factors at low and high concentration levels ranged within 96.9–
Ac ce p
98.9% for R-morinidazole and 101–102% for S-morinidazole, and the deviations from six batches of plasma were less than 3.8%. Obviously, ion suppression or enhancement caused by plasma matrix was negligible for the determination of morinidazole enantiomers under the present conditions. 3.3.5 Stability.
The stabilities of racemic morinidazole and major conjugated metabolites have
been evaluated in a previous study [7]. In the present study, the enantiomers were found to be stable under the following conditions: in plasma at room temperature for 8 h, in autosampler for 36 h post-extraction, in plasma after three freeze−thaw cycles, and in plasma at −20 °C for 56 days. The signal of the other enantiomer was not
detected after incubation of plasma samples containing one enantiomer at 37 °C for 12 h, suggesting that chiral inversion did not occur between R- and S-morinidazole during storage, sample preparation procedure and analysis. 13
Page 13 of 26
3.4 Method application The developed chiral LC–MS/MS method was successfully applied to the determination of plasma concentrations of morinidazole enantiomers in healthy
ip t
human subjects and patients with severe renal impairment after intravenous administration of 500 mg of racemic morinidazole. Figure 4 shows the mean plasma
cr
concentration–time profiles of morinidazole enantiomers. Table 4 lists the major pharmacokinetic parameters of R-morinidazole and S-morinidazole in humans.
us
After an intravenous administration of 500 mg of morinidazole racemate to healthy subjects, significant difference was observed between the pharmacokinetics of
an
the two enantiomers, with S-morinidazole providing slightly higher exposure than the R-enantiomer, i.e., the average AUC0-t ratio of the S-form to R-form was 1.71. The mean S/R plasma concentration ratio values gradually increased to 12 in healthy
M
subjects from the start of infusion time to 36.75 h. Mean elimination half-life (t1/2) of S-morinidazole was 1.6 times as long as R-morinidazole. The total plasma clearance
d
(CL) of R-morinidazole was higher than that of its antipode, and the CL ratio value
te
between two enantiomers was 1.72. Based on these findings, we speculated that the higher exposure of S-morinidazole was due to a lower clearance.
Ac ce p
Meanwhile, we determined the plasma concentrations of the main metabolites of
M7, M8-1, and M8-2 in these four healthy human subjects using the method previously described by Gao et al [7]. If the amount ratio of S-/R-morinidazole sulfate
in hepatocyte incubation was representative of that in plasma, we speculated that the sulfate conjugate M7 was mainly from S-morinidazole. Therefore, the sum of AUC values of S-morinidazole, glucuronide conjugate M8-1, and sulfate M7 was calculated (based on molar concentrations), and this sum was 1.28-fold higher than the sum of R-
morinidazole and its glucuronide conjugate M8-2. The ratio value of S-form to Rform drastically decreased, suggesting that the stereoselective metabolism of morinidazole was the key determinant for the stereoselective pharmacokinetics. Morinidazole and its main metabolites are eliminated primarily through kidney, with the excreted amount accounting for approximately 71% of the dose over the 0– 14
Page 14 of 26
36 h period [6]. Thus, the effect of renal function on the pharmacokinetics of morinidazole enantiomers was evaluated. A slight increase in systematic exposure of each enantiomer in patients with severe renal impairment was observed. The AUC values of R-morinidazole and S-morinidazole were 37% and 29% higher than those in
ip t
healthy subjects, respectively. However, the enantiomeric ratio of AUC (S to R) was not influenced by renal function, with a value of 1.60 in patients with severe renal
cr
impairment.
us
4. Conclusions
An enantioselective LC-MS/MS method determining morinidazole enantiomers
an
in human plasma using a deuterated IS by combining separation on a cellulose chiral stationary phase column was developed and validated for the first time. The LLOQ
M
was 5.00 ng/mL both for R-morinidazole and S-morinidazole using 50 μL of the plasma sample. No interference derived from the morinidazole conjugated metabolites was observed. Under the present conditions, no retention time drift and signal
d
intensity attenuation were found after about 100 injections in one analytical run. The
te
validated method was successfully applied to characterize the pharmacokinetic profiles of morinidazole enantiomers in healthy volunteers and patients with severe
Ac ce p
renal impairment after administration of therapeutic doses of racemic morinidazole, and the results verified the stereoselective pharmacokinetics of morinidazole in humans. These findings can help elucidate the stereoselective pharmacokinetics and the development of the single enantiomer of morinidazole.
15
Page 15 of 26
References [1] J.D. Sobel, P. Nyirjesy, W. Brown, Clin Infect Dis, 33 (2001) 1341-1346.
ip t
[2] J.A. Upcroft, L.A. Dunn, J.M. Wright, K. Benakli, P. Upcroft, P. Vanelle, Antimicrob Agents Chemother, 50 (2006) 344-347.
4210.
us
[4] A. Lv, China Patent CN 1981764A, (2007) 636-643.
cr
[3] J.R. Schwebke, F.J. Barrientes, Antimicrob Agents Chemother, 50 (2006) 4209-
[5] C. Zhang, X. Tao, Z. Teng, China Patent CN 1850086A, (2006).
(2012) 556-567.
an
[6] R. Gao, L. Li, C. Xie, X. Diao, D. Zhong, X. Chen, Drug Metab Dispos, 40
[7] R. Gao, D. Zhong, K. Liu, Y. Xia, R. Shi, H. Li, X. Chen, J Chromatogr B Analyt
M
Technol Biomed Life Sci, 908 (2012) 52-58.
[8] J. Liu, X. Tang, S.L. Harbeson, C.E. Masse, US Patent No. 0,160,247 (2010).
d
[9] S. Khaksar, A. Heydari, M. Tajbakhsh, H.R. Bijanzadeh, Journal of fluorine
te
chemistry, 131 (2010) 106-110.
[10] USFDA, Guidance for Industry: Bioanalytical Method Validation, (2001)
Ac ce p
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/ Guidances/ucm070107.pdf.
[11] P. Wang, S. Jiang, D. Liu, P. Wang, Z. Zhou, J Biochem Biophys Methods, 62 (2005) 219-230.
[12] D.T. Witte, J.P. Franke, F.J. Bruggeman, D. Dijkstra, R.A. De Zeeuw, Chirality, 4 (1992) 389-394.
[13] M. Jemal, Z. Ouyang, Y.Q. Xia, Biomed Chromatogr, 24 (2010) 2-19.
16
Page 16 of 26
Legends Fig. 1 Product ion spectra of [M + H]+ of morinidazole (A) and d4-morinidazole (B)
ip t
Fig. 2 Typical enantioselective MRM chromatograms of R-morinidazole (I), Smorinidazole (II), R-d4-morinidazole (III), and S-d4-morinidazole (IV) in human
plasma: (A) blank plasma sample; (B) plasma spiked with R-d4-morinidazole (100
cr
ng/mL) and S-d4-morinidazole (100 ng/mL); (C) plasma spiked with R-morinidazole
(5.00 ng/mL), S-morinidazole (5.00 ng/mL), R-d4-morinidazole (100 ng/mL) and S-
us
d4-morinidazole (100 ng/mL); and (D) plasma sample 2.75 h after a continuous
an
infusion injection of 500 mg of racemic morinidazole to a healthy volunteer
Fig. 3 Typical chromatograms of R-morinidazole and S-morinidazole from different enantiomeric ratios (total plasma concentration = 500 ng/mL): (A) S/R = 2:1; (B) S/R
M
= 4:1; and (C) S/R = 8:1. Peaks I, II, III and IV refer to R-morinidazole, S-
d
morinidazole, R-d4-morinidazole, and S-d4-morinidazole, respectively
te
Fig. 4 Plasma concentration versus time profiles (A) and semi-log scale plots (B) of R-morinidazole and S-morinidazole after a continuous infusion injection of 500 mg
Ac ce p
of racemic morinidazole to healthy volunteers and patients with severe renal impairment
18
Page 17 of 26
*Highlights (for review)
Research highlights: Baseline separation of morinidazole enantiomers was achieved on a cellulose chiral stationary phase column using a high proportion of organic phase. D4-morinidazole was synthesized as the internal standard to compensate for matrix effects and variables during sample preparation. The analysis time was short by using a flow rate gradient.
ip t
The method shows advantages of high selectivity and reproducibility.
The method was successfully applied to clinical stereoselective pharmacokinetics
Ac
ce pt
ed
M
an
us
cr
study of morinidazole.
Page 18 of 26
Ac
ce
pt
ed
M
an
us
cr
i
Figure 1
Page 19 of 26
Ac
ce
pt
ed
M
an
us
cr
i
Figure 2
Page 20 of 26
Ac
ce
pt
ed
M
an
us
cr
i
Figure 3
Page 21 of 26
Ac ce p
te
d
M
an
us
cr
ip t
Figure 4
Page 22 of 26
Tables
Table 1 Precision and accuracy data of the analysis of R-morinidazole, and S-morinidazole in
Added
Found
Intra-day Inter-day
5.00
5.14
5.1
4.0
10.0
10.6
3.4
4.0
500
503
1.0
2.3
0.7
4800
4835
2.1
2.1
0.7
5.00
4.87
6.4
3.7
-2.5
10.0
10.5
2.9
3.7
4.6
500
501
1.2
2.5
0.2
4800
4893
4.1
1.9
2.5
ip t
RE (%)
2.8 6.4
Ac
ce pt
ed
M
S-morinidazole
RSD (%)
cr
R-morinidazole
Concentration (ng/mL)
an
Analyte
us
human plasma (3 days with 6 replicates per day).
Page 23 of 26
Table 2 Precision and accuracy data of the analysis of morinidazole enantiomeric ratios in human plasma (3 days with 6 replicates per day).
Found 2.01 4.05 8.36
Intra-day 2.2 1.7 1.0
RE (%) Inter-day 1.0 1.2 0.6
0.3 1.2 4.5
Ac
ce pt
ed
M
an
us
cr
Added 2.00 4.00 8.00
RSD (%)
ip t
S/R ratios
Page 24 of 26
ip t cr us
Table 3
RSD (%) 3.0 0.7 3.3 3.6
Long-term (−20◦C for 56 days)
RSD (%) 3.2 4.7 3.3 3.6
RSD (%) 4.3 1.4 2.1 1.0
RSD (%)
RE (%)
9.50 3.50 4.10 1.50
0.6 -5.7 5.1 -2.9
M
RE (%) 11.2 3.4 6.7 1.6
d
S-morinidazole
(ng/mL) 10.0 4800 10.0 4800
3 Freeze–thaw (−20◦C to room temperature)
RE (%) 5.9 -0.8 1.9 1.6
RE (%) 3.9 0.3 1.0 2.9
ep te
R-morinidazole
Concentration
Autosampler for 36 h (4°C)
Ac c
Analyte
Short-term (room temperature for 8 h)
an
Stability of R-morinidazole, and S-morinidazole in human plasma under various storage conditions (n = 3).
Page 25 of 26
ip t cr us
Table 4
an
Mean pharmacokinetic parameters of R-morinidazole, and S-morinidazole after administration of 500 mg of racemic morinidazole by continuous intravenous infusion for 45 min to four healthy subjects and four patients with severe renal impairment.
T1/2 (h)
M
Analyte
AUC0-t (h*ng/mL)
CL (mL/h)
3.9 ± 0.2
0.7 ± 0.2
4975 ± 458
23751 ± 1715
10522 ± 724
S-morinidazole
6.4 ± 0.2
0.7 ± 0.2
5300 ± 556
40590 ± 1394
6128 ± 210
R-morinidazole
5.5 ± 1.7
0.9 ± 0.3
4503 ± 801
32671 ±8326
8140 ± 2763
S-morinidazole
7.8 ± 1.6
1.0 ± 0.3
4930 ± 856
52243 ± 7142
4782 ± 702
Ac c
ep te
Severe renal impairment
Cmax (ng/mL)
R-morinidazole
d
Healthy subjects
Tmax (h)
Page 26 of 26