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Abstract

In this research, as a part of the development of fast and reliable HPLC-MS/MS method for quantification of ibuprofen (IBP) enantiomers in human plasma, the possibility of IBP acylglucoronide (IBP-Glu) back-conversion was assessed. This involved investigation of in source and in vitro back-conversion. The separation of IBP enantiomers, its metabolite and rac-IBP-d3 (internal standard), was achieved within 6 min using Chiracel OJ-RH chromatographic column (150 × 2.1 mm, 5 μm). The followed selected reaction monitoring transitions for IBP-Glu (m/z 381.4 → 205.4, m/z 381.4 → 161.4 and m/z 205.4 → 161.4) implied that under the optimized electrospray ionization parameters, in source back-conversion of IBP-Glu was insignificant. The results obtained after liquid-liquid extraction of plasma samples spiked with IBP-Glu revealed that the amount of IBP enantiomers generated by IBP-Glu back-conversion was far <20% of lower limit of quantification sample. These results indicate that the presence of IBP-Glu in real samples will not affect the quantification of the IBP enantiomers; thereby reliability of the method was improved. Additional advantage of the method is the short analysis time making it suitable for the large number of samples. The method was fully validated according to the EMA guideline and was shown to meet all requirements to be applied in a pharmacokinetic study.

Introduction

Ibuprofen (IBP), an effective NSAID, is rapidly adsorbed after oral administration. The primary pathway of its metabolism is glucuronidation which results in formation of IBP acylglucoronide (IBP-Glu) (1, 2). The acylglucoronide metabolites are well known for their stability issues. These labile metabolites, present in the incurred samples, can convert back to their parent drug during sample preparation (in vitro back-conversion) and/or in the mass spectrometer (in source back-conversion), thereby jeopardizing accuracy and reproducibility of the bioanalytical method (3–6). Therefore, the possibility of bias introduced by metabolite back-conversion during the bioanalytical method development should be considered. The IBP enantiomers differ greatly in their pharmacological and pharmacokinetic properties; therefore, it is important to adopt stereospecific assay methodology. The stereospecific chromatographic separation could be achieved using derivatization reagent (indirect methods) or chiral stationary phase (direct methods). There are number of reports concerning determination of IBP enantiomers in plasma using high performance liquid chromatography with ultraviolet/fluorescent detection (HPLC-UV/FLD) (7–10) and HPLC with mass spectrometric (HPLC-MS) detection (11–16). Despite the importance of the metabolite interference testing, the reported HPLC-UV/FLD and HPLC-MS methods did not include investigation of the possibility of IBP-Glu back-conversion.

In addition, considering the large number of samples included in the bioequivalent studies, the use of high-throughput bioanalytical method is preferred. However, the existing literature data have shown that the reported high performance liquid chromatography with tandem mass detection (HPLC-MS/MS) did not provide short chromatographic runtime. Usually, the reported HPLC-MS/MS methods for analysis of IBP enantiomers in plasma that use chiral stationary phase have run times ranged from 12 (12) to 25 min (15). The indirect HPLC-MS/MS methods that use C18 chromatographic columns also did not provide separation of IBP enantiomers within short runtime and are ranged from 11 (17, 18) to 20 min (14).

In our previously published research, several critical steps that influence the quality of the data obtained from HPLC-MS/MS method for the analysis of IBP enantiomers in human plasma were investigated (16). The main objectives of this work were to further improve the method reliability and ruggedness through the investigation of the IBP-Glu back-conversion and to enhance the throughput of the existing method. The need for the detailed investigation of the IBP-Glu interference on the determination of IBP enantiomers in human plasma emerges from the fact that the metabolite interference has a great impact on the reliability of the method and currently is a hot topic. In addition, in HPLC-MS methods, it is highly preferred to use stable-isotope labeled internal standard (SIL-IS) because it not only possess similar properties as the analyte but also coelutes with the analyte (19). Therefore, to better meet the aim, SIL-IS was introduced instead of commonly used structural analog internal standard (SA-IS).

Experimental

Chemicals and reagents

Racemic IBP reference standard was purchased from EDQM (Strasbourg, France) and IBP D-acylglucoronide and racemic IBP-d3 reference standards were supplied from Sigma-Aldrich. Liquid chromatography–mass spectrometry grade methanol and water were supplied from Merck, Germany. Analytical grade acetic acid and ethylacetate were obtained from Sigma-Aldrich, Germany. The blank plasma samples were obtained from healthy volunteers.

HPLC-MS/MS conditions

The analyses were conducted on TSQ Quantum Discovery Max triple quadripole HPLC-MS/MS system (Thermo Scientific, USA) operated in electrospray ionization (ESI) (−) mode. A Chiracel OJ-RH column (150 × 2.1 mm, 5 μm) was used at the flow rate of 0.15 mL/min at 25 °C. A mixture of 0.1% (v/v) acetic acid in methanol/water (90:10, v/v) was used as a mobile phase. The injection volume was 10 μL and the chromatographic time was 6 min. The MS conditions were as follows: spray voltage 3.5 kV; sheet gas pressure 30 (in arbitrary units); capillary temperature 170oC and collision energy 9 V. The selected reaction monitoring (SRM) transitions (precursor and product ions) were m/z 381.4 → 205.4, m/z 381.4 → 161.4 and m/z 205.4 → 161.4 for IBP-Glu; m/z 205.4 → 161.4 for IBP enantiomers and m/z 207.4 → 164.4 for racemic IBP-d3.

Preparation of samples for in source and in vitro back-conversion experiments

Standard solution of IBP-Glu was prepared in concentration of 40 mg/L in 50% methanol in water. In order to investigate the in source back-conversion, IBP-Glu standard solution was injected in the HPLC-MS/MS system and analyzed under the optimized conditions.

In order to evaluate the in vitro back-conversion, blank plasma samples (n = 6) were spiked with IBP-Glu standard solution (40 mg/L) and followed by liquid-liquid extraction (LLE) procedure using 1 mL ethylacetate. The LLE procedure used for these experiments was previously described by our research group (16). Stability experiments were performed using blank plasma samples (n = 6) spiked with IBP-Glu standard solution (40 mg/L). The prepared samples were stored 18 h in dark cabinet, followed by 2 h exposition to light. After the respective storage time, samples were extracted and injected in the HPLC-MS/MS system. Also, stability after three freeze–thaw cycles of 24 h, each was determined. The amount of IBP enantiomers generated from the back-conversion of IBP-Glu was expressed as percent of lower limit of quantification (LLOQ) sample.

Preparation of calibration standards and quality control samples

The stock solution of rac-IBP (5,000 mg/L) and stock solution of IS in methanol (5,000 mg/L) were prepared and stored at −20°C. Working standard solutions of rac-IBP were prepared by dissolving appropriate amounts of rac-IBP stock solution in 50% methanol in water. The final concentration of R-IBP and S-IBP in calibration standards was 0.1; 0.2; 0.5; 1; 2; 5; 10; 20 and 50 mg/L. The concentration of R-IBP and S-IBP in quality control (QC) samples was as follows: 0.1 mg/L (LLOQ); 0.25 mg/L low QC (LQC); 8 mg/L medium QC (MQC) and 40 mg/L high QC (HQC).

IS working solution of 1.0 mg/L was prepared by dilution of IS stock solution in 50% methanol in water.

Method validation

Validation of the method for determination of IBP enantiomers was adhered to European Medicines Agency (2011) guideline (20).

Selectivity was evaluated by analyzing drug-free plasma samples derived from eight individual donors and human plasma spiked with rac-IBP and rac-IBP-d3 (IS). Matrix effect (ME) was determinated using drug-free plasma extracts (obtained from eight individual donors, including hyperlipidemic and hemolyzed plasma) spiked post-extraction with rac-IBP and IS. ME for IBP enantiomers was determined at LQC and HQC, whereas the ME for IS was determined at single concentration of 0.5 mg/L. Data obtained were used to calculate the relative matrix effect (RME) and its variability, expressed as coefficient of variation (CV, %).

Linearity was investigated using a nine-point calibration curve in the concentration range of 0.1−50 mg/L. The calibration curve was fitted to weighted (1/x) linear regression. Accuracy and precision of the method were evaluated by within-run (n = 5) and between-run (n = 30) assay using QC samples, and the results obtained were expressed as percent accuracy and CV (%).

Short-term and light stability tests were performed on three replicates of LQC and HQC samples that were thawed and left at room temperature (18 h in dark cabinet and 2 h exposed to light) and then extracted and analyzed. The post-preparative/autosampler stability was assessed using LQC and HQC samples that were extracted, reconstituted and stored for 96 h at −20°C (no light exposure) and 24 h at 10 °C (in autosampler) until analysis. Freeze and thaw stability was determined after three freeze and thaw cycles each lasting 24 h. The QC samples were extracted and analyzed after the third cycle. All stability results were expressed as mean percent accuracy against nominal (spiked) concentrations together with the CV (%).

Results

Fast chromatographic separation plays an important role in obtaining high sample throughput. This is of relevance especially for pharmacokinetic and bioequivalent studies where a large number of samples are present. In order to obtain better throughput, short cellulose-based column having smaller internal diameter was used (150 × 2.1 mm, 5 μm particle size). The retention time of R-IBP and S-IBP obtained on this stationary phase was 4.8 and 5.3 min, respectively (Figure 1a). The method was initially developed using ketoprofen (SA-IS) as an internal standard. The chromatographic separation required around 10 min. In order to obtain shorter analysis runtime and to improve the ruggedness of the method, SIL-IS instead of SA-IS was used. Considering that the optimized chromatographic conditions allowed stereospecific separation, two separate peaks from rac-IBP-d3 (R-IBP-d3 and S-IBP-d3) at almost same retention time as for the IBP enantiomers were obtained (Figure 1b). In addition, considering that the type of IS has a significant influence of the variability of ME, comparison between SIL-IS and SA-IS on the magnitude of the variability of the RME was done (Figure 2).
Figure 1.
SRM chromatogram of: (a) IBP enantiomers and (b) IS (rac-IBP-d3) under the optimized chromatographic conditions.
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SRM chromatogram of: (a) IBP enantiomers and (b) IS (rac-IBP-d3) under the optimized chromatographic conditions.

Figure 2.
Comparison between SIL-IS (rac-IBP-d3) and SA-IS (ketoprofen) on the variability of the RME.
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Comparison between SIL-IS (rac-IBP-d3) and SA-IS (ketoprofen) on the variability of the RME.

Due to the high temperature environment in the ESI source, fragmentation of the bonds between the IBP and glucoronic acid can occur, producing the same SRM transitions as IBP enantiomers (Figure 3). In high-throughput HPLC-MS/MS analysis, chromatographic separation between the analyte and the metabolite can be compromised, resulting in overestimated measurements (6). Therefore, it was necessary to verify whether, under the optimized chromatographic conditions, IBP enantiomers are chromatographically separated from the IBP-Glu. The results obtained after the analysis of solution containing standards of rac-IBP and IBP-Glu showed that besides the bad peak shape of IBP-Glu, separation between the IBP-Glu and IBP enantiomers was achieved (Figure 4a). Since the proposed method was intended for IBP enantiomers quantification, further modification of the optimized chromatographic conditions towards improvement of IBP-Glu peak shape was not necessary. Considering our interest towards investigation of the potential in source metabolite back-conversion, solution containing only IBP-Glu standard was analyzed. As it can be seen from the SRM chromatograms presented in Figure 5, the peak area of the transition m/z 381.4 → 205.4 (IBP-Glu to IBP parent ion) (Figure 5b) was significantly bigger than the peak area obtained from the SRM transition m/z 381.4 → 161.4 (IBP-Glu to IBP product ion) (Figure 5c). This suggests that under the optimized ESI parameters, most of the IBP-Glu molecular ions (m/z 381) will survive the collision. In order to ascertain whether the optimized ESI conditions lead to IBP-Glu back-conversion, the SRM transition m/z 205.4 → 161.4 was included. The presence of a peak at the retention time of 3.2 min, obtained from the transition m/z 205.4 → 161.4, indicated that some in source conversion of IBP-Glu might occurs (Figure 5a).
Figure 3.
Structure of the followed SRM transitions of IBP-Glu.
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Structure of the followed SRM transitions of IBP-Glu.

Figure 4.
SRM chromatograms of: (a) solution containing rac-IBP (0.25 mg/L) and IBP-Glu (40 mg/L) reference standards, (b) solution obtained after LLE of blank human plasma spiked with IBP-Glu standard solution (40 mg/L).
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SRM chromatograms of: (a) solution containing rac-IBP (0.25 mg/L) and IBP-Glu (40 mg/L) reference standards, (b) solution obtained after LLE of blank human plasma spiked with IBP-Glu standard solution (40 mg/L).

Figure 5.
SRM chromatogram of IBP-Glu standard solution: (a) m/z 205.4 > 161.4 (IBP parent ion to IBP product ion), (b) m/z 381.4 > 205.4 (IBP-Glu to IBP parent ion), (c) m/z 381.4 > 161.4 (IBP-Glu to IBP product ion).
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SRM chromatogram of IBP-Glu standard solution: (a) m/z 205.4 > 161.4 (IBP parent ion to IBP product ion), (b) m/z 381.4 > 205.4 (IBP-Glu to IBP parent ion), (c) m/z 381.4 > 161.4 (IBP-Glu to IBP product ion).

Acylglucoronide metabolites show different pH and temperature stability (3). Therefore, it was necessary to investigate the possibility of metabolite back-conversion during the sample preparation process. Plasma samples (obtained from six healthy volunteers) were spiked with IBP-Glu standard solution (40 mg/L). As it can be seen from the SRM chromatogram presented in Figure 4b, peaks at the retention time of IBP enantiomers (4.8 and 5.3 min) were observed. This indicated that certain in vitro metabolite back-conversion occurs. Therefore, the amount of IBP enantiomers generated by back-conversion of IBP-Glu was compared with LLOQ sample. The results have shown that the amount of R-IBP was <6% of LLOQ and <5% of LLOQ for S-IBP (Table I). Additionally, influence of the storage conditions on metabolite back-conversion was investigated. For that purpose, short-term (18 h in dark cabinet followed by 2 h exposition to light and then extracted) and freeze/thaw stability experiments were performed using plasma samples spiked with IBP-Glu. The amount of IBP enantiomers generated from the IBP-Glu obtained during those stability experiments was <8% of the LLOQ sample.

Table I.

IBP enantiomers obtained from in vitro IBP-Glu back-conversion, expressed as percent from LLOQ sample

Sample (n = 6)Area R-IBPR-IBP (%) of LLOQArea S-IBPS-IBP (%) of LLOQ
1 161 4.7 154 4.3 
2 152 4.4 143 4.0 
3 123 3.6 139 3.9 
4 89 2.6 176 5.0 
5 175 5.1 152 4.3 
6 202 5.9 129 3.6 
Sample (n = 6)Area R-IBPR-IBP (%) of LLOQArea S-IBPS-IBP (%) of LLOQ
1 161 4.7 154 4.3 
2 152 4.4 143 4.0 
3 123 3.6 139 3.9 
4 89 2.6 176 5.0 
5 175 5.1 152 4.3 
6 202 5.9 129 3.6 
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Table I.

IBP enantiomers obtained from in vitro IBP-Glu back-conversion, expressed as percent from LLOQ sample

Sample (n = 6)Area R-IBPR-IBP (%) of LLOQArea S-IBPS-IBP (%) of LLOQ
1 161 4.7 154 4.3 
2 152 4.4 143 4.0 
3 123 3.6 139 3.9 
4 89 2.6 176 5.0 
5 175 5.1 152 4.3 
6 202 5.9 129 3.6 
Sample (n = 6)Area R-IBPR-IBP (%) of LLOQArea S-IBPS-IBP (%) of LLOQ
1 161 4.7 154 4.3 
2 152 4.4 143 4.0 
3 123 3.6 139 3.9 
4 89 2.6 176 5.0 
5 175 5.1 152 4.3 
6 202 5.9 129 3.6 
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The method was validated following the European Medicines Agency (2011) guideline (20), by assessing selectivity, matrix effect, linearity, accuracy, precision and stability. SRM chromatograms for IBP enantiomers in blank, LLOQ and real study sample are presented in Figure 6. As it can be seen, an acceptable enantioselective separation was achieved. The results of the validation parameters, summarized in Table II, were always found to be within the acceptable limits (±15%, ±20% for LLOQ), indicating that the proposed method will generate reliable data during the real sample analysis.
Figure 6.
SRM chromatogram of: (a) IBP enantiomers (m/z 205.4 > 161.4) in blank human plasma (top), LLOQ sample (0.1 mg/L, middle) and real study sample (bottom) (b) internal standard (m/z 207.4 > 164.4) in blank human plasma (top), LLOQ sample (0.1 mg/L, middle) and real study sample (bottom).
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SRM chromatogram of: (a) IBP enantiomers (m/z 205.4 > 161.4) in blank human plasma (top), LLOQ sample (0.1 mg/L, middle) and real study sample (bottom) (b) internal standard (m/z 207.4 > 164.4) in blank human plasma (top), LLOQ sample (0.1 mg/L, middle) and real study sample (bottom).

Table II.

Results for the validation parameters (matrix effect, accuracy, precision and stability) of the proposed HPLC-MS/MS method

ME investigationR-IBPR-IBP-d3R-IBPS-IBPS-IBP-d3R-IBP
ME (%)ME (%)RME (%)ME (%)ME (%)RME (%)
LQC Mean (n = 8) 88.5 95.5 92.7 88.4 93.4 94.6 
SD 5.6 5.6 2.6 4.7 4.6 2.5 
CV (%) 6.3 5.8 2.8 5.3 4.9 2.7 
HQC Mean (n = 8) 89.4 94.5 94.6 91.5 96.0 95.3 
SD 4.3 5.6 2.8 4.5 5.9 3.3 
CV (%) 4.8 5.9 2.9 4.9 6.1 3.4 
ME investigationR-IBPR-IBP-d3R-IBPS-IBPS-IBP-d3R-IBP
ME (%)ME (%)RME (%)ME (%)ME (%)RME (%)
LQC Mean (n = 8) 88.5 95.5 92.7 88.4 93.4 94.6 
SD 5.6 5.6 2.6 4.7 4.6 2.5 
CV (%) 6.3 5.8 2.8 5.3 4.9 2.7 
HQC Mean (n = 8) 89.4 94.5 94.6 91.5 96.0 95.3 
SD 4.3 5.6 2.8 4.5 5.9 3.3 
CV (%) 4.8 5.9 2.9 4.9 6.1 3.4 
Accuracy and precisionWithin-run assay (n = 5)Between-run assays (n = 30)
R-IBPS-IBPR-IBPS-IBP
Accuracy (%)Precision (CV%)Accuracy (%)Precision (CV%)Accuracy (%)Precision (CV%)Accuracy (%)Precision (CV%)
LQC 102.6 5.8 103.0 5.0 104.7 7.1 105.2 7.2 
MQC 106.9 5.0 106.0 3.5 104.6 5.0 104.7 5.2 
HQC 101.6 2.8 101.8 4.5 100.3 5.5 99.7 4.4 
Accuracy and precisionWithin-run assay (n = 5)Between-run assays (n = 30)
R-IBPS-IBPR-IBPS-IBP
Accuracy (%)Precision (CV%)Accuracy (%)Precision (CV%)Accuracy (%)Precision (CV%)Accuracy (%)Precision (CV%)
LQC 102.6 5.8 103.0 5.0 104.7 7.1 105.2 7.2 
MQC 106.9 5.0 106.0 3.5 104.6 5.0 104.7 5.2 
HQC 101.6 2.8 101.8 4.5 100.3 5.5 99.7 4.4 
Stability experiments (n = 3)LQCHQC
R-IBPS-IBPR-IBPS-IBP
Accuracy (%)CV (%)Accuracy (%)CV (%)Accuracy (%)CV (%)Accuracy (%)CV (%)
Short-term/light stability 104.7 0.6 103.4 4.5 104.2 0.6 101.5 1.6 
Freeze–thaw stability 105.0 4.1 105.4 2.2 104.1 1.6 103.7 1.1 
Post-preparative/autosampler stability 107.2 3.4 108.7 3.8 105.7 2.2 103.3 2.8 
Stability experiments (n = 3)LQCHQC
R-IBPS-IBPR-IBPS-IBP
Accuracy (%)CV (%)Accuracy (%)CV (%)Accuracy (%)CV (%)Accuracy (%)CV (%)
Short-term/light stability 104.7 0.6 103.4 4.5 104.2 0.6 101.5 1.6 
Freeze–thaw stability 105.0 4.1 105.4 2.2 104.1 1.6 103.7 1.1 
Post-preparative/autosampler stability 107.2 3.4 108.7 3.8 105.7 2.2 103.3 2.8 
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Table II.

Results for the validation parameters (matrix effect, accuracy, precision and stability) of the proposed HPLC-MS/MS method

ME investigationR-IBPR-IBP-d3R-IBPS-IBPS-IBP-d3R-IBP
ME (%)ME (%)RME (%)ME (%)ME (%)RME (%)
LQC Mean (n = 8) 88.5 95.5 92.7 88.4 93.4 94.6 
SD 5.6 5.6 2.6 4.7 4.6 2.5 
CV (%) 6.3 5.8 2.8 5.3 4.9 2.7 
HQC Mean (n = 8) 89.4 94.5 94.6 91.5 96.0 95.3 
SD 4.3 5.6 2.8 4.5 5.9 3.3 
CV (%) 4.8 5.9 2.9 4.9 6.1 3.4 
ME investigationR-IBPR-IBP-d3R-IBPS-IBPS-IBP-d3R-IBP
ME (%)ME (%)RME (%)ME (%)ME (%)RME (%)
LQC Mean (n = 8) 88.5 95.5 92.7 88.4 93.4 94.6 
SD 5.6 5.6 2.6 4.7 4.6 2.5 
CV (%) 6.3 5.8 2.8 5.3 4.9 2.7 
HQC Mean (n = 8) 89.4 94.5 94.6 91.5 96.0 95.3 
SD 4.3 5.6 2.8 4.5 5.9 3.3 
CV (%) 4.8 5.9 2.9 4.9 6.1 3.4 
Accuracy and precisionWithin-run assay (n = 5)Between-run assays (n = 30)
R-IBPS-IBPR-IBPS-IBP
Accuracy (%)Precision (CV%)Accuracy (%)Precision (CV%)Accuracy (%)Precision (CV%)Accuracy (%)Precision (CV%)
LQC 102.6 5.8 103.0 5.0 104.7 7.1 105.2 7.2 
MQC 106.9 5.0 106.0 3.5 104.6 5.0 104.7 5.2 
HQC 101.6 2.8 101.8 4.5 100.3 5.5 99.7 4.4 
Accuracy and precisionWithin-run assay (n = 5)Between-run assays (n = 30)
R-IBPS-IBPR-IBPS-IBP
Accuracy (%)Precision (CV%)Accuracy (%)Precision (CV%)Accuracy (%)Precision (CV%)Accuracy (%)Precision (CV%)
LQC 102.6 5.8 103.0 5.0 104.7 7.1 105.2 7.2 
MQC 106.9 5.0 106.0 3.5 104.6 5.0 104.7 5.2 
HQC 101.6 2.8 101.8 4.5 100.3 5.5 99.7 4.4 
Stability experiments (n = 3)LQCHQC
R-IBPS-IBPR-IBPS-IBP
Accuracy (%)CV (%)Accuracy (%)CV (%)Accuracy (%)CV (%)Accuracy (%)CV (%)
Short-term/light stability 104.7 0.6 103.4 4.5 104.2 0.6 101.5 1.6 
Freeze–thaw stability 105.0 4.1 105.4 2.2 104.1 1.6 103.7 1.1 
Post-preparative/autosampler stability 107.2 3.4 108.7 3.8 105.7 2.2 103.3 2.8 
Stability experiments (n = 3)LQCHQC
R-IBPS-IBPR-IBPS-IBP
Accuracy (%)CV (%)Accuracy (%)CV (%)Accuracy (%)CV (%)Accuracy (%)CV (%)
Short-term/light stability 104.7 0.6 103.4 4.5 104.2 0.6 101.5 1.6 
Freeze–thaw stability 105.0 4.1 105.4 2.2 104.1 1.6 103.7 1.1 
Post-preparative/autosampler stability 107.2 3.4 108.7 3.8 105.7 2.2 103.3 2.8 
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Discussion

Our previously published direct method for determination of IBP enantiomers in human plasma was initially developed using long cellulose-based chiral column (250 × 4.6 mm, 5 μm particle size) (16). Although the optimized chromatographic conditions allowed good resolution between the enantiomers, the chromatographic separation required 13 min. The use of shorter column with smaller internal diameter allowed significant decrease of the chromatographic runtime. In addition, the replacement of ketoprofen with rac-IBP-d3 as an internal standard enabled the whole analysis runtime to be completed within 6 min, thereby improvement on throughput of at least of 50% was achieved (12, 14–18).

The variation of the RME obtained using SIL-IS was <4% for LQC and HQC samples. In comparison, the CV of RME using ketoprofen as an IS was <5% for LQC and <8% for HQC samples. These results confirmed the expectation that the use of SIL-IS compared to a SA-IS enabled reduction of magnitude and variability of the matrix effect, thereby improving ruggedness of the method. Considering that the RME obtained using SA-IS was within the acceptable limit of ±15% (20), ketoprofen could be also used as an IS in case of unavailability of SIL-IS. However, the chromatographic separation in that case would require ~10 min.

Metabolite interference can be perceived during the incurred sample reanalysis (21). However, using this approach the potential back-conversion during sample processing cannot be ruled out (20). Therefore, the possibility of in source and in vitro back-conversion of IBP-Glu was assessed in the course of method development. The conducted experiments towards determination of in source metabolite back-conversion revealed that the peak area of R-IBP and S-IBP generated from the IBP-Glu was up to or <1% of LLOQ. Considering that the in source IBP-Glu back-conversion was insignificant and a chromatographic separation between the analyte and the metabolite was achieved, there was no need for further optimization/modification of the ESI parameters and/or the chromatographic conditions.

The literature data for determination of IBP-Glu in plasma/urine indicated that this metabolite is stable in acidic environment (2, 22–24). In order to prevent hydrolysis of the acylglucoronide metabolite, acidic buffer (0.15 M HCl) was used during the LLE of IBP enantiomers from human plasma. However, considering that hydrolysis of IBP-Glu could occur during the different steps of the sample preparation process, it was necessary to investigate whether the proposed LLE procedure leads to metabolite back-conversion in addition to the insignificant in source back-conversion. The findings indicated that during the sample preparation process, metabolite back-conversion occurs; therefore, assessment of the amount of IBP-Glu back-conversion was needed. The obtained results confirmed that the amount of back-conversion during the sample preparation process was far <20% of the LLOQ sample. In addition, it should be emphasized that the concentration of IBP-Gluc (40 mg/L) used for the in vitro back-conversion experiments was significantly higher than the metabolite concentration (0.55 mg/L) found in human plasma after treatment with IBP 800 mg capsule (22). Despite the high metabolite concentration spiked, the amount of back-conversion was insignificant. Based on the findings above, we confirm that the presence of IBP-Glu in real samples will not affect the quantification of IBP enantiomers.

Conclusions

In this research, for the first time the influence of IBP-Glu back-conversion on the HPLC-MS/MS determination of IBP enantiomers in human plasma was investigated. The optimized HPLC-MS/MS conditions allow separation between IBP enantiomers and IBP-Glu; therefore, metabolite chromatographic interference was prevented. The obtained results from the in source and in vitro metabolite back-conversion experiments imply that the presence of IBP-Glu in real samples will not affect the quantification of the IBP enantiomers, thereby the improved reliability of the method was confirmed. Further improvement of the reliability of the method was achieved using SIL-IS instead of SA-IS. Another advantage of the method is short analysis runtime (6 min), which is especially important for bioequivalent studies where a large number of samples are present. Considering the advantages of the proposed method, one can conclude that this method could be successfully applied for the fast determination of R-IBP and S-IBP levels in human plasma and to provide reliable data from pharmacokinetic and bioequivalent studies of this drug.

References

1

American hospital formulary service (AHFS)
; Drug information, American Society of Healthy-System Pharmacists, Inc. (
1995
).

2

Castillo
,
M.
,
Smith
,
P.C.
;
Disposition and reactivity of ibuprofen and ibufenac acylglucoronides in vivo in the rhesus monkey and in vitro in human serum albumin
;
Drug Metabolism Disposition
, (
1995
);
23
:
566
–
572
.

Google Scholar

PubMed
OpenURL Placeholder Text

 
3

Shipkova
,
M.
,
Armstrong
,
V.W.
,
Oellerich
,
M.
,
Wieland
,
E.
;
Acyl glucuronide drug metabolites: Toxicological and analytical implications
;
Therapeutic Drug Monitoring
, (
2003
);
25
:
1
–
16
.

Google Scholar

Crossref
Search ADS
PubMed

 
4

Dell
,
D.
;
Labile metabolites
;
Chromatographia
, (
2004
);
59
:
S139
–
S148
.

Google Scholar

Crossref
Search ADS

 
5

Garofolo
,
F.
,
Savoie
,
N.
,
Bergenon
,
A.
,
Amestroy
,
C.
,
Lefebvre
,
M.
,
Caille
,
L.
, et al. .;
Importance of metabolite testing in regulated bioanalysis
;
Bioanalysis
, (
2010
);
2
:
1171
–
1174
.

Google Scholar

Crossref
Search ADS
PubMed

 
6

Li
,
W.
,
Zhang
,
J.
,
Tse
,
F.L.S.
;
Strategies in quantitative LC-MS/MS analysis of unstable small molecules in biological matrices
;
Biomedical Chromatography
, (
2011
);
25
:
258
–
277
.

Google Scholar

Crossref
Search ADS
PubMed

 
7

Wright
,
M.R.
,
Sattari
,
S.
,
Brocks
,
D.R.
,
Jamali
,
F.
;
Improved high-performance liquid chromatographic assay method for the enantiomers of ibuprofen
;
Journal of Chromatography
, (
1992
);
583
:
259
–
265
.

Google Scholar

Crossref
Search ADS
PubMed

 
8

Lemko
,
C.H.
,
Caillé
,
G.
,
Foster
,
R.T.
;
Stereospecific high-performance liquid chromatographic assay of ibuprofen: Improved sensitivity and sample processing efficiency
;
Journal of Chromatography
, (
1993
);
619
:
330
–
335
.

Google Scholar

Crossref
Search ADS
PubMed

 
9

Naidong
,
W.
,
Lee
,
J.W.
;
Development and validation of a liquid chromatographic method for the quantification of ibuprofen enantiomers in human plasma
;
Journal of Pharmaceutical and Biomedical Analysis
, (
1994
);
12
:
551
–
556
.

Google Scholar

Crossref
Search ADS
PubMed

 
10

Canaparo.
,
R.
,
Muntoni
,
E.
,
Zara
,
G.P.
,
Della Pepa
,
C.
,
Berno
,
E.
,
Costa
,
M.
, et al. .;
Determination of ibuprofen in human plasma by high-performance liquid chromatography: validation and application in pharmacokinetic study
;
Biomedical Chromatography
, (
2000
);
14
:
219
–
226
.

Google Scholar

Crossref
Search ADS
PubMed

 
11

Bonato
,
P.S.
,
Perpetua
,
M.
,
Del Lama
,
F.M.
,
de Carvalho
,
R.
;
Enantioselective determination of ibuprofen in plasma by high-performance liquid chromatography-electrospray mass spectrometry
;
Journal of Chromatography B
, (
2003
);
796
:
413
–
420
.

Google Scholar

Crossref
Search ADS

 
12

Mendes
,
G.D.
,
Mendes
,
F.D.
,
Dominguesm
,
C.C.
,
de Oliveiram
,
R.A.
,
da Silvam
,
M.A.
,
Chen
,
L.S.
, et al. .;
Comparative bioavailability of three ibuprofen formulations in healthy human volunteers
;
International Journal of Clinical Pharmacology and Therapy
, (
2008
);
36
:
309
–
318
.

Google Scholar

Crossref
Search ADS

 
13

Reddy
,
R.
,
Chandiran
,
I.S.
,
Jayaverra
,
K.N.
,
Divi
,
K.R.
;
Quantification of ibuprofen in human plasma by high throughput liquid chromatography-tandem mass spectrometric method and its application in pharmacokinetics
;
Archives of Apllied Science Research
, (
2010
);
2
:
101
–
111
.

Google Scholar

OpenURL Placeholder Text

 
14

Szeitz
,
A.
,
Edginton
,
A.N.
,
Peng
,
H.T.
,
Cheung
,
B.
,
Riggs
,
K.W.
;
A validated enantioselective assay for the determination of ibuprofen in human plasma using ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS)
;
American Journal of Analytical Chemistry
, (
2010
);
2
:
47
–
58
.

Google Scholar

Crossref
Search ADS

 
15

Cardoso
,
J.L.C.
,
Lanchote
,
V.L.
,
Pereira
,
M.P.M.
,
de Moraes
,
N.V.
,
Lepera
,
J.S.
;
Analysis of ibuprofen enantiomers in rat plasma by liquid chromatography with tandem mass spectrometry
;
Journal of Separation Science
, (
2014
);
37
:
944
–
949
.

Google Scholar

Crossref
Search ADS
PubMed

 
16

Nakov
,
N.
,
Petkovska
,
R.
,
Ugrinova
,
L.
,
Kavrakovski
,
Z.
,
Dimitrovska
,
A.
,
Svinarov
,
D.
;
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
);
992
:
67
–
75
.

Google Scholar

Crossref
Search ADS

 
17

Ogawa
,
S.
,
Tadokoro
,
H.
,
Sato
,
M.
,
Hanawa
,
T.
,
Higashi
,
T.
;
S)-1-(4-Dimethylaminophenylcarbonyl)-3-aminopyrrolidine: A derivatization reagent for enantiomeric separation and sensitive detection of chiral carboxylic acids by LC/ESI-MS/MS
;
Journal of Chromatography B
, (
2013
);
940
:
7
–
14
.

Google Scholar

Crossref
Search ADS

 
18

Ogawa
,
S.
,
Tadokoro
,
H.
,
Sato
,
M.
,
Higashi
,
T.
;
Enantioselective determination of ibuprofen in saliva by liquid chromatography/tandem mass spectrometry with chiral electrospray ionization-enhancing and stable isotope-coded derivatization
;
Journal of Pharmaceutical and Biomedical Analysis
, (
2014
);
98
:
387
–
392
.

Google Scholar

Crossref
Search ADS
PubMed

 
19

Mulvana
,
D.
;
Critical topics in ensuring data quality in bioanalytical LC-MS method development
;
Bioanalysis
, (
2010
);
2
:
1051
–
1072
.

Google Scholar

Crossref
Search ADS
PubMed

 
20

European Medical Agency
; Guideline on validation of bioanalytical methods. Committee for Medical products for human use; EMEA/CHMP/EWP/192217/2009; (
2011
).

21

Jemal
,
M.
,
Ouyang
,
Z.
,
Xia
,
Y.-Q.
;
Systematic LC-MS/MS bioanalytical method development that incorporates plasma phospholipids risk avoidance, usage of incurred sample and well thought-out chromatography
;
Biomedical Chromatography
, (
2010
);
24
:
2
–
19
.

Google Scholar

Crossref
Search ADS
PubMed

 
22

Castillo
,
M.
,
Smith
,
P.C.
;
Direct determination of ibuprofen and ibuprofen acyl glucoronide in plasma by high-performance liquid chromatography using solid-phase extraction
;
Journal of Chromatography
, (
1993
);
614
:
109
–
116
.

Google Scholar

Crossref
Search ADS
PubMed

 
23

Ikegawa
,
S.
,
Murao
,
N.
,
Oohashi
,
J.
,
Goto
,
J.
;
Separatory determination of diastereomeric ibuprofen glucoronides in human urine by liquid-chromatography/electropray ionization-mass spectrometry
;
Biomedical Chromatography
, (
1998
);
12
:
317
–
321
.

Google Scholar

Crossref
Search ADS
PubMed

 
24

Smith
,
R.W.
,
Toutoungib
,
D.
,
Reynoldsa
,
J.
,
Bristowc
,
A.
,
Rayc
,
A.
,
Saged
,
A.
, et al. .;
Enhanced performance in the determination of ibuprofen 1-O-acyl glucuronide in urine by combining high field asymmetric waveform ion mobility spectrometry with liquid chromatography-time-of-flight mass spectrometry
;
Journal of Chromatography A
, (
2013
);
1278
:
76
–
81
.

Google Scholar

Crossref
Search ADS
PubMed

 
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Liquid Chromatography
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