MS and its application in a study of pharmacokinetics

Accepted Manuscript Title: Simultaneous determination of nimesulide and its four possible metabolites in human plasma by LC-MS/MS and its application in a study of pharmacokinetics Author: Xiao Sun Kai-Lu Xue Xin-Yue Jiao Qian Chen Li Xu Heng Zheng Yu-Feng Ding PII: DOI: Reference:

S1570-0232(16)30313-0 http://dx.doi.org/doi:10.1016/j.jchromb.2016.05.008 CHROMB 20039

To appear in:

Journal of Chromatography B

Received date: Revised date: Accepted date:

28-12-2015 30-4-2016 6-5-2016

Please cite this article as: Xiao Sun, Kai-Lu Xue, Xin-Yue Jiao, Qian Chen, Li Xu, Heng Zheng, Yu-Feng Ding, Simultaneous determination of nimesulide and its four possible metabolites in human plasma by LC-MS/MS and its application in a study of pharmacokinetics, Journal of Chromatography B http://dx.doi.org/10.1016/j.jchromb.2016.05.008 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.

Simultaneous determination of nimesulide and its four possible metabolites in human plasma by LC-MS/MS and its application in a study of pharmacokinetics Xiao Suna,b, Kai-Lu Xuea, Xin-Yue Jiaob, Qian Chena, Li Xub , Heng Zhenga,*, Yu-Feng Dinga,** a

Department of Pharmacy, Tongji Hospital of Tongji Medical College, Huazhong

University of Science and Technology, 1095 Jiefang Avenue, Wuhan 430030, China b

College of Pharmacy, Tongji Medical College, Huazhong University of Science and

Technology, Wuhan 430030, China *

Corresponding author. Tel.: +86 27 83662498; fax: +86 27 83663643.

E-mail address: [email protected] (H. Zheng) **

Corresponding author. Tel.: +86 27 83663641; fax: +86 27 83663641.

E-mail address: [email protected] (Y.F Ding)

Highlights:  A LC-MS/MS method for determination of nimesulide and its metabolites in plasma was developed.  The method was fully validated and successfully applied in clinical practice.  A new conjugated metabolite of nimesulide in plasma was determined and discussed firstly.

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Abstract In this study, it was the first time that we simultaneously quantified nimesulide and its possible metabolites M1, M2, M3 and M4 by employing liquid chromatography-tandem mass spectrometry (LC-MS/MS). Nimesulide-d5 was used as internal standard (IS) for validation. Analytes and IS were recovered from human plasma by protein precipitation with acetonitrile. Prepared plasma samples were analyzed under the same LC–MS/MS conditions, and chromatographic separation was realized by using an Ultimate C18 column, with run time being 5 min for each sample. Our results showed that various analytes within their concentration ranges could be quantified accurately by using the method. Mean intra- and inter-day accuracies ranged from -4.8% to 4.8% (RE), and intra- and inter-assay precision ≤6.2% (RSD). The following parameters were validated: specificity, recovery, matrix effects, dilution integrity, carry-over, sample stability under a variety of storage and handling conditions (room temperature, freezer, freeze-thaw and post-preparative) and stock solution stability. Pharmacokinetics of nimesulide and its metabolites were calculated based on the analysis of samples collected from twelve Chinese healthy volunteers after single oral dose of 100 mg nimesulide tablets. By applying the pharmacokinetic determination into human samples, we preliminarily detected a new metabolite of nimesulide (M4*), and the concentration of M4* was relatively higher in plasma. Furthermore, we predicted part of conceivable metabolism pathway in plasma of after oral administration of 100 mg nimesulide tablets. This research provided an

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experimental basis for further studies on metabolic activation and biotransformation of nimesulide, and for more comprehensive conjecture of its metabolic pathways.

Keywords: Nimesulide; metabolites; LC-MS/MS; pharmacokinetics 1. Introduction Nimesulide, N-(4-nitro-2-phenoxyphenyl)methanesulfonamide (pKa = 6.4, as shown in Fig. 1), is a non-steroidal anti-inflammatory drug (NSAID) and at recommended doses (100 mg), possesses antipyretic and analgesic properties [1] with a good tolerability[2]. Over the past years, it has been extensively used for the treatment of inflammatory and painful conditions[3]. This drug have relatively severe hepatotoxicity[4-6], but the exact mechanism associated with and factors responsible for this toxicity remain poorly understood. To understand the underlying mechanisms of its hepatotoxicity, multiple studies examined the metabolic pathways of nimesulide, with an attempt to identify its reactive metabolites [1, 3, 6, 7]. In fact, researchers have found and characterized a number of nimesulide metabolites and reactive intermediates, which assisted us to not only find the reason of nimesulide idiosyncratic hepatotoxicity but also study on toxication of its metabolites. Upon absorption, drug molecules undergo phase I (e.g. oxidation, reduction) and phase II (e.g. glucuronidation, sulphation) metabolic reactions. Safety of phase I metabolites is more important since phase I metabolites are likely to be more pharmacologically active. It is of great importance to investigate potential toxicity of active metabolites when the major or specific metabolites in human body bear active

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functional groups. Labile intermediate products can seldom be detected due to their short half-life time, but they can be indirectly identified by detecting their metabolites which are more stable. Inactivity of drug metabolites on target receptors does not necessarily mean this drug is non-toxic. For example, while glucuronidated or sulphated metabolites of phase II are usually less active, more water-soluble and more actively excreted, some, such as paracetamol, isoniazid, clozapine, are also toxic[8-11]. When glucuronidated or sulphated metabolites (e.g. phthalidyl glucuronide) are as active as their substrates, toxicological evaluation of the metabolites is essential. Investigations on the toxicity of drug metabolites provide basis for further studies on drug toxication, and pave the way to the development of new drugs[12]. Thus, more metabolites of nimesulide are expected to be discovered by analyzing human plasma samples, and the method development process is the key to find appropriate analytical conditions. In this study, we developed a bioanalytical method for the simultaneous determination of nimesulide and its possible metabolites M1, M2, M3 and M4 (Fig. 1). Among the four metabolites, M1 and M3 had been previously reported[1-3,6,7,13,14]. M1 was the major metabolite of the parent drug. M3 was detected in urine, its presence in plasma has not been confirmed. M2 is an analogue of M3 and is obtained by methylation of the phenolic hydroxyl of M3 (in Fig. 1). Methylation is one of the reactions in drug metabolism. Additionally, sulphation is also a ubiquitous metabolic reaction in human body. Therefore, as a sulphated metabolite of M1, M4 should also be taken into account in toxicity evaluation. If all the four compounds in plasma were

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definitely confirmed to be present in plasma, we could move forward to further assess their toxicity. Reported in this study was the simultaneous quantification of nimesulide, M1, M2, M3 and M4 in human plasma and urine by using LC-MS/MS. Different from previously

reported

techniques[1-3,

13]

used

in

pharmacokinetics[14],

bioequivalence[15-18] or bioavailability[19] studies, this method, for the first time, separated and determined six compounds (including IS) simultaneously within 5 min. Moreover, isotope IS nimesulide-d5 was used in the assay. The method and validation results were detailed. The utility and suitability of the assay were illustrated by a brief summary of the analysis of pharmacokinetic plasma samples collected from 12 healthy volunteers receiving 100 mg nimesulide tablets. Furthermore, a new conjugated metabolite was determined in human plasma and its mass spectrometric confirmation was mentioned. 2. Experimental 2.1. Chemical and reagents Nimesulide (Batch No.100555-201202, 100.0% purity) and internal standard (IS) Nimesulide-d5 (Lot# 25-GHZ-13-1, chemical purity 98.0%, isotopic purity 99.1%) were provided by National Institutes for Food and Drug Control (Beijing, China). Other metabolites of nimesulide, M1 (N-(2-(4-hydroxyphenoxy)-4-nitrophenyl) methanesulfonamide, 99.81% purity), M2 (N-(3-(4-methoxyphenoxy)-4-(methyl sulfonamido) phenyl) acetamide, 99.47% purity), M3 (N-(3-(4-hydroxyphenoxy) -4-(methylsulfonamido)

phenyl)

acetamide,

5

99.54%

purity)

and

M4

(2-hydroxyl-5-(2-(methylsulfonamido)-5-nitrophenoxy)phenyl

hydrogen

sulfate,

99.74% purity) were obtained from Wuhan Humanwell Pharmaceutical CO., LTD (Wuhan, China). Fig. 1 shows the structures. HPLC-grade acetonitrile was purchased from Fisher Scientific (Fair Lawn, NJ, USA). Ultrapure water was produced by Milli-Q® reagent-grade water system (Millipore, MA, USA). Formic acid and ammonium acetate were procured from Sigma-Aldrich (St. Louis, MO, USA). Other chemicals and reagents were of analytical grade. Human plasma was provided by the Blood Center of Tongji Hospital of Tongji Medical College (Wuhan, China) and was stored at −80 °C.

2.2. Apparatus and operation conditions 2.2.1. Liquid chromatography The liquid chromatographic analysis was performed on Shimadzu LC system (Chiyoda-Ku, Kyoto, Japan), consisting of two LC-20AD pumps, DGU-20A3 online degasser,

SIL-20ACHT

autosampler,

and

CTO-20AC

thermostatic

column

compartment. A Welch Ultimate XB-C18 column (5 μm, 50 mm×2.1 mm, Maryland, USA) protected by a Phenomenex ODS guard column (5 μm, 4.0 mm×3.0 mm, Torrance, CA, USA) was applied for the separation and the mobile phase was consisted of water containing 1 mM ammonium acetate and 0.1% formic acid (A) acetonitrile (B) at a flow rate of 0.45 mL/min. The sample injection volume was 5 μL

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with the autosampler conditioned at 4 °C. The column temperature was maintained at 40 °C. The total LC analysis time lasted 5 min for each injection. Gradient elution was performed under the follow program: 80% A for 0.01 min, decreased linearly to 5% A in 3 min and maintained for 0.5 min, then increased linearly to 80% A in 0.1 min and maintained for another 1.4 min. 2.2.2. Mass spectrometry A QTrap5500 MS/MS system (Applied Biosystems, Foster City, CA, USA) with a turbo ion spray source was used for the LC-MS/MS analysis under the negative electrospray ionization (ESI) mode with Multi Reaction Monitor (MRM). Analyst 1.6.2 software package was employed for data-processing. The optimal ESI-MS/MS parameters were as follows: Ion Spray Voltage: -4500 V, Temperature: 450 °C, Ion Source Gas1: 40 psi, Ion Source Gas2: 40 psi, Collision Gas: medium, Curtain Gas: -30 psi. Other optimal parameters are shown in the Table 1. 2.3. Preparation of the standard solutions, quality control (QC) samples and IS Standard stock solutions and QC working solutions were prepared separately. Both standard stock solutions and QC stock solutions of nimesulide, M1, M2, M3, M4 and IS were individually prepared in acetonitrile/water (50:50, v/v) at 1,000,000 ng/mL. The standard stock solutions were serially diluted with 50% acetonitrile to produce combined standard working solutions at concentrations of 200, 400, 1000, 2000, 10,000, 20,000, 32,000 and 40,000 ng/mL for M1 and M4, 100, 200, 500, 1000, 5000, 10,000, 16,000 and 20,000 ng/mL for M2 and M3, and 400, 800, 2000, 4000, 20,000, 40,000, 64,000 and 80,000 ng/mL for nimesulide. The QC stock solutions

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were also diluted with 50% acetonitrile to produce combined QC solutions at lower limit of quantification (LLOQ), low, middle, high and dilution concentrations of 200, 600, 15000, 30000, 80000 ng/mL for M1 and M4, 100, 300, 7500, 15000, 40000 ng/mL for M2 and M3 and 400, 1200, 30000, 60000, 16,0000 ng/mL for nimesulide. The IS working solution was diluted with acetonitrile/water (50:50, v/v) to 34 ng/mL. Calibration samples were freshly prepared by spiking 20 μL of the working solutions into 180 μL blank plasma, so as to obtain the final concentrations in the range of 10-2000 ng/mL for M2 and M3, 20-4000 ng/mL for M1 and M4 and 40-8000 ng/mL for nimesulide. The QC samples were also prepared with blank plasma at LLOQ, low, middle, high and dilution concentrations of 20, 60, 1500, 3000, 8000 ng/mL for M1 and M4, 10, 30, 750, 1500, 4000 ng/mL for M2 and M3 and 40, 120, 3000, 6000, 16,000 ng/mL for nimesulide. All aforementioned solutions were stored at 4 °C. 2.4. Sample preparation The human plasma samples (200 μL) and 20 μL of IS (34 ng/mL) were added to a 1.5 mL plastic vial and vortex-mixed thoroughly before and after mixing with precipitant acetonitrile (600 μL). Then the mixture was centrifuged at 14,700 × g for 10 min. After centrifugation, 200 μL of the supernatant was transferred into another plastic vial and then diluted with mobile phase A/B (80:20, v/v) at the ratio of 1:4 (v/v). Finally, 5 μL of the diluent was injected into the LC-MS/MS system for analysis. 2.5. Method validation 2.5.1. Specificity

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Six individual sources of the appropriate blank matrices were used for the specificity verification based on the evaluation of interference near the retention time. Interfering components were acceptable if the response value was less than 20% of the LLOQ for the analytes and less than 5% for IS. 2.5.2. Interference among analytes and IS The interference check among different analytes was performed by using processed samples with each analyte at the upper limit of quantification (ULOQ) in at least triplicates. In addition, IS was investigated at the concentration used in the assay (34 ng/mL) in at least triplicates to check its interference with analytes. 2.5.3. Linearity The five calibration curves, using weighted (1/x2) least squares regression analysis, with different linearity ranges were generated with the peak area (A) ratios (Aanalyte / AIS) as vertical axis (y) and the known concentrations as horizontal axis (x). For all five curves, required correlation coefficient (r) should be 0.99 or greater. 2.5.4. Intra- and inter-day accuracy and precision Relative error (RE) and relative standard deviation (RSD) were the parameters for accuracy and precision evaluation. Six samples per level at four QC concentrations (LLOQ, low QC, middle QC, high QC) on the same day was analyzed to assess the intra-day accuracy and precision, and on three consecutive days, the four QC samples were analyzed for evaluating the inter-day accuracy and precision. The acceptable RE and RSD should not deviate by 20% for each LLOQ sample while the mean value should be within ±15% and 15% for other QC samples.

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2.5.5. Recovery, matrix effect, dilution integrity and carry-over Recovery was performed at low QC, middle QC and high QC levels (n=6) by comparing the response of analytes or IS for precipitated QC samples with standards spiked after precipitation at the same concentration level. Matrix effect was investigated with different blank matrices from individual donors. Matrix factor (MF), the evaluation index of matrix effect, was obtained as the ratio of the peak area of analyte and IS spiked after precipitation from different matrices to neat solution of the analyte at equivalent concentration (low QC and high QC levels).The IS- normalized MF was also calculated by: IS- normalized MF(%)= MFanalyte / MFIS×100% The calculated Coefficient of Variance (CV) of the IS-normalized MF from six individual matrices should be less than 15%. Dilution integrity was assessed by analyzing six replicates of a four-fold dilution of the dilution QC sample with blank plasma prior to protein precipitation. Carry-over was evaluated by analyzing extracted samples of blank plasma immediately after ULOQ sample (n=6). Carry-over in the blank sample should not be greater than 20% of LLOQ and 5% of IS. 2.5.6. Stability Stability was tested by using triplicates of QC samples stored under different conditions. The autosampler stability was evaluated by analyzing extracted QC samples kept under autosampler condition (4 °C) for 24 h. Room temperature stability was assessed by using untreated QC samples kept at room temperature for 24 h and

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long-term stability was assessed by using samples stored at −80 °C for 38 days. The freeze-thaw stability of the analytes was determined over three freeze-thaw cycles. With each cycle, the samples were frozen and stored at −80 °C for 24 h and then thawed at room temperature. Samples were considered to be stable if their calculated values were within 15% error of the nominal values. The stability of stock solutions and working solutions of analytes and IS (with an appropriate dilution) were evaluated by comparing the peak area of the stock solutions and working solutions kept at 4 °C for 38 days with that of the freshly prepared solutions. The solutions were considered to be stable if their calculated values were within ±10% error of freshly prepared solutions. 2.6. Pharmacokinetic study The whole pharmacokinetic study was carried out in full compliance with the principles of the Declaration of Helsinki and all its amendments. The clinical protocol of the study was approved by the local ethics committee and written informed consents were obtained from 12 healthy volunteers (6 males, 6 females) after they had been well-informed of the nature and details of the study. Upon physical examination, volunteers were enrolled in this trial. Any subject would be excluded if they didn’t satisfy the criteria listed in our study protocol and the Declaration of Helsinki. Blood samples (3 mL ) were collected at pre-dose (0 h) and at 0.5 h，1 h，2 h， 2.5 h，3 h，3.5 h，4 h，4.5 h，5 h，6 h，7 h，8 h，10 h，12 h，15 h，24 h post-dosing. Plasma was immediately separated by centrifugation at 3000 rpm for 10min and stored at −80 °C until analysis.

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The data were pharmacokinetically analyzed by using DAS 3.2.7 software (Mathematical Pharmacology Professional Committee of China). The maximum plasma concentration (Cmax) and the time of maximum plasma concentration (Tmax) were obtained directly from the concentration-time curve. The area under the plasma concentration-time curve (AUC) was estimated against the trapezoidal rule. The terminal elimination rate constant (Ke) was determined by linear least-squares regression of the terminal portion of the plasma concentration-time curve and the elimination half-time (t1/2) was calculated by using the equation: t1/2= ln2/Ke. 3. Results and discussion 3.1. Optimization of LC-MS/MS conditions and sample preparation In order to optimize mass spectrometric conditions, the solutions containing nimesulide, M1, M2, M3, M4 and IS at the concentration of 1000 ng/mL in 50% acetonitrile were directly infused into the mass spectrometer at a flow rate of 10 μL/min via a syringe pump, respectively. According to the result of ion mode optimization, the responses of precursor ions of analytes and IS in negative-ion mode were much higher than those in positive-ion mode. The Q1 MS full scan spectra contained protonated precursor [M-H]- ions at m/z 312.0, 307.0, 323.0, 349.0, 335.0, 419.0 and the most abundant product ions were m/z 234.0, 229.0, 245.0, 269.0, 255.0, 259.0 for IS, nimesulide, M1, M2, M3, M4, respectively. A dwell time of 80 ms was adopted according to the number of ion pairs and peak width. The m/z transitions are detailed in Fig. 2. Other parameters were optimized and are listed in Table 1. In the assay, gradient elution mode was chosen for the separation of the six

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compounds with a relatively short running time 5 min (Fig. 3). In order to obtain a sharp and symmetrical peak, we tried different proportion of formic acid and ammonium acetate to mobile phase A. Eventually, 0.1% formic acid containing 1 mM ammonium acetate was selected. The appropriate proportion of formic acid and ammonium acetate promoted the ionization efficiency of analytes. Plasma protein precipitation was adopted in this assay. Compared with liquid-liquid extraction (LLE) and solid-phase extraction (SPE), protein precipitation was less time-consuming with higher reproducibility and recovery. Acetonitrile and methanol are two commonly used precipitation agents. To obtain better precipitation results, methanol, methanol–acetonitrile (50:50, v/v) and acetonitrile were investigated. Finally, acetonitrile was proved to be the best precipitant due to its lower interferences, higher yields and consequently cleaner samples, and the results was consistent with the previously reported findings[20]. To reduce the solvent effect and maintain the peak shape, we diluted the supernatant with the mobile phase A/B (80:20, v/v) in a 1:4 (v/v) ratio. 3.2. Selection of IS Because of similar chemical properties and the identical retention time with the analytes, generally, the isotope IS was the first choice for the LC-MS/MS analysis. Nimesulide-d5, the isotope of nimesulide and the analog of nimesulide metabolites, was adopted in this experiment. It successfully offset the matrix effect in the ionization and eliminated the differences during the sample pretreatment. 3.3. Method validation

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A thorough and complete method validation was carried out in accordance with the Guideline on Bioanalytical Method Validation[21]. 3.3.1. Specificity As shown in Fig. 3, the retention time of nimesulide, M1, M2, M3, M4 and IS was 2.78 min, 2.34 min, 2.09 min , 1.58 min, 2.50 min and 2.78 min, respectively. All of the analytes were well separated. Endogenous interferences were less than 20% LLOQ for analytes and less than 5% for IS. Fig. 4(1) shows the blank sample. The LLOQ for every analyte is shown in Fig. 4(2). 3.3.2. Interferences among analytes and IS Analysis of the active LLOQ calibrators showed that the mean peak areas at the expected retention time of nimesulide, M1, M2, M3 and M4 were ≤20% of the mean peak area. The IS sample without any analyte, the mean peak area at the expected retention time of the internal standard was ≤5% of the mean peak area of internal standard from calibration curve. Fig. 4(3) shows the chromatogram of each analyte at the concentration of ULOQ. 3.3.3. Calibration curve The calibration curves were evaluated by using a least-squares linear regression analysis. The equations of calibration curves were weighted by 1/x2. The calibration curves of the analytes showed excellent linearity over the studied concentration range (10–2000 ng/mL for M2 and M3, 20–4000 ng/mL for M1 and M4, and 40-8000 ng/mL for nimesulide). The correlation coefficients (r) of all five analytes were ≥ 0.9972, and the deviation of each point on calibration curve was less than 5%. The

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regression equations for calibration curves were as follows: y=0.00449x+0.50883 (r=0.9992)

for

nimesulide,

y=0.00433x+-0.00384

(r=0.9981)

for

M1,

y=0.00322x+0.0011 (r=0.9991) for M2, y=0.0166x+-0.0213 (r=0.9989) for M3, y=0.00201x+0.0092 (r=0.9972) for M4. 3.3.4. Intra- and inter-day accuracy, precision of LLOQ and QC samples The LLOQ values for nimesulide, M1, M2, M3 and M4 were 40 ng/mL, 20 ng/mL, 10 ng/mL, 10 ng/mL and 20 ng/mL, each showing a good sensitivity. The details of intra-and inter-day precision and accuracy for the five analytes are presented in Table 2. The results demonstrated that the LC-MS/MS method was accurate, sensitive and reproducible. 3.3.5. Recovery, matrix effect, dilution integrity and carry-over Table 3 presents the recoveries and IS-normalized matrix effect of different compounds. The mean recovery values of five analytes and IS were ≥87.58%, and the RSD recovery values for all compounds at three QC concentrations were ≤6.4%. The CV values of the calculated IS-normalized MF from six lots of blank matrices were ≤5.7%, which met the requirements of the guidance[21]. And the results indicated that ion suppression or enhancement from plasma matrix was negligible and no co-eluting endogenous substances interfered with the ionization of the analytes. The RSD and RE values of DQC samples (n=6) after dilution were 1.7% and -2.7 to 2.2%, respectively. The results met the criterion that the precision should not exceed 15% and accuracy within ±15% of the nominal value. The maximal peak area of carryover samples were ≤3.3% for all analytes and

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0.1% for IS of the minimum peak area of LLOQ, respectively. As a result, the carry-over did not affect the LC-MS/MS determination. 3.3.6. Stability The results of the stability test are summarized in Table 4. All of the analytes showed good stability and didn’t influence the concentration of the analytes. The stock solutions diluted by 100,000 folds were considered to be stable because their mean RE values were -1.2%, 1.9%, 2.3%, -3.9%, 2.1%, 3.5% for nimesulide, M1, M2, M3, M4 and IS, respectively. The working solutions were also deemed to be stable with the RE values being within ±5%. 3.4. Pharmacokinetic study at plasma level The

validated

LC-MS/MS

method

was

successfully

applied

to

the

pharmacokinetic study of nimesulide and its four possible metabolites in 12 healthy Chinese volunteers who were orally given 100 mg nimesulide tablets. LC-MS/MS detection showed that nimesulide and the hydroxylated metabolite M1 were the main existing forms in human plasma, and the result was consistent previously published findings[2, 14, 18]. However, M3, which was detectable in human urine following single oral administration of 100 mg nimesulide, was not detected in plasma. As we know, M3 results from four different metabolic steps, i.e., hydroxylation, nitro group reduction, N-acetylation and conjugation[1]. The possible reason might be that the metabolic conjugation didn't take place in human plasma. What is more, M2 wasn’t found in plasma either. We hypothesized that the biotransformation pathways of nimesulide in plasma couldn’t produce the methoxyl

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products. Another reason might be that its level was too low to be detected within the range of calibration curve. Finally, M4, the sulfated product of M1 in vivo, was observed as a minor metabolite in human plasma. Its retention time was 2.50 min with the validated method (Fig. 5(a)), however, greater retention time 2.59 min was observed in the plasma samples (Fig. 5(b)). On the basis of this shift in retention time, we inferred that the sulfated metabolite in human plasma might be an isomer of M4. To verify our speculation, we added 20 μL of M4 (2000 ng/mL) into 200 μL human plasma sample and then vortex-mixed them for 1 min after mixing with 600 μL precipitant acetonitrile. The mixture was centrifuged at 14,700 × g for 10min. After centrifugation, the supernatant liquid (200 μL) was transferred to a sample vial for later LC-MS/MS analysis. A prolonged chromatographic separation was conducted on a Welch Ultimate XB-C18 column (5 μm, 50 mm× 2.1 mm, Maryland, USA) protected by a Phenomenex ODS guard column (5 μm, 4.0 mm×3.0 mm, Torrance, CA, USA) with a mobile phase of acetonitrile-water (20:80, v/v) containing 1mM ammonium acetate and 0.1% formic acid at a flow rate of 0.6 mL/min for a extending LC analysis time (10 min). Fig. 5(c) showed that, the complete separation was difficult, but the LC separation result, with two peaks appearing under exactly the same mass spectrometric conditions, proved that the compound in plasma was the isomer of M4. Studies on the exact structure of M4* (isomer of M4) are now underway in our laboratory and the researches will further reveal whether or not M4 is a new metabolite of nimesulide. The proposed metabolism pathway in human plasma was shown in Fig. 6, with previously reported metabolism pathways [6, 7] provided

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The mean plasma concentration-time profiles of nimesulide, M1, M4* were shown in Fig. 7, and the related pharmacokinetic parameters obtained using DAS 3.2.7 software package were listed in Table 5. Based on the findings, the pharmacokinetic parameters of nimesulide and hydroxylated M1 were coincident with other previously reported results [22]. However, according to Chinese Pharmacopoeia 2015, blood collection duration should be 72 h or longer for more accurate evaluation. In this study, since the duration lasted only for 24 hours, the time was not long enough for adequate assessment of exposure level of M4. Inasmuch as we used the M4 as reference standard for the determination of M4*, the result only reflected the in vivo tendency of concentration change. In order to get relatively accurate pharmacokinetic parameters, standard M4* need be synthesized for the future research. 4. Conclusion In this study, the LC–MS/MS method for simultaneous quantification of nimesulide and its four synthesized metabolites of human plasma was successfully validated. The sensitivity and utility of the assay was demonstrated by detecting the plasma samples from 12 healthy volunteers after oral administration of 100 mg nimesulide tablets. On the basis of the pharmacokinetic study, we are led to reach the following conclusions (1) Nimesulide and the hydroxylated metabolite M1 were the main forms in plasma, and their pharmacokinetic parameters were close to previously reported results; (2) M3 and M2 were not detectable in plasma, and the reasons might be that the metabolic conjugation didn't take place in human plasma or the biotransformation pathways of nimesulide in plasma couldn’t produce the methoxyl

18

products; (3) M4* in human plasma had the same mass transition behaviors as M4; (4) One more metabolite of nimesulide was discovered by analyzing human plasma samples. The research provided experimental basis for further research on metabolic activation of nimesulide, and for more comprehensive conjecture of its metabolic pathways.

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N-(3-Arylaminopyridin-4-yl)alkanesulfonamides as pyridine analogs of nimesulide: Cyclooxygenases inhibition, anti-inflammatory studies and insight on metabolism, Eur. J. Med. Chem. 74 (2014) 12-22. [13] P. Ferrario, M. Bianchi, Simultaneous determination of nimesulide and hydroxynimesulide in rat plasma, cerebrospinal fluid and brain by liquid chromatography using solid-phase extraction, J. Chromatogr. B 785 (2003) 227-236. [14] A. Bernareggi, Clinical pharmacokinetics of nimesulide, Clin. Pharmacokinet. 35 (1998) 247-274. [15] R.E. Barrientos-Astigarraga, Y.B. Vannuchi, M. Sucupira, R.A. Moreno, M.N. Muscara, G. De Nucci, Quantification of nimesulide in human plasma by high-performance liquid chromatography/tandem mass spectrometry. Application to

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bioequivalence studies, J. Mass. Spectrom. 36 (2001) 1281-1286. [16] F.R. Cui, H.F. Wang, Z. Li, M. Ji, S.A. Khan, Pharmacokinetics and bioequivalence study of nimesulide tablets in healthy human volunteers, Lat. Am. J. Pharm. 32 (2013) 892-896. [17] C.M.B. Rolim, V. Porta, S. Storpirtis, Quantitation of nimesulide in human plasma by high-performance liquid chromatography with ultraviolet absorbance detection and its application to a bioequivalence study, Arzneimittel-forsch. 57 (2007) 537-541. [18] N.A. Alekseev, A.M. Drobyshevsky, D.A. Rozhdestvensky, Structure of chemical compounds, methods of analysis and process control solid-phase extraction and hplc determination of nimesulide and its active metabolite in human blood serum, Pharm. Chem. J. 44 (2011) 697-701. [19] S. Chandran, P. Ravi, P.R. Jadhav, R.N. Saha, A simple, rapid, and validated LC method for the estimation of nimesulide in human serum and its application in bioavailability studies, Anal. Lett. 41 (2008) 2437-2451. [20] I. Sora, T. Galaon, V. David, A. Medvedovci, Determination of nimesulide and its active metabolite in plasma samples based on solvent deproteinization and HPLC-DAD analysis, Rev. Roum. Chim. 52 (2007) 499-507. [21] European Medicines Agency Guideline on bioanalytical method validation, http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2011/ 08/WC500109686.pdf. [22] M. Chen, Y. Lu, T. Hang, Y. Ding, L. Li, P. Ma, Clinical pharmacokinetics and

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bioequivalence of nimesulide dispersible tablets by LC-MS/MS method, Chin. J. Pharm. Anal. 33 (2013) 30-33,38.

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Figure captions

Fig. 1. The structures of nimesulide, M1, M2, M3, M4 and IS.

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Fig. 2. The details of : (a) nimesulide ; (b) M1; (c) M2 ; (d) M3 ; (e) M4 ; (f) nimesulide-d5 (IS) m/z transitions.

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Fig. 3. The LC separated chromatogram of analytes.

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Fig. 4. Typical chromatograms of blank plasma (1); blank plasma sample spiked with five analytes at LLOQ (2); blank plasma sample spiked with five analytes at ULOQ (3); (A): nimesulide, (B): M1, (C): M2, (D): M3 and (E): M4.

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Fig. 5. Different retention time of (a) M4 in standard curve; (b) M4* in one of the human plasma samples; (c) the LC separation chromatogram of M4 and isomer M4*.

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Fig. 6. The reported and conjecture metabolism pathways of nimesulide in human plasma.

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Fig. 7. Mean plasma concentration-time profiles of nimesulide, M1 and M4* in twelve Chinese healthy volunteers after oral administration of 100 mg nimesulide tablets.

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Table 1 The optimal ESI-MS/MS parameters, including MRM parameters, collision energy (CE), declustering potential (DP), entrance potential (EP) and cell exit potential (CXP) of five analytes and IS. Analyte

Q1 Mass (Da)

Q3 Mass (Da)

Dwell Time (msec)

Collision Energy (eV)

Declustering Potential (eV)

Entrance Potential (eV)

Cell Exit Potential (eV)

IS Nimesulide M1 M2 M3 M4

312.0 307.0 323.0 349.0 335.0 419.0

234.0 229.0 245.0 269.0 255.0 259.0

80 80 80 80 80 80

-25 -55 -40 -25 -25 -31

-50 -50 -50 -50 -50 -50

-10 -10 -10 -10 -10 -10

-10 -10 -10 -10 -10 -10

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Table 2 Intra-day and Inter-day precision and accuracy. Analyte

Nimesulide

M1

M2

M3

M4

Nominal concentration(ng/ml)

Intra-day (n=6)

Inter-day (n=3)

Calculated concentration (ng/ml)a

RSD (%)

RE (%)

Calculated concentration (ng/ml)a

RSD (%)

RE (%)

40

39.5 ± 2.4

6.1

-1.2

40.1 ± 2.5

6.2

0.3

120

121.1 ± 5.3

4.4

0.9

117.9 ± 5.6

4.8

-1.7

3000

2962 ± 60

2.0

-1.3

2964 ± 96

3.2

-1.2

6000

5920 ± 89

1.5

-1.3

5841 ± 215

3.7

-2.7

20

19.6 ± 1.0

5.1

-2.1

20.6 ± 0.8

3.7

3.2

60

60.4 ± 1.7

2.9

0.7

60.5 ± 2.1

3.5

0.8

1500

1485 ± 28

1.9

-1.0

1514 ± 47

3.1

1.0

3000

2859 ± 56

2.0

-4.7

2893 ± 85

3.0

-3.6

10

10.4 ± 0.4

4.0

3.8

9.9 ± 0.6

6.0

-1.0

30

29.2 ± 1.0

3.3

-2.8

29.8 ± 1.2

3.9

-0.7

750

742.1 ± 19.0

2.6

-1.1

745.3 ± 22.1

3.0

-0.6

1500

1524 ± 104

3.1

1.6

1484 ± 72

4.8

-1.1

10

9.6 ± 0.4

4.6

-4.2

9.5 ± 0.4

4.5

-4.8

30

30.7 ± 0.4

1.3

2.3

30.6 ± 0.7

2.4

1.8

750

762.1 ± 19.4

2.5

1.6

752.4 ± 31.9

4.2

0.3

1500

1442 ± 22

1.5

-3.9

1440 ± 31

2.1

-4.3

20

19.9 ± 0.6

2.9

-0.4

19.8 ± 1.2

6.0

-1.1

60

60.0 ± 1.2

2.0

0.0

60.6 ± 2.1

3.5

1.0

1500

1572 ± 68

4.3

4.8

1513 ± 77

5.1

0.9

3000

3066 ± 94

3.1

2.2

2961 ± 119

4.0

-1.3

a: (mean ± SD) ng/ml 32

Table 3 Recovery and matrix effect. Analyte

Nimesulide

M1

M2

M3

M4 IS

Recovery(n=5)

Nominal concentration(ng/ml)

(mean accuracy ± SD)%

120 3000 6000 60 1500 3000 30 750 1500 30 750 1500 60 1500 3000 34

89.3 ± 3.7 89.3 ± 3.6 90.2 ± 4.1 86.6 ± 4.2 88.7 ± 3.2 87.6 ± 2.0 92.5 ± 4.2 94.0 ± 3.7 95.0 ± 2.4 91.9 ± 3.5 93.0 ± 4.0 91.3 ± 2.6 89.9 ± 1.5 90.2 ± 2.6 91.4 ± 5.9 89.6 ± 3.4

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IS-Normalized Matrix Effect(n=6) RSD (%) 4.2 4.0 4.6 4.8 3.7 2.3 4.5 3.9 2.6 3.8 4.3 2.9 1.7 2.8 6.4 3.7

(mean accuracy ± SD)%

98.1 ± 3.5 — 98.1 ± 2.0 90.3 ± 5.1 — 96.6 ± 3.2 113.6 ± 2.6 — 110.7 ± 6.3 111.9 ± 1.9 — 112.2 ± 5.2 113.4 ± 5.3 — 112.5 ± 4.2 —

RSD (%) 3.6 — 2.0 5.6 — 3.3 2.3 — 5.7 1.7 — 4.7 4.7 — 3.7 —

Table 4 Stability of nimesulide and its metabolites in human plasma (n=3). Stability in different storage condition (mean ± SD) % (n= 3) Nominal concentration Analyte 24h at room (ng/ml) 24h at autosampler 3 freeze-thaw cycles 38 days at -80 oC temperature 120 99.6 ± 4.9 103.0 ± 1.1 99.1 ± 2.9 98.1 ± 2.8 Nimesulide 3000 98.5 ± 2.4 100.0 ± 1.9 96.7 ± 2.7 97.8 ± 7.8 6000 96.6 ± 4.1 98.6 ± 2.4 98.5 ± 1.1 100.7 ± 3.1 60 102.4 ± 0.4 97.2 ± 1.1 100.6 ± 1.6 101.8 ± 5.1 M1 1500 100.8 ± 2.1 101.2 ± 3.0 103.1 ± 2.9 100.3 ± 2.5 3000 98.2 ± 1.0 101.7 ± 1.5 103.5 ± 4.6 96.4 ± 3.1 30 104.7 ± 3.7 97.4 ± 3.1 97.0 ± 3.7 95.4 ± 6.0 M2 750 99.5 ± 3.5 102.6 ± 5.5 99.7 ± 4.0 101.0 ± 3.2 1500 99.4 ± 7.6 101.0 ± 4.0 101.1 ± 4.0 99.4 ± 1.9 30 99.8 ± 4.7 96.5 ± 7.3 98.3 ± 2.7 99.3 ± 0.9 M3 750 97.2 ± 1.7 100.9 ± 4.0 97.6 ± 1.8 98.5 ± 6.2 1500 100.4 ± 4.5 98.7 ± 4.3 97.4 ± 5.3 99.0 ± 1.8 60 99.7 ± 2.7 101.1 ± 2.3 102.6 ± 5.3 99.8 ± 2.2 1500 97.3 ± 1.7 97.0 ± 5.8 98.9 ± 2.7 99.2 ± 3.2 M4 3000 104.3 ± 0.9 101.7 ± 4.8 97.9 ± 0.4 98.2 ± 2.6

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Table 5 Pharmacokinetic parameters of nimesulide,M1 and M4* in 12 healthy Chinese subjects after oral administration of 100mg nimesulide tablets (n=12, mean ± SD). Parameters t1/2 (h) Cmax (ng/ml) Tmax (h) Vz/F (L) CLz/F (L/h) AUC0-t (μg/Lh) AUC0-∞ (μg/Lh) Nimesulide 4.8 ± 1.7 5099 ± 1675 3.7 ± 0.8 16.0 ± 4.6 2.7 ± 1.4 45000 ± 23000 49000 ± 26000 M1 7.9 ± 4.2 1560 ± 679 5.7 ± 1.9 52.3 ± 27.6 4.9 ± 1.7 19000 ± 6500 23000 ± 7900 M4* 11.8 ± 6.2 1106 ± 302 8.7 ± 3.1 69.2 ± 31.2 4.4 ± 1.3 16248 ± 3280 24013 ± 6745

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