MS method for direct determination of ibuprofen enantiomers in human plasma

Accepted Manuscript Title: Critical development by design of a rugged HPLC-MS/MS method for direct determination of ibuprofen enantiomers in human plasma Author: Natalija Nakov Rumenka Petkovska Liljana Ugrinova Zoran Kavrakovski Aneta Dimitrovska Dobrin Svinarov PII: DOI: Reference:

S1570-0232(15)00241-X http://dx.doi.org/doi:10.1016/j.jchromb.2015.04.029 CHROMB 19420

To appear in:

Journal of Chromatography B

Received date: Revised date: Accepted date:

8-10-2014 27-2-2015 19-4-2015

Please cite this article as: N. Nakov, R. Petkovska, L. Ugrinova, Z. Kavrakovski, A. Dimitrovska, D. Svinarov, Critical development by design of a rugged HPLC-MS/MS method for direct determination of ibuprofen enantiomers in human plasma, Journal of Chromatography B (2015), http://dx.doi.org/10.1016/j.jchromb.2015.04.029 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.

Highlights  Fully validated LC-MS/MS method for analysis of ibuprofen enantiomers in plasma

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 Enantiomers were chromatographically separated without derivatization  Matrix effect investigation showed that LLE yields cleaner extracts than the SPE

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 Confirmation of the absence of R/S interconversion during sample processing

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 The method was successfully applied on volunteers using only 50μL sample volume

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Critical development by design of a rugged HPLC-MS/MS method for direct

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determination of ibuprofen enantiomers in human plasma

Natalija Nakov1,*, Rumenka Petkovska1, Liljana Ugrinova1, Zoran Kavrakovski1, Aneta

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Faculty of Pharmacy, University Ss Cyril and Methodius, 1000 Skopje, Macedonia

Alexander University Hospital, Faculty of Medicine, Medical University, 1431 Sofia,

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Dimitrovska1, Dobrin Svinarov2

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*The corresponding author:

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Bulgaria

M Sci Natalija Nakov

Institute of Applied Chemistry and Pharmaceutical Analysis, Faculty of Pharmacy, University “Ss. Cyril and Methodius”, Mother Tereza 47, 1000 Skopje, Macedonia Tel.: +389 023126024 Fax: +389 023223143 E-mail address: [email protected], [email protected]

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Abstract Development and validation of a HPLC-MS/MS method for direct determination of R- and S-ibuprofen (IBU) in human plasma without a need of derivatization or other complexities

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such as postcolumn infusion of solvents or reagents was performed. Critical steps were investigated during method development using experimental design to achieve a reliable and

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rugged assay. The LC-MS/MS separation of R-Ibu and S-Ibu was obtained on Lux Cellulose chiral column utilizing 0.1 % (v/v) acetic acid in mixture of methanol and water (90:10 % v/v)

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as a mobile phase. Two types of extraction procedure for IBU and Ketoprofen (internal

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standard, IS) were optimized using Full factorial 32 design (LLE) and D-Optimal Experimental Design (SPE). Excellent recovery values, 80% (mean) and 95% (mean) for

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LLE and SPE respectively, were obtained using 50 μL plasma. The matrix effect was assessed for both of the extraction procedures, including hyperlipidaemic and haemolyzed

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plasma. The extensive investigation of matrix effect showed that LLE yields cleaner extracts

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than the SPE. The result of the investigation of in vitro interconversion of R-Ibu and S-Ibu showed that it does not occur under the influence of pH, temperature, and in the overall

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analytical procedure. The validation data, adhered to EMA guideline for validation of bioanalytical methods, showed that the proposed method provides accurate and reproducible results in range of 0.1-50 mg/L with a lower limit of detection of 0.02 mg/L. The applicability of the method was demonstrated through determination of R-Ibu and S-Ibu in human plasma after oral administration of 400 mg rac-Ibu. Keywords: ibuprofen enantiomers, LC-MS/MS, sample preparation, experimental design, matrix effect, in vitro chiral interconversion

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1. Introduction Rac-ibuprofen (rac-Ibu) was introduced in the late sixties as a safe NSAID drug for

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treatment of wide range of medical ailments, including pain, fever, inflammation and arthritis. It has been established that S-(+)enantiomer of ibuprofen (S-Ibu) is almost entirely

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responsible for the anti-inflammatory action of rac-Ibu and the inactive R-(-)enantiomer

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(R-Ibu) in vivo undergoes a unidirectional metabolic inversion to its active form [1-4]. Ibuprofen (Ibu) is present on the market only as a racemic mixture because the production of

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single-isomer product and its enantiomerical purification has been limited by process difficulties [5].

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The enantiomers of Ibu differ greatly in their pharmacological and pharmacokinetic properties, so it is important to adopt stereospecific assay methodology. Stereospecific

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chromatographic separation requires conversion of enantiomers either to diastereoisomers

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using homochiral reagent (indirect methods) or to diastereomeric complexes using chiral

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stationary phase (direct methods). The reported indirect HPLC methods with UV [6] or FLD detection [7-9] require large sample volume (0.5 mL plasma) and extended sample preparation time due to derivatization process and are poorly suited for bioequivalent studies where large number of samples are included. Generally direct HPLC methods are more convenient for chromatographic separation because the drawbacks of the derivatization process are avoided. However, the reported direct HPLC-UV methods for determination of Ibu [10-12] also have some disadvantages such as large sample volumes and poor selectivity. In the recent years tandem mass spectrometry is widely used for quantitative determination of drugs. Reddy’s et al. [13] and Mendez et al. [14] described a LC-MS/MS method for quantification of Ibu in human plasma, but these methods are not stereoselective. Most of the chiral columns require mobile phases which are not compatible with the MS 4

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system; therefore most of the published LC-MS bioanalytical methods for determination of Ibu enantiomers use derivatization process [15-17]. Bonato et al. [18] reported direct HPLCMS/MS method where the problem of the compatibility of the mobile phase with the MS

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system was overcome with the post-column addition of NH4OH solution delivered by a syringe pump. However the baseline separation of the Ibu enantiomers was not achieved (Rs

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= 1.25) and the plasma volume required for extraction was 0.5 mL. An HPLC-MS/MS method involving direct determination of Ibu enantiomers in rat plasma was described by

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Cardoso et al. [19]. Even though the method showed good recovery values, the

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chromatographic separation was within 25 min, the sample volume was 0.2 mL and the extraction procedure was time-consuming using large volume of organic solvent (5 mL).

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Though several bioanalytical methods employing HPLC-MS/MS have been published, the evaluation of matrix effect is lacking [13, 14 and 18]; the investigation is not in accordance to

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EMA guideline for bioanalytical method validation [15] or hiperlipidaemic plasma was not

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included [19]. In addition, the previously reported HPLC/MS methods for indirect [13-15] and direct [18, 19] determination of Ibu enantiomers in biological fluids did not include

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investigation of the possibility of chiral interconversion during the sample preparation process. The GC/MS bioanalytical methods for analysis of Ibu presented in the literature have same limitations such as lack of enantioselectivity [20], time-consuming derivatization procedure [21, 22], large sample volume or insufficient sensitivity [20, 22]. In this manuscript LC-MS/MS method for determination of Ibu enantiomers in human

plasma without a need of derivatization or postcolumn infusion was presented. The aim of this study was to investigate the critical steps that influence the quality of the data obtained from the bioanalytical method during the method development. The approach of incorporation quality by design in course of method development was emphasized to ensure

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the reliability and ruggedness of the validated method once applied in practice for pharmacokinetic and bioequivalent studies. Considering that sample preparation is one of the most critical steps in bioanalysis, it was

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necessary to develop simple extraction procedure that generates good recovery values and clean extracts. For that purpose two types of sample preparation procedures, liquid-liquid

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extraction and solid-phase extraction (LLE and SPE), were optimized using experimental design. The matrix effect (ME), which is the second critical topic for ensuring data quality in

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LC-MS/MS, was extensively investigated for both of the extraction procedures. Additionally,

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comparison between LLE and SPE procedure in terms of extraction recovery and ME was done. The possibility of chiral interconversion during the sample preparation is one of the

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most critical steps in enantioselective assays and should be considered in time of enantioselective bioanalytical method development. Therefore the influence of pH and

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temperature on the in vitro interconversion of R-Ibu and S-Ibu was investigated.

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Once the critical steps were assessed, validation of the proposed method in accordance with the EMA guideline for bioanalytical method validation was conducted. The applicability

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of the validated method was confirmed by determining the concentrations of R-Ibu and S-Ibu in human plasma after oral administration of ibuprofen capsule on healthy volunteers.

2. Materials and methods

2.1 Materials

R-(-)ibuprofen and S-(+)ibuprofen reference standards were supplied from Sigma-Algrich and Rac-ibuprofen and ketoprofen reference standards were obtained from EDQM (Strasbourg, France). Analytical grade formic acid, acetic acid, ethylacetate and isopropanol were supplied from Sigma-Aldrich, Germany. MS grade methanol and water were from Merck, Germany. Whole blood obtained from healthy volunteers was collected into EDTA 6

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test tubes and centrifuged at 5500 rpm for 5 min in order to produce blank plasma source. 2.2 Instrumentation LC-MS/MS equipment consisted of Surveyor Autosampler, Surveyor LC Pump and TSQ

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Quantum Discovery Max triple quadrupole mass spectrometer (Thermo Scientific, USA). 2.3 LC conditions

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The chromatographic separation was performed using Lux Cellulose 3 (250 x 4.6mm, 5µm particle size) purchased from Phenomenex and maintained at 25°C. Mobile phase

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consisted of a 0.1 % (v/v) acetic acid in mixture of methanol and water in ratio 90:10.

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Analyses were conducted at a flow rate of 0.6 mL/min and the autosampler temperature was set at 10°C.The injection volume was 10 μL and the chromatographic time was 14 minutes.

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Methanol and water in ratio 50:50 (v/v) was used as a sample solvent. 2.4 MS conditions

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The mass spectrometer was operated in the selected reaction monitoring (SRM) mode

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using negative electrospay ionization, ESI(-). The parameters for the SRM analysis were: spray voltage 5500 V, sheet gas pressure 60 Arb units, ion sweep gas pressure 7 Arb units,

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auxiliary gas pressure 1 Arb units and capillary temperature 330°C. The SRM transitions of precursor to parent ions used for quantification were as follows: m/z 205.1→161.3 for Ibu and m/z 253.2→209.2 for ketoprofen (internal standard, IS). The collision energy was 8 V for Ibu and 10 V for the IS.

2.5 Preparation of calibration standard (CS) and quality control (QC) samples The stock solution of rac-Ibu in methanol (2 mg/mL) and the stock solution of IS in

methanol (1 mg/mL) were prepared and stored at -20°C. The stock solution of rac-Ibu was further diluted with solvent to obtain series of working standards solutions. The calibration standards (CS) and quality control (QC) samples were prepared by spiking 50 μL of rac-Ibu working standard solutions to 950 μL blank human plasma. The final concentration of R-Ibu

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and S-Ibu in the CS was in the range from 0.1-50 mg/L. IS working solution of 5 mg/L was prepared by dilution of IS stock solution in 50% methanol in water. 2.6 Extraction procedure

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2.6.1 Optimization of LLE and SPE procedure using Experimental Design Full factorial 32 design and D-Optimal experimental design were applied for optimization

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of LLE and SPE procedure, respectively. MODDE 10.1 Software (Umetrics, Umea, Sweden) [23] was used for design of experiments and optimization. The designed experiments were

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conducted on blank plasma spiked with rac-Ibu and IS in concentration of 2 mg/L and 5 mg/L respectively.

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For the optimization of the LLE procedure, using Full factorial 32 design, nine

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experiments were performed evaluating two experimental factors (type of buffering solvent and type of organic solvent) at three factor levels (Table 1).

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For the optimization of SPE procedure D-Optimal experimental design was employed.

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The influence of type of buffering solvent, type and volume of wash solvent and type and volume of elution solvent was investigated. In order to conduct estimation of the influence of

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the type and volume of wash solvent and type and volume of elution solvent, using two types of buffering solvent (0.15 M HCl or 0.2 M KH2PO4 at pH 2), two sets with 25 experiments were performed. The difference in the sets was regarding the type of buffering solvent added to the plasma before the loading step. The studied experimental factors and their levels are presented in Table 2.

2.6.2 Optimized LLE procedure Plasma samples (50 μL) were spiked with 50 μL IS working solution of 5 mg/L, and

50 μL of 0.15 M hydrochloric acid. The samples were vortex-mixed for 30 s, 1 mL ethylacetate (EtAc) was added, and extraction was performed by vortex-mixing for 10 min. Then samples were centrifuged at 10500 rpm for 5 min, the upper organic layer was

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transferred to another tube and evaporated in vacuum centrifuge for 30 min. The dried residues were dissolved in 100 μL 50% methanol in water and 10 µl were injected into the LC-MS/MS system.

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2.6.3 Optimized SPE procedure The SPE procedure was performed on manual manifold using Strata-X-Drug N Polymer

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RP cartridge (Phenomenex, 60mg/3mL). Plasma samples (50 μL) were spiked with 50 μL IS working solution of 5 mg/L and 50 μL of 0.15 M hydrochloric acid, followed by 30 s vortex-

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mixing. The SPE procedure was carried out according to following steps: a) loading the

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sample; b) washing with 1 mL mixture of methanol and water in ratio 15:85 (v/v); e) drying for 3 min under vacuum; c) elution with 1 mL mixture of ethylacetate and isopropanol

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(EtAc/IPA) in ratio 80:20 (v/v). The eluate was evaporated in vacuum centrifuge for 60 min. The dried residues were dissolved in 100 μL 50% methanol in water and 10 µl were injected

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2.7 Matrix effect investigation

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into the LC-MS/MS system.

ME was determined by comparing the responses of blank plasma extracts (obtained from

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eight different lots, including hyperlipidaemic and haemolyzed plasma) spiked postextraction with analytes and IS with response of analytes from neat standard solution (analytes and IS in solvent). The matrix factor (MF) for Ibu was determined at low QC (LQC) and high QC (HQC), whereas the MF for IS was determinate at single concentration of 5 mg/L. The IS normalized MF was obtained by dividing the Ibu MF by the MF of IS. The ME has been evaluated for both sample preparation procedures (LLE and SPE) and comparison between them has been made. 2.8 In vitro investigation of chiral interconversion The influence of pH value on chiral interconversion at different time points (0; 0.5; 1; 2 and 24h) was investigated. The R-Ibu and S-Ibu solutions were prepared at 400 mg/L

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separately in three different solvents: 0.1% acetic acid in 50% methanol (pH 3.5), 10 mM ammonium acetate buffer at pH 5.0 and 10 mM ammonium acetate buffer at pH 8.0. The prepared solutions were stored at room temperature (RT). After the respective storage time,

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each of the solutions was diluted 1:10 with solvent and injected in the LC-MS/MS system. The concentration of enantiomers yield in the final solutions was 40 mg/L.

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Experiments for investigation of temperature influence on interconversion were performed on blank plasma, spiked with Ibu enantiomers (40 mg/L) stored at 4°C and 25°C.

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After 5h and 24h, the samples were extracted according the LLE protocol described in 2.5.2.

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Also, the possibility of post preparative interconversion was investigated on plasma extracts, kept for 18h in autosampler at 10°C.

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For S-Ibu, the percentage of interconversion was expressed as AreaS-Ibu / Area(S-Ibu + R-Ibu) x 100, obtained from R-Ibu solution. For R-Ibu, the percentage of interconversion was

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2.9 Method validation

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expressed as Area R-Ibu / Area (R-Ibu + S-Ibu) x 100, obtained from S-Ibu solution.

A validation according to the EMA guideline was performed for the determination of

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ibuprofen enantiomers in human plasma (EMA 2011) [24]. For selectivity assessment, blank samples from eight individual donors, including

hyperlipidaemic and haemolyzed plasma, were tested using the proposed method. The obtained results were compared with the lower limit of quantification (LLOQ) samples (0.1mg/L).

The linearity of the method was assessed in the range from 0.1-50 mg/L for each Ibu

enantiomer. A calibration curve of seven concentration levels was fitted to weighted (1/x2) linear regression. The accuracy and precision of the method were evaluated by within-run (n=5) and between-run (n=30) assay using QC samples at concentrations of 0.1mg/L (LLOQ); 0.2 mg/L (low QC, LQC), 20mg/L (medium QC, MQC) and 40 mg/L (high QC,

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HQC) and the results obtained were expresses as percent accuracy and coefficient of variation (CV, %). Stability of rac-Ibu spiked in human plasma was evaluated by analyzing replicates (n=3)

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that were exposed to different conditions at LQC and HQC. Short term stability studies included investigation of stability on spiked plasma at RT and at 4°C, left for 5h and 24h.

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Post preparative stability of extracts included 18h resident time in autosampler, in order to verify if they can be analyzed in long batch series and 8h at RT. Stability after three freeze-

2.10 Applicability of the proposed method

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accuracy against theoretical (spiked) concentrations.

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thaw cycles of 24 h each was also determined. Stability results were expressed as percent

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For the needs of examination of the applicability of the method, five adult volunteers, each receiving single dose of 400 mg rac-Ibu were recruited. Subjects had no history of drug

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ingestion in the preceding 2 weeks. Venous blood samples were collected from the subjects

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into heparinised tubes immediately before and at the time of 20, 40, 60, 75, 90, 120, 150, 180, 210, 240, 270, 360, 480, 600, 720 and 1400 minutes after oral administration of Ibu capsule.

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After centrifugation, the plasma samples were transferred into clean tubes and kept at -20ºC until analysis. Maximum plasma concentrations (Cmax) of R-Ibu and S-Ibu were recorded as observed.

3. Results and discussion

3.1 Optimization of LC-MS/MS conditions Most of the chiral columns, protein-based or polysaccharide-based, require mobile

phases which are not suitable for the MS system. Based on this, we selected Lux Cellulose 3, a cellulose based stationary phase which can be used under reverse-phase conditions. The use of ammonium formate, which is a common component of mobile phase in ESI(-) mode, resulted in losing separation between the enantiomers. Although chiral resolution was 11

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achieeved with addition of formic acid in methanol/water mobile phase, the presence of formic acid lead to formation of adduct ion of Ibu with m/z 251 and in addition, the molecular ion of Ibu (m/z 205) could not be obtained. To avoid this problem, we replaced, formic acid

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with acetic acid, and thus Ibu molecular ion, which was further fragmented to its major product ion (m/z 161), was obtained. The selected mobile phase is compatible with the MS

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system and the chromatographic separation is within 13 min, in contract to 25 min reported by Cardoso et al. [19]. In addition, good resolution between the enantiomers (Rs = 3) was

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obtained (Fig.1a).

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The quality of the internal standard is one of the important factors in a quantitative method [27]. In this study we had no access to stable isotope labeled enantiomers of Ibu, and

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therefore several structural analogs (ketoprofen, diclofenac and naproxen) were evaluated during the selection of the IS. Diclofenac and naproxen could not experience the same

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ionization suppression/enhancement as the enantiomers because their retention times were

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25 min and 40 min, respectively. Ketoprofen was chosen as IS because it elutes close to the Ibu enantiomers (Fig. 1b), and in addition, its extraction recovery was similar to those of the

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enantiomers.

3.2 Development of liquid-liquid extraction (LLE) and solid-phase extraction (SPE) procedure using experimental design Sample preparation procedure is first of the critical steps in bioanalytical method

development and should be systematically assessed in order to obtain optimal recovery, good reproducibility and to minimize matrix effect. In addition, sample preparation is most laborintensive and error prone process in overall bioanalytical methodology and accounts up to 80% of the total bioanalysis time [28]. The most widely used extraction techniques in bioanalysis are LLE and SPE, and depending on the analyte, they exhibit different extraction recoveries and matrix effect. Therefore, in our study these two procedures were optimized

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using experimental design and afterwards comparison in terms of recovery and ME was done. Process efficiency (PE, %) of the enantiomers was chosen as a response parameter for evaluation of the influence of experimental factors as it reflects both extraction recovery and

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ME. In the optimization of LLE, Full factorial 32 design was applied. The critical

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parameters: type of buffering solvent added to plasma before extraction (0.15M HCl, 0.2M HCl and saturated solution of NaH2PO4), and type of organic solvent (1-chlorobutan, EtAc,

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and mixture of EtAc/IPA in ratio 95/5) used for extraction, were evaluated (described in

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section 2.6.1). In order to define the optimal conditions for obtaining good PE, Response Surface Methodology (RSM) for the enantiomers was employed. The 2D Response Contour

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diagrams of R-Ibu and S-Ibu (Fig. 2) have shown that the salting-out effect obtained with the addition of saturated solution of NaH2PO4, regardless the organic solvent polarity, provided

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PE around 60%. The results obtained from this contour diagram also indicated that the use of

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nonpolar extraction solvent (1-chlorobutan), independently from the buffering solvent, also led to low PE. As can be seen from Fig.2, PE above 90% could be obtained using HCl

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(0.15M HCl or 0.2M HCl) as a buffering solvent and more polar extraction solvent (EtAc or mixture of EtAc/IPA). The presence of IPA in the extraction solvent, compared to pure EtAc, did not improve PE significantly but it increased the evaporation time with 15 min. Based on that, pure EtAc was chosen as an extraction solvent. The SPE procedure was performed on Strata-X-Drug N Polymer cartridge (polymeric

mixed-mode sorbent) where the conditioning step was not required. Preliminary investigations have shown that the use of nonpolar elution solvent (1-chlorbutan) resulted in low PE. Several essential factors for development of SPE procedure, such as type and volume of the wash solvent and type and volume of the elution solvent were examined (detailed description in section 2.6.1). In this evaluation D-Optimal experimental design was

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employed. In order to investigate the influence of the buffering solvent (0.15 M HCl or 0.2 M KH2PO4 at pH 2) added to plasma before the loading step, two independent experimental sets were performed. Fig. 3a and 3b present the 4D Response Contour diagrams of R-Ibu obtained

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using 0.15 M HCl and 0.2 M KH2PO4 at pH 2, respectively. PE for S-Ibu from both experimental sets was similar to those of R-Ibu (diagrams not shown). Results obtained using

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this RSM (Fig. 3) showed that higher PE could be obtained with addition of acid instead of a buffer to the plasma before the SPE. Contour diagram of R-Ibu using 0.15 M HCl as a

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buffering solvent (Fig. 3a) showed that PE around 90% may be obtained when 1 mL of 15 %

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methanol is used for the washing step and 1 mL mixture of EtAc/IPa (80/20, v/v) is used as an elution solvent. Results presented in Fig. 3b, indicated that the use of buffer resulted in PE

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lower than 60% in all cases.

Calculation of extraction recovery for LLE and SPE was carried out by spiking of

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blank plasma extract with rac-Ibu and IS standard solutions at LQC and HQC (Table 3). The

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extraction yields with LLE procedure for R-Ibu at LQC and HQC were 83.3% and 74.4%, and for S-Ibu 85.9% and 72.8%, respectively. The previously reported LLE procedures gave

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similar (83%, 73%) [15, 18] or lower (69%, 64.9%) [13, 9] extraction recovery values using 0.1 mL [13, 15], 0.2 mL [19] or 0.5 mL [7, 9, 18] sample volume. The LLE procedure described in this study required only 50 μL of plasma and volume of organic solvent was reduced from 5 mL [19] to 1 mL, thereby shortening sample preparation time. The SPE seemed to give higher recovery than LLE ranging 92.2% to 98.8% for R-Ibu and 93.2 to 97.9% for S-Ibu. Recovery of the IS was similar to those of Ibu enantiomers, but still a bit higher for SPE. The CV % for both extraction procedures was lower than 10%, thus confirming acceptable repeatability. 3.3 Investigation and comparison of matrix effect for LLE and SPE procedure

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In general, ME is directly related to the sample clean-up procedure; so along with extraction recoveries investigation of ME is essential before choosing the optimal sample extraction technique. Therefore extensive investigation of ME for LLE and SPE procedure

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was performed using six lots of normal plasma and one lot of hyperlipidaemic and one lot of haemolyzed plasma at two concentration levels (LQC and HQC). The same plasma volume

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(50 μL) for both procedures was used, and dry residues obtained from LLE and SPE were reconstituted with same solvent (100 μL 50% methanol).

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Criteria used for comparison of LLE and SPE were not just the variability of the

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IS-normalized MF as recommended in EMA guideline [24], but also the ME itself [27]. Variation of the IS-normalized matrix factor for R-Ibu and S-Ibu obtained from LLE and SPE

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was within the acceptable limit of ±15% (Table 4 and Table 5, respectively) which indicated that MF for both procedures is consistent and not affected by the different plasma. However,

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in order to assess the cleanliness of the extracts obtained, it was necessary to evaluate the

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magnitude of ME itself and not just its variation. ME originating from SPE at LQC ranged 112.5-119.0% for R-Ibu, 115.1-137.6% for S-Ibu and 133.5-146.6% for IS, indicating that the

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SPE procedure generates ME in terms of ionization enhancement above 20% for the S-Ibu and above 30% for the IS. In contrast, ME obtained from LLE at LQC was between 88.397.6% for R-Ibu, 86.7-103.6% for S-Ibu, and from 107.3-114.3% for IS. ME of hyperlipidaemic and haemolyzed plasma was similar to that obtained from normal plasma for both procedures.

In this study the experimental data indicated that LLE gave cleaner extracts (less ME)

than SPE and provided sufficient extraction recovery. Therefore, it was chosen as an optimal procedure for extraction of Ibu enantiomers from human plasma. 3.4 In vitro investigation of chiral interconversion

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All chiral compounds may undergo in vitro chiral conversion occurring at physiological pH and temperature or under extreme pH and elevated temperature [28]. Therefore, investigation of the possibility for in vitro interconversion during sample

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processing is one of the critical steps in enantioselective method development. In this study, R-Ibu and S-Ibu were dissolved in acetic acid solution at pH 3.5;

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ammonium acetate buffer at pH 5.0 and ammonium acetate buffer at pH 8.0. These solutions were stored at RT and interconversion was assessed after 0.5; 1; 2 and 24h. Results at the

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investigated pH values indicated percent interconversion for R-Ibu of less than 0.5% and

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around 1% for S-Ibu, similar to percent interconversion obtained for the enantiomers when dissolved in 50% methanol in water.

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The influence of temperature was investigated on blank plasma spiked with R-Ibu and S-Ibu, separately. Samples kept at 25°C and at 4°C, were extracted after the 5h and

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24h and were compared to samples extracted immediately after preparation. In addition, the

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possibility for post-preparative interconversion was investigated on plasma extracts kept at 10°C for 18h in autosampler. Influence of temperature was found to be insignificant: in all

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cases the percent interconversion for R-Ibu and S-Ibu, was around 0.5% and 1%, respectively.

It could be summarized that in vitro interconversion of R-Ibu and S-Ibu does not

occur under the influence of pH, temperature, and the overall analytical procedure. Therefore the method proposed is appropriate to assess the extent of in vivo interconversion without a bias.

3.5 Method validation Validation experiments were performed on spiked plasma QCs extracted according the protocol for LLE procedure (section 2.6.2).

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Acceptable selectivity was demonstrated by response in blank matrix that were less than 12% at LLOQ for Ibu enantiomers, and less than 2% for the IS. LLOQ was determined in blank plasma (n=3) spiked with R-Ibu and S-Ibu at 0.1 mg/L. The signal/noise ratio of the

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enantiomers was higher than 10-times the response obtained from blank plasma. The established lower limit of detection (LLOD) of 0.02 mg/L was obtained using only 50 μL

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plasma. Typical SRM chromatograms of blank plasma, LLOQ and LLOD samples are presented in Figure 4.

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Calibration curves were linear (1/x2 weighted regression model) with correlation

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coefficient >0.995 for both enantiomers in the concentration range 0.1 - 50 mg/L. The back calculated concentrations for all calibration standards, including the LLOQ, were within

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acceptable limits of ± 15% of the nominal value. Accuracy and precision meet the acceptance criteria of ± 15% (Table 6).

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Considering the results obtained during the in vitro investigation of the chiral

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interconversion, the stability experiments during the validation process were conducted using racemic QCs. Stability data of spiked plasma and extracts (Table 7) indicate that Ibu

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enantiomers are stable under all investigated storage conditions and that no stability related problems would be expected during study sample analysis. 3.6 Applicability of the method on real study samples The applicability of the method described in this study was demonstrated through

determination of R-Ibu and S-Ibu in human plasma after oral administration of 400 mg racIbu. The mean human plasma concentration-time profile of volunteers is presented in Figure 5. The concentration of enantiomers increased rapidly after oral absorption, reaching maximum plasma concentrations after 120 minutes. The peak plasma concentration (mean ± SD, n=5) for R-Ibu and for S-Ibu were 14.54 ± 3.18 mg/L and 13.37 ± 2.28 mg/L. After this time point, the plasma concentration of R-Ibu and S-Ibu decreased gradually and after

17

Page 17 of 40

720 min was 0.46 mg/L and 0.80 mg/L, respectively. At the final sampling time (1400 min) plasma concentrations of enantiomers of Ibu were not observed. In each time point the concentration of S-Ibu exceeded the R-Ibu concentration, which was in line with the

ip t

literature data [9, 15, 18]. 4. Conclusions

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In this research, critical steps that influence the quality of the data obtained from direct HPLC-MS/MS for determination of Ibu enantiomers in human plasma were investigated, thus

us

highly reliable method was developed. The proposed method enables separation of Ibu

an

enantiomers using MS compatible mobile phase without splitting the column effluent or postcolumn addition of solvent. Two types of sample preparation procedures, LLE and SPE, were

M

optimized using experimental design and the selection of the optimal extraction procedure was based on the extraction recovery and the cleanliness of the extracts. Excellent recovery

d

values were obtained for both extraction procedures (80% for LLE and 95% for SPE).

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However, the ME investigations have shown that LLE yields cleaner extracts than the SPE, thus it was chosen as an optimal procedure for extraction of ibuprofen enantiomers from

Ac ce p

human plasma. Investigation of interconversion have shown that in vitro interconversion of R-Ibu and S-Ibu does not occur under the influence of different pH values or different temperature, thus bias caused by interconversion during the sample preparation was prevented. The major advantages of the proposed method are: enantioselectivity, small sample volume (50μL), simplicity of the extraction, no matrix effect and absence of chiral interconversion during the overall analytical procedure. Validation data showed that the proposed method provides accurate and reproducible results in range of 0.1-50 mg/L with a LLOD of 0.02 mg/L. The applicability of the method was demonstrated through determination of Ibu enantiomers in human plasma obtained after oral administration of rac-Ibu. 18

Page 18 of 40

Considering all of the advantages of the proposed method and the benefits gained with the used approach of incorporation a quality during the method development, it can be concluded that the method generate reliable bioanalytical data and could be successfully applied for

ip t

determination of ibuprofen enantiomers in human plasma for the needs of pharmacokinetic and bioequivalent studies.

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References

of ibuprofen, Xenobiotica 3 (1973) 589-598.

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[1] R.F.N. Mills, S.S.Adams, E.E. Cliffe, W. Dickinson, J.S. Nicholson, The metabolism

an

[2] S.S. Adams, P. Bredloff, G.C. Mason, Pharmacologycal differences between optical isomers of ibuprofen: Evidence for metabolic inversion of the (-) Isomer, J.

M

Pharmac. Pharmacol. 28 (1976) 156-157.

[3] J. Caldwell, A.J.Hutt, S. Gigleux-Fournel, The metabolic chiral inversion and

d

disposition of the 2-arylpropyonic acids and their biological consequences,

te

Biochem. Pharmacol. 37 (1988) 105-114. [4] J.M.Mayer, B. Testa, Pharmacodynamics, pharmacokinetics and toxicity of ibuprofen

Ac ce p

enantiomers, Drugs Fut. 22 (1997) 1347-1366.

[5] P.O.Carnavalho, Q.B. Cass, S.A. Calafatti, F.J. Contesini, R. Bizaco, ReviewAlternatives for the separation of drug enantiomers: Ibuprofen as a model compound, Braz. J. Chem. Eng. 23 (2006) 291-300.

[6] M.R.Wright, S.Sattari, D.R. Brocks, F.Jamali, Improved high-performance liquid chromatographic assay method for the enantiomers of ibuprofen, J.Chromatogr. 583 (1992) 259-265. [7] C.H. Lemko,G. Caillé, R.T. Foster, Stereospecific high-performance liquid chromatographic assay of ibuprofen: improved sensitivity and sample processing efficiency, J.Chromatogr. 619 (1993) 330-335. 19

Page 19 of 40

[8] S.C.Tan, S.H.D. Jackson, C.G. Swift, A.J. Hutt, Enantiospecific analysis of ibuprofen by high-performance liquid chromatography: Determination of free and total drug enantiomer concentrations in serum and urine, Chromatographia 46 (1997) 23-32.

ip t

[9] R.Canaparo, E.Muntoni, G.P. Zara, C. Della Pepa, E. Berno, M. Costa, M. Eandi, Determination of ibuprofen in human plasma by high-performance liquid

cr

chromatography: validation and application in pharmacokinetic study, Biomed. Chromatogr. 14 (2000) 219-226.

us

[10] S.Menzel-Soglowek, G. Geisslinger, K. Brune, Stereoselective high-performance

an

liquid chromatographic determination of ketoprofen, ibuprofen and fenoprofen in plasma using a chiral alpha 1-acid glycoprotein column, J. Chromatogr. A 532

M

(1990) 295-303.

[11] K.J. Pettersson, A.Olsson, Liquid chromatographic determination of the enantiomers

te

414-418.

d

of ibuprofen in plasma using a chiral AGP column, J. Chromatogr. A 563 (1991)

[12] W. Naidong, J.W. Lee, Development and validation of a liquid chromatographic

Ac ce p

method for the quantification of ibuprofen enantiomers in human plasma, J Pharm. Biomed. Anal. 12 (1994) 551-556.

[13] R. Reddy, I.S. Chandiran, K.N. Jayaverra, K.R.Divi, Quantification of ibuprofen in human plasma by high troughput liquid chromatography-tandem mass spectrometric method and its application in pharmacokinetics, Arch.Apll.Sci.Res. 2 (2010)101111.

[14] G.D. Mendes, F.D. Mendes, C.C. Domingues, R.A. de Oliveira, M.A. da Silva, L.S. Chen, J.O.Ilha, C.E. Fernandes, G. De Nucci, Comparative bioavailability of three ibuprofen formulations in healthy human volunteersInt. J Clin PharmacolTher. 36 (2008) 309-318.

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[15] A. Szeitz, A.N. Edginton, H.T.Peng, B. Cheung, K.W. Riggs, A validated enantioselective assay for the determination of ibuprofen in human plasma using ultra performance liquid chromatography tandem mass spectrometry (UPLC-

[16] S.

Ogawa,

H. Tadokoro,

M. Sato, T.

Hanawa, T.

ip t

MS/MS), AJAC 2 (2010)47-58. Higashi,

(S)-1-(4-

cr

Dimethylaminophenylcarbonyl)-3-aminopyrrolidine: A derivatization reagent for enantiomeric separation and sensitive detection of chiral carboxylic acids by

us

LC/ESI-MS/MS, J Chromatography B, 940 (2013) 7-14.

an

[17] S. Ogawa, H. Tadokoro, M. Sato, T. Higashi, Enantioselective determination of ibuprofen in saliva by liquid chromatography/tandem mass spectrometry with chiral

M

electrospray ionization-enhancing and stable isotope-coded derivatization, J Pharm and Biomed Anal, 98 (2014) 387-392.

d

[18] P.S. Bonato, M. Perpetua, F.M. Del Lama, R. de Carvalho, Enantioselective

te

determination of ibuprofen in plasma by high-performance liquid chromatographyelectrospray mass spectrometry, J Chromatogr.B 796 (2003) 413-420.

Ac ce p

[19] J.L.C. Cardoso, V.L. Lanchote, M.P.M. Pereira, N.V. de Moraes, J.S. Lepera, Analysis of ibuprofen enantiomers in rat plasma by liquid chromatography with tandem mass spectrometry. J. Sep. Sci. 37 (2014) 944-949.

[20] B.A. Way, T.R. Wilhite, C.H. Smith, M. Landt, Measurments of plasma ibuprofen by gas chromatography-mass spectrometry, J Clin Lab Anal. 11 (1997) 336-339.

[21] M. Zhao, C. Peter, M. Holtz, N. Hugenell, J. Koffel, L. Jung, Gas-chromatographymass spectrometric determination of ibuprofen enantiomers in human plasma using R(-)-2,2,2-trifluoro-1-(9-anthryl)ethanol as derivatizing reagent, J Chromatogr.B 656 (1994) 441-446. [22] P. Seideman, F. Lohrer, G.G. Graham, M.W. Duncan, K.M. Williams, R.O. Day, The

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stereoselective disposition of the enantiomers of ibuprofen in blood, blister and synovial fluid, Br J ClinPharmac. 38 (1994) 221-227. [23] http://www.umetrics.com/products/modde.

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[24] European Medical agency. Guideline on Validation of Bioanalitical Methods. Committee for Medical products for human use, 2011. Avaible from:

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http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2011/ 08/WC500109686.pdf

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development,Bioanalysis 2 (2010) 1051-1072.

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[25] D.Mulvana, Critical topics in ensuring data quality in bioanalytical LC-MS method

[26] P.L. Kole, G. Venkatesh, J. Kotecha, R. Sheshala, Recent advantages in sample

M

preparation techniques for effective bioanalytical methods, Biomed. Chromatogr. 25 (2011) 199-217.

d

[27] B.K.Matuszewski, M.L. Constanzer, C.M. Chavez-Eng, Stategies for the assessment

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of the matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS,

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Anal.Chem. 75 (2003) 3019-3030. [28] C.J. Briscoe, D.S. Hage, Factors affecting the stability of drugs and drug metabolites in biological matrices, Bioanalysis 1 (2009) 205-220.

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Figure captions Fig.1 SRM chromatogram of: a) Ibu enantiomers and b) IS (ketoprofen) under the optimized conditions: 0.1 % (v/v) acetic acid in mixture of methanol and water in ratio 90:10; flow rate

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of 0.6 mL/min.

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Fig. 2 2D Response contour diagram of R-Ibu and S-Ibu. [organic solvent: 90=chlorobutan,

0.2=0.2M HCl, 1=saturated solution of NaH2PO4]

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95=mixture of EtAc/IPA in ratio 95/5, 100= EtAc; buffering solvent: 0.15=0.15M HCl,

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Fig. 3 4D Response contour diagram of R-Ibu using buffering solvent: a) 0.15M HCL and b) 0.2 M KH2PO4 at pH 2.[wash (%) = methanol in water (%, v/v); Vwash (mL) = volume of

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wash solvent (mL); elution solvent = 80/20 % EtAc/IPA, 90/10 %EtAc/IPA, 100 % EtAc;

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Velution (mL) = volume of elution solvent (mL)]

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Fig.4 SRM chromatograms of: a) blank plasma; b) plasma spiked with Ibu enantiomers

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0.1 mg/L (LLOQ sample) and c) plasma spiked with Ibu enantiomers 0.02 mg/L (LLOD)

Fig.5 Mean plasma-time concentration profile obtained from five volunteers after single oral administration of 400 mg rac-Ibu

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Table 1 Evaluated factors and their levels for the optimization of LLE procedure Experimental factors

1-chlorobutan

Level 3 0.20 M HCl EtAc/ IPA (95/5, v/v)

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d

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Type of buffering solvent Type of organic solvent

Factor levels Level 2 NaH2PO4 saturated solution Ethylacetate (EtAc)

Level 1 0.15 M HCl

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Table 2 Evaluated factors and their levels for the optimization of SPE procedure

Wash solvent (methanol in water, % v/v) Volume of wash solvent Elution solvent

Level 3

10

15

20

1 x 1 mL EtAc/ IPA (80/20, v/v) 0.3

1 x 2 mL EtAc/ IPA (90/10, v/v) 0.5

2 x 1 mL EtAc (100, v/v) 1

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Volume of elution solvent (mL)

Factor levels Level 2

Level 1

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Experimental factors

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Page 25 of 40

Compound R-Ibu S-Ibu

0.2 40 0.2 40 5

Mean recovery (%) (n=6) LLE SPE 83.3 98.8 74.4 92.2 85.9 97.9 72.8 93.2 82.2 90.2

Coefficient of variation (CV, %) LLE SPE 1.5 4.7 4.7 6.5 4.1 5.6 2.9 7.1 5.4 7.1

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IS (ketoprofen)

Concentration (mg/L)

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Table 3 Absolute recovery values obtained from LLE and SPE procedure

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Table 4 ME investigation for LLE procedure High QC ( 40 mg/L)

IS ME (%)

R-Ibu IS norm. MF (%)

S-Ibu IS norm. MF (%)

R-Ibu ME (%)

S-Ibu ME (%)

IS ME (%)

88.3 93.8 91.2 94.8 92.3 97.6 95.9 108.7

86.7 103.0 96.1 96.1 98.5 103.6 101.4 107.8

107.3 112.2 108.9 114.3 112.1 113.7 108.9 118.5 Mean SD CV %

82.3 83.6 83.8 82.9 82.3 85.9 88.0 91.7 85.1 3.3 3.9

80.8 91.8 88.3 84.0 87.9 91.1 93.1 91.0 88.5 4.2 4.8

91.7 89.6 88.1 92.8 99.7 99.9 105.0 99.7

90.5 86.2 87.7 94.3 95.5 91.4 99.0 97.2

109.1 95.2 88.9 113.7 111.6 110.2 105.2 104.6 Mean SD CV %

R-Ibu IS norm. MF (%)

84.1 94.1 99.1 81.6 89.3 90.7 99.8 95.3 91.8 6.6 7.2

S-Ibu IS norm. MF (%)

83.0 90.5 98.6 82.9 85.6 83.0 94.1 92.9 88.8 6.1 6.8

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S-Ibu ME (%)

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Lot 1 2 3 4 5 6 lipidaemic haemolized

Low QC ( 0.2 mg/L) R-Ibu ME (%)

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LLE

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Page 27 of 40

Table 5 ME investigation for SPE procedure IS ME (%)

R-Ibu IS norm. MF (%)

S-Ibu IS norm. MF (%)

112.5 117.6 118.0 125.2 122.5 119.0 116.3 122.2

115.1 119.8 123.1 131.2 137.6 123.0 125.3 129.5

133.5 138.7 134.4 145.7 142.3 146.6 132.3 124.3 Mean SD CV %

84.0 84.8 87.8 85.9 86.1 81.1 87.9 98.4 87.0 5.1 5.9

86.3 86.4 91.6 90.0 96.7 83.8 94.7 104.2 91.7 6.7 7.3

R-Ibu ME (%)

92.0 91.9 96.5 97.2 99.0 92.0 94.5 98.9

S-Ibu ME (%)

82.0 84.0 88.8 90.0 92.3 85.3 89.0 95.3

IS ME (%)

R-Ibu IS norm. MF (%)

122.3 129.2 124.6 136.8 129.5 120.6 112.0 118.0 Mean SD CV %

75.2 71.2 77.5 71.1 76.4 76.3 84.4 83.8 77.0 5.0 6.5

S-Ibu IS norm. MF (%)

67.0 65.0 71.3 71.3 71.3 70.7 79.5 80.8 71.4 5.9 8.3

Ac ce p

te

d

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1 2 3 4 5 6 lipidaemic haemolized

High QC ( 40 mg/L)

S-Ibu ME (%)

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Lot

Low QC ( 0.2 mg/L) R-Ibu ME (%)

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SPE

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Page 28 of 40

Table 6 Within-run and between-run accuracy and precision for Ibu enantiomers in human plasma Between-run assays (n=30) R-Ibu S-Ibu Accuracy Precision Accuracy Precision (%) (CV %) (%) (CV %) 92.0 8.1 86.8 3.9 99.7 4.4 94.3 5.2 106.1 3.3 106.6 3.9 102.3 6.9 103.1 6.3

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te

d

M

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LLOQ LQC MQC HQC

Within-run assay (n=5) R-Ibu S-Ibu Accuracy Precision Accuracy Precision (%) (CV %) (%) (CV %) 91.8 6.6 88.2 2.4 99.2 2.8 94.1 1.4 106.2 1.2 108.3 3.0 105.7 3.4 107.5 2.5

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Quality control samples

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Page 29 of 40

Table 7 Stability of Ibu enantiomers in human plasma (n=3) 40 mg/L R-Ibu S-Ibu Accuracy CV Accuracy CV (Mean, %) (%) (Mean, %) (%)

1.9 3.1 1.6 1.3 2.0

104.4 97.0 103.8 102.0 104.0

3.3 0.2 1.0 3.7 1.9

97.5 97.5 98.8 99.8 106.9

91.6 105.1

3.3 1.2

93.2 105.5

1.9 2.8

93.2 105.2

1.4 3.1 2.8 1.7 1.9

94.0 98.2 99.1 101.1 108.3

1.5 1.5 2.7 2.3 1.7

1.3 1.5

91.7 105.4

2.6 1.7

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103.8 102.2 98.3 101.3 104.9

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d

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Short-term 5h at RT 24h at RT 5h at 4°C 24h at 4°C Freeze-thaw Post-preparative 18h at 10°C 8h at RT

0.2 mg/L R-Ibu S-Ibu Accuracy CV Accuracy CV (Mean, %) (%) (Mean, %) (%)

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Nominal concentration

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Page 30 of 40

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Figure 1A

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Figure 1B

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Figure 2

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Figure 3A

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Figure 3B

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Figure 4A

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Figure 4B

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Figure 4C

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Table 1

Table 1 Evaluated factors and their levels for the optimization of LLE procedure Experimental factors

1-chlorobutan

Factor levels Level 2 NaH2PO4 saturated solution Ethylacetate (EtAc)

Level 3 0.20 M HCl EtAc/ IPA (95/5, v/v)

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ce pt

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Type of buffering solvent Type of organic solvent

Level 1 0.15 M HCl

Page 40 of 40