Journal of Pharmaceutical and Biomedical Analysis 92 (2014) 193–202

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Ultrasound-assisted emulsification microextraction combined with ultra-high performance liquid chromatography–tandem mass spectrometry for the analysis of ibuprofen and its metabolites in human urine Sylwia Magiera a,∗ , S¸efika Gülmez b a b

Department of Analytical Chemistry, Silesian University of Technology, 7M. Strzody Str., 44-100 Gliwice, Poland Department of Chemistry, Pamukkale University, 20020 Denizli, Turkey

a r t i c l e

i n f o

Article history: Received 29 September 2013 Received in revised form 11 January 2014 Accepted 13 January 2014 Available online 24 January 2014 Keywords: Ultrasound-assisted emulsification microextraction Ultra-high performance liquid chromatography Ibuprofen Metabolite Human urine

a b s t r a c t In this study, a fast, simple and efficient method based on ultrasound-assisted emulsificationmicroextraction (USAEME) coupled with ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) was successfully developed for the determination of ibuprofen (IBU) and its four metabolites (1-hydroxyibuprofen (1-HIBU), 2-hydroxyibuprofen (2-HIBU), 3-hydroxyibuprofen (3-HIBU), carboxyibuprofen (CIBU)) in human urine. For this purpose, the influence of the different parameters affecting the USAEME procedure was evaluated in order to optimize the efficiency of the process. The optimum conditions were found to be: 100 ␮L of 1-octanol as extraction solvent, 2 mL of urine sample, 15% (w/v) NaCl to control the ionic strength, ultrasonication for 10 min; and centrifugation for 5 min at 6500 rpm. After sample preparation, chromatographic separation was achieved on a Zorbax Rapid Resolution High Definition (RRHD) SB-C18 column using the mobile phase consisting of 0.1% formic acid in water and acetonitrile in an elution gradient. Detection was performed in a triple quadrupole tandem mass spectrometer using the multiple reaction monitoring (MRM) mode and negative ionization. The proposed method showed satisfactory linearity over a wide concentration range (correlation coefficients over 0.9994). The lower limit of quantification (LLOQ) was 0.0005 ng/mL for IBU and its metabolites. The intra- and inter-day precisions were in the range of 2.19–10.8% and the accuracies were between −5.93% and 6.29%. The mean recovery of analytes ranged from 90.7 to 104%. As a result, this method has been successfully applied for the sensitive determination of IBU and its metabolites in human urine samples. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Ibuprofen (IBU) is a non-steroidal anti-inflammatory drug (NSAID) that has been widely used in the treatment of pain and inflammation in rheumatic disease and other musculoskeletal disorders [1]. Pharmacokinetic studies have shown that about 66% of the drug is excreted in the urine whereas about 34% is excreted in the feces (biliary excretion). Recovery studies revealed that 60% of the given dose was excreted within the first 24 h [2]. Oxidative metabolism is the major route for biotransformation of IBU and four oxidative metabolites (1-hydroxyibuprofen (1-HIBU), 2-hydroxyibuprofen (2-HIBU), 3-hydroxy-ibuprofen (3-HIBU),

∗ Corresponding author. Tel.: +48 32 237 1396; fax: +48 32 237 1396. E-mail address: [email protected] (S. Magiera). 0731-7085/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2014.01.012

carboxyibuprofen (CIBU)) have been identified in urine and plasma samples obtained from humans after the oral intake of ibuprofen. In humans, the parent drug, as well as the metabolites, is found to be conjugated with glucuronic acid [3]. The two major metabolites, 2-HIBU and CIBU, and their glucuronic acid conjugates were found to account for approximately 58% of the given dose of ibuprofen, whereas the two minor metabolites, 1-HIBU and 3-HIBU, and their glucuronic acid conjugates were found to be present in the urine in only very small concentrations [4]. For the diagnosis or, more importantly, the differential diagnostic exclusion of cases of acute overdose or chronic abuse, an analytical procedure is necessary for the determination of the drug and its metabolites in biological fluids. The chromatographic methods that are currently available to measure IBU include gas chromatography (GC) [5,6], capillary electrophoresis (CE) [7–10], high-performance thin-layer chromatography (HPTLC)

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[11] and high-performance liquid chromatography (HPLC) [12–26]. IBU was isolated from biological fluids by deproteination [10,23], liquid–liquid extraction [8,11,14,21,25,26], solid-phase extraction (SPE) [8,12,18,22] solid-phase microextraction (SPME) [13,17], hollow fiber-based liquid phase microextraction (HF-LPME) [15,16] and hollow-fiber liquid membrane-protected solid-phase microextraction (HFLM-SPME) [6]. Three papers have described methods for the simultaneous determination of IBU and its metabolites (only selected two metabolites) [12,14,17]. Recently, much attention is being paid to the development of miniaturized, more efficient and environmentally friendly extraction techniques which could greatly reduce the consumption of organic solvents. The ultrasound-assisted emulsificationmicroextraction method (USAEME) is an effective technique among these microextraction methods. This approach is based on the emulsification of a microvolume of organic extractant in an aqueous sample by ultrasound radiation and further separation of both liquid phases by centrifugation. The application of ultrasonic radiation accelerates the mass-transfer process between two immiscible phases, which, together with the large surface of contact between both phases, leads to an increment in the extraction efficiency in a minimal amount of time. In this way, USAEME can be employed as a simple, fast and efficient extraction and preconcentration procedure for organic compounds in aqueous samples [27]. The aim of the present work is to investigate and develop a rapid and efficient USAEME method coupled with ultra-highperformance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) for the analysis of IBU and its metabolites in human urine samples. It is to be noted that this is the first report on the simultaneous determination of IBU and its four metabolites in human urine using USAEME, thus paving the way as a good alternative for routine analysis with the advantages of simplicity, reliability, cost effectiveness and minimized matrix interferences. The effect of various experimental conditions on the extraction of analyzed compounds is investigated and discussed. The optimized procedure was successfully applied to determination of the target analyte in human urine samples. 2. Experimental 2.1. Chemicals and reagents Ibuprofen (IBU; purity >98.1%), 1-hydroxyibuprofen (1-HIBU; purity >98.5%), 2-hydroxyibuprofen (2-HIBU; purity >98.5%), 3-hydroxyibuporfen (3-HIBU; purity >98.5%), carboxyibuprofen (CIBU; purity >98.5%) and naproxen (NAP, used as an internal standard (IS); purity >98.3%) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Acetonitrile (HPLC–MS grade), methanol (HPLC–MS grade) and water (HPLC grade) were purchased from Merck (Darmstadt, Germany). Formic acid, 1-decanol, 1-octanol, n-decane, n-undecane and n-hexadecane were obtained from Sigma–Aldrich (St. Louis, MO, USA). Sodium hydroxide (NaOH) was purchased from Stanlab (Lublin, Poland). Hydrochloric acid (HCl) was obtained from Chempur (Piekary Slaskie, Poland). Sodium chloride (NaCl) was purchased from Merck (Darmstadt, Germany). 2.2. Preparation of standard solutions, calibration standards and quality control (QC) solutions The standard stock solutions of IBU and its metabolites from independent weighting were prepared in methanol at a concentration of 1.0 mg/mL. Working standard solutions of IBU and its metabolites ranging from 0.5 ng/mL to 5000 ng/mL were prepared by diluting the stock solutions with methanol. Finally, urine calibration standards (CS) were made at concentrations in the range

of 0.0005–250 ng/mL for IBU, 1-HIBU, 2-HIBU, 3-HIBU and CIBU by spiking working standard solution to free-drug urine samples (blank sample). Also, quality control (QC) samples were prepared at four concentrations: lower limit of quantitation (LLOQ): 0.0005 ng/mL for IBU and its metabolites, low concentration quality control (LQC): 0.005 ng/mL for IBU and its metabolites, middle concentration quality control (MQC): 75 ng/mL for IBU and its metabolites, and high concentration quality control (HQC): 200 ng/mL for IBU and its metabolites. Similarly, an IS stock solution (20 ␮g/mL) was also prepared in methanol and diluted with methanol to give an IS working solution (200 ng/mL). All solutions were stored in a refrigerator (4–8 ◦ C) until used. 2.3. Sample preparation Drug-free human urine samples used for the preparation of calibration and validation standards were collected from six different healthy subjects who were drug free. Additionally, urine samples were collected from ten patients being treated with IBU. All urine samples were stored in a freezer at −20 ◦ C. Human urine samples were prepared using the new USAEME method. The USAEME procedure was performed using five different extraction solvents: 1-decanol, 1-octanol, n-decane, n-undecane and n-hexadecane. In the optimization step, 1-octanol was selected, as the best solvent to extract the target analytes and this was used for all experiments. Before the USAEME extraction, the hydrolysis reactions were performed according to Tan et al. [20] during 30 min. Urine samples (2 mL) containing IS (20.0 ng/mL) were alkalinized with 400 ␮L of 1 M NaOH for the hydrolysis of acyl glucuronic acid conjugates. The hydrolysis reaction was left to proceed for 30 min at room temperature and the hydrolyzed urine samples were then neutralized with 400 ␮L of 1 M HCl; the extraction was carried out as described in the following section. After hydrolysis, urine samples were placed in glass centrifuge tubes and the ionic strength and pH of the solutions were adjusted to an appropriate level (sodium chloride, 15% (w/v); pH 2.0). Then, 100 ␮L of 1-octanol (extraction solvent) was quickly injected into the sample solution, and the tube was shaken manually for 10 s and so that small droplets of the extraction solvent formed an emulsion with the sample solution. The tube was immersed in an ultrasonic water bath, sonicated for 10 min and shaken manually for 10 s. The formation of tiny droplets greatly enlarges the contact area between the extraction solvent and aqueous phase, which enhances the extraction efficiency. Then, the emulsion was centrifuged at 6500 rpm (without 4912.5 × g) for 5 min in order to disrupt the emulsions and separate both phases (the organic phase remained at the bottom of the tube). After centrifugation, 50 ␮L of the extraction solvent floating on the surface was collected with a Hamilton microsyringe, from which 10 ␮L was dissolved in 90 ␮L of methanol. Finally, 5 ␮L of the obtained mixture was injected into the UHPLC–MS/MS system for subsequent analysis. Fig. 1 shows a scheme of the USAEME procedure. 2.4. Instrumentation and LC–MS/MS analytical conditions Liquid chromatography was performed on a Dionex UPLC system (Dionex Corporation, Sunnyvale, CA, USA) consisting of an UltiMate 3000 RS (Rapid Separation) pump with on-line vacuum degasser, an UltiMate 3000 autosampler, an UltiMate 3000 column compartment with a thermostable column area and an UltiMate 3000 variable wavelength detector, all of which were operated using Dionex ChromeleonTM 6.8 software. The chromatographic separation was performed on a Zorbax Rapid Resolution High Definition (RRHD) SB-C18 column

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Fig. 1. Schematic representation of the ultrasound-assisted emulsification microextraction (USAEME) procedure for urine sample preparation.

(50 mm × 2.1 mm, 1.8 ␮m, Agilent Technologies, Waldbronn, Germany). The gradient elution mobile phase composed of 0.1% formic acid in water (pH = 3.0) (solvent A) and acetonitrile (solvent B). The gradient was controlled as follows: 0–0.5 min, 70% A (0.5 mL/min), 0.5–0.7 min, 50% A (0.5 mL/min), 0.7–1.0 min, 10% A (from 0.5 mL/min to 1.0 mL/min), 1.0–1.8 min, 10% A (1.0 mL/min). The column temperature was maintained at 35 ◦ C and autosampler was kept at 5 ◦ C. A 5.0 ␮L aliquot was injected into the UHPLC–MS/MS system and the total analytical run time was 1.8 min. Mass spectrometric analyses were performed using an AB SCIEX 4000 Q TRAP triple quadrupole mass spectrometer (Applied Biosystems/MDS SCIEX, Foster City, CA, USA) equipped with an electrospray ionization (ESI) source. Mass spectrometry with a TurboIonSpray source was performed in negative ion mode. Crucial detector parameters such as ionization voltage (ISV), collision assisted dissociation (CAD) gas, turbo-gas temperature (TEM), ion source Gas 1 (nebulizer) (GS1), turbo ion source Gas 2 (heater) (GS2) and curtain gas (CUR) were optimized by flow injection analysis (FIA) to obtain better ionization. Tuning and optimization of the compound-dependent parameters (declustering potential (DP), collision energy (CE), entrance potential (EP), collision cell exit potential (CXP)) were performed for the analytes and IS by the direct infusion of a 1 ␮g/mL standard solution into the ion source using a Harvard syringe pump at a flow rate of 10 ␮L/min. All quantifications were performed using the multiple reaction monitoring (MRM) mode. Continuous mass spectra were obtained by scanning from m/z 50 to 300. Quadrupoles Q1 and Q3 were maintained at unit resolution with a dwell time of

100 ms per channel. Analyst 1.5.1 software (Applied Biosystems, Foster City, CA, USA) was applied for instrumental control, data acquisition and quantitative analysis. 2.5. Method validation The method validation assays were performed according to the United States Food and Drug Administration (FDA) guidelines [28]. The description was placed in Electronic Supplementary Material, ESM. 3. Results and discussion 3.1. UHPLC–MS/MS method developed In order to establish a sensitive and selective quantitative method, the chromatographic conditions and mass spectrometry parameters were optimized. A standard solution along with the mobile phase was directly infused into the mass spectrometer in order to optimize mass conditions. The electrospray interface (ESI) was employed to obtain good sensitivity and fragmentation. The ionization mode was optimized in both the positive- and negative-ion modes. For all analyzed compounds, the response observed was much better in the ionization negative mode than in the positive mode. Mass spectrometric source conditions (curtain gas, collision gas, spray voltage, source temperature, and source gases) were then optimized for IBU and its metabolites. The detector conditions were as follows: ISV = −4000 V; CAD = high; TEM = 500 ◦ C; GS1 = 90 psi;

Table 1 MS/MS parameters for IBU and its metabolites. Analyte IBU 1-HIBU 2-HIBU 3-HIBU CIUBU NAP (IS) a b c d e f

Parent ion. Fragment ion. Declustering potential. Entrance potential. Collision energy. Cell exit potential.

Q1a (m/z) 205.036 221.070 221.070 221.070 235.005 229.083

Q3b (m/z) 160.780 114.913 176.960 158.953 176.960 158.953 176.960 158.953 190.971 72.957 185.051 169.060

DPc (V) −45 −50 −50 −50 −50 −40

EPd (V)

CEe (V)

CXPf (V)

−10 −8 −10 −7 −10 −8 −10 −8 −10 −7 −3 −5

−10 −30 −10 −16 −10 −16 −10 −16 −10 −22 −10 −36

−7 −7 −7 −1 −9 −1 −7 −1 −5 −3 −5 −9

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Fig. 2. Mass spectra of product ions of (A) IBU, (B) NAP (IS), (C) 1-HIBU, (D) 2-HIBU, (E) 3-HIBU and (F) CIBU in negative electrospray ionization mode.

GS2 = 60 psi; CUR = 10 psi. To obtain the maximum sensitivity for the [M−H]− ions, the declustering potential (DP) was optimized. A suitable collision energy (CE) was obtained by finding the maximum response for the MS/MS fragment ion. Entrance potential (EP) and collision cell exit potential (CXP) did not greatly affect the analyte response. The optimal conditions for all of the analyzed compounds are presented in Table 1. The MRM mode was used to carry out the quantitative analysis due to the high selectivity and sensitivity achieved. For each compound, two MRM transitions were monitored to provide additional confidence in identification (Table 1). The ratio of these ions (qualifier ion to quantitation ion) was calculated. For all of the compounds investigated, ion ratios were found to be very reproducible with variation (as %CV) less than 8%. Full-scan product ion spectra of the [M−H]− ions and the fragmentation pathways of IBU, its metabolites and IS are shown in Fig. 2. NAP was selected as IS due to the similarity of its retention time and extraction efficiency with the analytes and its efficient ionization in the negative ionization mode. The UHPLC conditions, such as the stationary phase, mobile phase composition, column temperature and flow rate were investigated after the optimization of MS/MS parameters. With the aim

of achieving short retention times and symmetric peak shapes, two columns (Zorbax RRHD SB-C18; 50 mm × 2.1 mm, 1.8 ␮m and Hypersil GOLDTM , 100 mm × 2.1 mm, 1.9 ␮m) were investigated under the same UHPLC gradient program and mobile phase composition. It was found that the MS/MS signals of the analyzed compounds were decreased by a factor of 2–4 using the Zorbax RRHD SB-C18 column when compared to the Hypersil GOLDTM column and a shorter retention time was obtained. Therefore, the Zorbax RRHD SB-C18 column was selected as the analytical column. As for the choice of strong elution mobile phase, methanol and acetonitrile were considered as two candidates. Results showed that the responses of analytes with acetonitrile as the mobile phase were higher than those with methanol under the ESI negative mode. At the start of the analysis, a pure water and acetonitrile was used and then the addition of acetic acid, formic acid and ammonium acetate, to the water phase was tested. The analyzed compounds and IS were found to have the highest response and the best peak shapes in the mobile phase containing 0.1% formic acid. Hence, the optimal chromatography conditions for the elution of IBU and its metabolites were achieved using acetonitrile and 0.1% formic acid in water as mobile phase in the gradient profile, as

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Fig. 3. Representative MRM chromatograms of IBU, 1-HIBU, 2-HIBU, 3-HIBU, CIBU, and NAP (IS): (A, C, E, G) blank urine after the USAEME procedure, (B, D, F, H) blank urine spiked with analytes at the LLOQ and IS after the USAEME procedure.

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Table 2 Intra- and inter-day precision and accuracy for the determination of IBU and its metabolites in human urine samples (n = 6). Analyte

Cnominal (ng/mL)

Intra-day a

ERc (%)

Inter-day b

a

MEd (%)

b

RSD (%)

RE (%)

IBU

5 × 10−4 5 × 10−3 75.0 200

6.30 2.19 2.38 4.63

−4.22 3.28 −1.52 0.845

8.60 6.40 7.30 5.68

−5.67 6.12 4.31 −2.69

92.5 93.7 96.0 97.0

5.99 1.02 −4.23 −2.30

1-HIBU

5 × 10−4 5 × 10−3 75.0 200

5.59 5.40 4.17 2.88

3.69 5.06 1.22 3.20

9.72 7.70 6.00 4.95

1.57 5.13 3.21 4.67

97.1 98.5 98.4 99.0

−5.03 −4.85 −3.96 −3.33

2-HIBU

5 × 10−4 5 × 10−3 75.0 200

6.71 4.01 4.65 3.01

−2.19 2.46 −1.61 −1.76

7.78 5.23 5.84 5.13

5.10 2.43 −2.09 1.86

90.9 92.8 90.7 91.7

−4.97 −2.72 −2.24 −1.32

3-HIBU

5 × 10−4 5 × 10−3 75.0 200

5.27 3.89 3.77 3.16

10.8 8.79 5.74 5.04

3.64 −3.95 −2.08 1.86

92.0 93.8 93.1 99.5

1.93 −5.38 −4.19 −4.04

CIBU

5 × 10−4 5 × 10−3 75.0 200

6.64 7.98 3.90 4.22

8.19 8.53 7.69 5.37

6.29 −5.93 3.30 2.26

a b c d

2.99 1.92 0.727 1.56

RSD (%)

4.81 −1.64 3.01 2.17

RE (%)

104 103 103 102

−2.49 4.67 2.84 1.80

Relative standard deviation. Relative error. Extraction recovery. Matrix effect.

described in Section 2.4, a flow-rate in the range of 0.6–1.0 mL/min and a column temperature of 35 ◦ C. The separation of five analytes and IS was completed within only 1.8 min per sample. 2-HIBU, CIBU, 3-HIBU, 1-HIBU, NAP (IS) and IBU were eluted at 0.94, 1.02, 1.04, 1.20, 1.39 and 1.52 min, respectively. 3.2. Optimization of the extraction parameters Before extraction, the hydrolysis of acyl glucuronic acid conjugates was performed. This step was taken from the literature [20], only shorter hydrolysis time was used (30 min), which was sufficient to obtain satisfactory results.

In order to obtain high levels of sensitivity and precision, several parameters of the USAEME method were considered and optimized. These included the effects of ultrasound extraction time, extraction solvent volume, ionic strength, pH and volume of sample solution, centrifugation time and speed. All determinations were performed three times for each optimized extraction parameter in three independent experiments. Extraction recovery was measured as a response to the processed spiked urine sample, which was expressed as a peak area and finally calculated as the mean of experiments. The obtained results (description and figures) were placed in Electronic Supplementary Material, ESM.

Table 3 Stability of IBU and its metabolites in human urine samples (n = 6). Analyte

Cnominal (ng/mL)

−4

Short-term stability

Long-term stability

Freeze–thaw stability

Post-preparative stability

RSDa (%)

RSDa (%)

REb (%)

RSDa (%)

REb (%)

RSDa (%)

REb (%)

REb (%)

IBU

5 × 10 5 × 10−3 75.0 200

5.18 4.89 2.84 3.99

5.13 3.43 2.66 0.621

7.59 6.08 2.92 4.51

−6.10 −5.68 −1.71 −5.56

5.54 4.14 6.64 6.57

−9.04 −2.49 −2.40 −5.72

5.08 4.08 2.21 4.27

4.41 0.625 2.19 −1.91

1-HIBU

5 × 10−4 5 × 10−3 75.0 200

5.81 3.25 5.53 4.19

−2.58 −1.57 −0.793 1.94

6.02 4.80 5.85 1.59

−7.67 −5.82 −2.30 3.39

7.94 6.31 3.44 4.81

−8.79 −6.35 −5.80 4.94

6.06 1.62 4.20 5.75

2.35 2.81 5.22 2.08

2-HIBU

5 × 10−4 5 × 10−3 75.0 200

5.70 3.35 4.55 3.73

8.37 6.60 1.23 2.95

5.84 5.46 4.02 4.23

−7.54 −6.47 −7.71 −6.33

6.46 4.26 7.17 5.88

−6.70 −7.63 −8.36 0.66

5.08 6.23 2.60 4.34

−4.97 −7.31 −5.82 −4.19

3-HIBU

5 × 10−4 5 × 10−3 75.0 200

7.62 4.94 6.94 2.62

3.79 0.94 −2.34 −1.02

9.33 3.87 5.01 1.92

−9.76 −8.55 −7.40 −6.16

9.86 6.22 2.30 1.56

−4.19 −3.80 −3.94 −4.39

7.89 6.27 5.01 2.52

6.28 −3.54 1.93 −4.03

CIBU

5 × 10−4 5 × 10−3 75.0 200

6.09 6.10 3.47 5.53

−4.25 −3.63 −0.562 0.103

6.90 8.78 3.24 3.63

−7.57 −3.73 8.10 8.88

4.43 3.13 2.33 5.64

−8.00 −6.51 −1.55 7.97

4.78 4.99 2.57 4.73

−5.86 −2.44 4.17 2.57

a b

Relative standard deviation. Relative error.

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3.3. Method validation 3.3.1. Selectivity The selectivity of the method was determined by comparing the MRM chromatograms of IBU, its metabolies and IS obtained from a blank urine samples and the corresponding spiked urine samples. As shown in Fig. 3, no significant interference from endogenous substances was observed in the chromatograms of drug-free urine at the retention times of the analytes and IS. The six lots spiked with the respective LLOQ concentrations of IBU, 1-HIBU, 2-HIBU, 3-HIBU and CIBU showed accuracies (%RE) between −2.6% and −10.0% while the precision (%RSD) ranged between 4.5% and 11.2%. All of the peaks for analytes and IS were detected with good peak shapes (IBU: As = 1.05, 1-HIBU: As = 1.10, 2-HIBU: As = 1.02, 3-HIBU: As = 1.08, CIBU: As = 0.99, IS: As = 1.01). These data show that the methodology was highly selective and there were no endogenous substances or contaminants interfering with the quantification. 3.3.2. Linearity and sensitivity The calibration curves in the urine samples were found to be linear in the concentration range of 0.0005–250 ng/mL for IBU and its metabolites. The correlation coefficient was 0.9996, 0.9998, 0.9994, 0.9998 and 0.9996 for all IBU, 1-HIBU, 2-HIBU, 3-HIBU and CIBU, respectively, on three independent validation batches. The accuracy (%RE) of the calibration curve standards ranged from −7.14 to 5.27%, whereas the precision (%RSD) ranged from 1.12 to 6.53%. The lowest concentration with an RSD < 20% was considered as LLOQs and those for IBU and its metabolites were found to be

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0.0005 ng/mL. This level of sensitivity is unlikely ever to be required in this application (ibuprofen in urine), but it may be useful if the method is adapted to small plasma samples from e.g. pediatric studies. Both the precision and the accuracy of the five analytes at the LLOQ were less than 12.5%, which indicates that this method is sufficiently sensitive for the quantitative evaluation of the five compounds. 3.3.3. Precision and accuracy The intra-day and inter-day accuracy and precision were evaluated at four concentrations levels (LLOQ, LQC, MQC, HQC), as described in Section 2.5.3 (in ESM) Data for intra- and inter-day precision and accuracy of the method for IBU and its metabolites are presented in Table 2. The RSD and RE values for intra-day precision and accuracy were in the ranges 2.19–7.98% and −4.22 to 5.06%, respectively. In contrast, the corresponding inter-day values were 4.95–10.8% and −5.93 to 6.29%, respectively. The results suggested that the method was accurate and precise for the determination of IBU and its metabolites in human urine samples. 3.3.4. Stability of the analytes The stability of analytes in human urine at four QC concentration levels (LLOQ, LQC, MQC, LQC) in six replicates was evaluated after subjecting samples to different times and temperatures that could be encountered during regular analysis. The results shown in Table 3 indicate that the five analytes were stable in human urine samples through three freeze–thaw cycles (RE in the range −9.04% to 7.97%) and at a temperature of −20 ◦ C for 4 weeks (RE in the

Fig. 4. Representative MRM chromatograms of the extracted urine sample obtained from a patient 7 h after the administration of IBU at a dose of 200 mg.

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range −9.76% to 8.88%). The analytes were also found to be stable for at least 24 h at room temperature (RE in the range −4.25% to 8.37%) and for 12 h in the autosampler after sample preparation (RE in the range −7.31% to 6.28%). All of the results showed that no significant degradation of analytes was observed over the stability duration and the conditions described. 3.3.5. Extraction recovery and matrix effect The extraction recovery of analytes from human urine was determined by comparing the peak responses of urine samples spiked before USAEME extraction with those of urine samples spiked after USAEME extraction. As shown in Table 2, the extraction recoveries of the investigated analytes were in the range from 90.7 to 104% with an RSD of less than 8.13%, which indicates that the USAEME method developed could offer good extraction recovery for these analytes in urine matrices. The matrix effects for analytes, calculated according to Section 2.5.5 (in ESM) ranged from −5.38 to 5.99% with an RSD of less than 4.81%. The results demonstrated that no significant matrix effect was observed for analytes, indicating that the ionization competition between the analyte and the endogenous co-elutions was negligible. The data are summarized in Table 2. In this study directly injecting urine (after hydrolysis) onto their UHPLC–MS/MS was also performed. The obtained results have

shown that the matrix effect was increased without extraction procedure (without extraction procedure ME ranged from −8.11 to 15.9% with an RSD of less than 7.46%). For this reason new extraction procedure, which allowed to remove interference from human urine, was developed. The recovery of IS at 20 ng/mL was 92.5% and the matrix effect of IS was 2.82%.

3.3.6. Dilution integrity and carry-over effect For the present study, the urine samples analyzed to date were outside the concentration range of the method and required dilution; therefore, the method was validated for dilution integrity. Dilution integrity experiments were carried out at 10 and 25 times the ULOQ concentration for all five analytes. The results indicated that the precision was less than 2.51% and 4.13% for dilution QC samples after 10- and 25-fold dilutions, respectively. The accuracy (%RE) after 10- and 25-fold dilutions was −4.45% and −7.17%, respectively. Carry-over is one of the most commonly encountered problems in LC–MS/MS method development. It can affect the accuracy and precision of a method and should be evaluated during method validation. For IBU, 1-HIBU, 2-HIBU, and 3-HIBU no carry-over was observed, whereas for CIBU the carry-over detected in the first double blank sample was 3.41% and 2.21% of the response detected in

Table 4 Comparison of the proposed method with other methods. Method

Analyt

LOD/LLOQ

Linear range

Recovery (%)

Extraction solvent volume

Extraction time/analysis time

Ref.

LLE-HPLC–UV LLE-HPLC–UV LLE-HPLC–UV

Ibuprofen Ibuprofen enantiomers Carboxyibuprofen enantiomers Hydroxyibuprofen enantiomers Ibuprofen enantiomers Ibuprofen Ibuprofen 2-Hydroxyibuprofen Carboxyibuprofen Ibuprofen 21 Other drugs Ibuprofen enantiomers Ibuprofen enantiomers Carboxyibuprofen enantiomers 2-Hydroxyibuprofen enantiomers Ibuprofen Diclofenac Naproxen Ibuprofen Diclofenac Acetylosalicylic acid

–/1.0 ␮g/mL –/0.1 ␮g/mL –/10 ␮g/mL

1–40 ␮g/mL 0.1–50 ␮g/mL 10–320 ␮g/mL

99.2–100 85.2–99.1 90.5–97.1

1.5 mL 0.1 mL 5 mL

10 min/14 min 12 min/30 min 25 min/21 min

[23] [26] [14]

–/0.12 ␮g/mL –/1.56 ␮g/mL –/4–17 ␮g/mL

0.12–90.0 ␮g/mL 0.78–100 ␮g/mL 5–300 ␮g/mL

59.8–84.1 93.0 85.2–103

3 mL 0.5 mL 0.5 mL

25 min/12 min –/5 min –/34 min

[21] [22] [12]

0.2 ng/L/–

0.6–5000 ng/L

89.0–102

2 mL

17 min/30 min

[5]

0.25 ␮g/mL/0.5 ␮g/mL –/0.25 ␮g/mL –/5.0 ␮g/mL

0.5–250 ␮g/mL 0.25–25 ␮g/mL 5–50 ␮g/mL

90.0–98.0 19.1–19.8 1.4–5.7

2 mL 200 ␮L 200 ␮L

20 min/20 min –/16 min 30 min/40 min

[8] [13] [17]

–/0.08 ng/mL

0.08–400 ng/mL

90.3–98.5

6 ␮L

80 min/30 min

[6]

40.6 ng/mL (DAD) 1.9 ng/mL (FLD)/–

83.2–85.3

50 ␮L

15 min/13 min

[15]

98.4–100

50 ␮L

15 min/13 min

[16]

90.7–104

100 ␮L

15 min/1.8 min

This work

LLE-HPLC–MS/MS SPE-HPLC–UV SPE-HPLC–UV

SPE-GC–MS SPE/LLE-CE-UV SPME-HPLC–UV SPME-HPLC–UV

HFLM-SPME-GC–FID

HF-LPME-FIA-CL

Ibuprofen

0.03 ␮g/mL/–

0.135–10 ␮g/mL (DAD) 6.3–50 ng/mL (FLD) 0.1–20 ␮g/mL

USAEME-UHPLC–MS/MS

Ibuprofen 1-Hydroxyibuprofen 2-Hydroxyibuprofen 3-Hydroxyibuprofen Carboxyibuprofen

–/0.0005 ng/mL

0.0005–250 ng/mL

HF-LPME-HPLC–DAD/FLD

CE: capillary electrophoresis, CL: chemiluminescence, DAD: diode array detection, FIA: flow-injection analysis, FID: flame-ionization detection, FLD: fluorescence detection, GC: gas chromatography, HFLM: hollow-fiber liquid membrane, HF-LPME: hollow fiber-based liquid-phase microextraction, HPLC: high performance liquid chromatography, LLE: liquid–liquid extraction, MS: mass spectrometry, SPE: solid-phase extraction, SPME: solid-phase microextraction, UHPLC: ultra high-performance liquid chromatography, USAEME: ultrasound-assisted emulsification-microextraction, UV: ultraviolet.

S. Magiera, S¸. Gülmez / Journal of Pharmaceutical and Biomedical Analysis 92 (2014) 193–202

an LLOQ sample. Therefore, the carry-over effect was found to be negligible from previously concentrated samples. 3.4. Method applicability Under the optimized experimental conditions, the proposed USAEME-UHPLC–MS/MS method was successfully used to analyze IBU and its metabolites in human urine samples. The applicability of the method has been demonstrated by the quantification of IBU and its phase I metabolites, 1-HIBU, 2-HIBU, 3-HIBU and CIBU in urine samples for ten patients receiving a single oral dose of 200 mg of ibuprofen. Urine samples were collected between 0 h and 36 h after drug administration. The quantification of drugs was carried out using calibration curves. Most urine samples collected from 1 h up to 20 h were diluted (1:10 or 1:25) by blank urine to be within the range of the calibration curve. IBU was detected at levels ranging from 18.18 ± 2.01 to 1563 ± 15.1 ng/mL, 1-HIBU at levels ranging from 12.08 ± 1.15 to 1316 ± 20.1 ng/mL, 2-HIBU at levels ranging from 117.6 ± 21.22 to 5526± 85.5 ng/mL, 3-HIBU at levels ranging from 9.01 ± 0.53 to 385.1 ± 9.15 ng/mL and CIBU at levels ranging from 181.2 ± 8.52 to 4167 ± 25.89 ng/mL. The extracted amount of the given dose in the interval from 0 to 36 h for the analyzed compounds was as follows: IBU: 5.66–6.85%, 1-HIBU 4.68–5.85%, 2-HIBU 27.41–28.25%, 3-HIBU 1.47–2.16%, CIBU 25.24–26.78% and the total amount excreted was 60.29–64.52%. The obtained date are comparable to an average of 67% as reported earlier [12]. The results obtained for metabolites of IBU are also consistent with the data in the literature; the two major metabolites 2-HIBU and CIBU were found in human urine at higher concentrations and the two minor metabolites 1-HIBU and 3-HIBU were found in urine in only very small concentrations [12,20]. Fig. 4 shows single ion chromatograms following the analysis of a urine sample extracted from a patient 7 h after IBU intake. A urine concentration-time profile of IBU and its metabolites for one of the patients is presented in Fig. 5.

201

organic solvent and a reduced sample preparation time. Moreover, it showed relatively lower LLOQ and wider linear ranges than previous methods. The developed methods also showed good recovery of IBU and its metabolites. These merits emphasize the fact that the proposed method is highly cost-effective, environmentally friendly and rapid. 4. Conclusions In this paper, a novel USAEME method coupled with UHPLC–MS/MS has been developed for the determination of IBU and its metabolites in human urine. For this purpose, the USAEME procedure was optimized by evaluating the influence of different parameters on the recoveries of the target compounds. Then, the analytical performance of the optimized method was evaluated achieving satisfactory linearity and precision and LLOQ of the target analytes. The proposed USAEME-UHPLC–MS/MS procedure was an efficient, simple, rapid, sensitive, cost-effective and environmentally friendly extraction and analysis method for the determination of IBU and its metabolites. The results showed good applicability of the proposed method for the determination of selected compounds in human urine. Acknowledgements I would like to thank Prof. Irena Baranowska for her very useful comments and suggestions during the preparation of this manuscript. This project was supported by funds from the Ministry of Science and Higher Education (grant no. IP 2011 032271 for 2012–2013, Warsaw, Poland). The research was performed with LC–MS/MS equipment purchased under the Silesian BIO-FARMA Project (Poland). Appendix A. Supplementary data

3.5. Comparison of the proposed method with other reported methods

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jpba.2014.01.012.

The comparison of the proposed method with other methods for the determination of IBU and/or its metabolites in biological fluids is summarized in Table 4. It can be deduced that the proposed method allows the achievement of quantitative extraction efficiencies for IBU and its metabolites by using the lowest amount of

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Fig. 5. Mean concentration-time profile for IBU and its metabolites in human urine after administration of a single 200 mg oral dose to a patient.

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