Analysis of Aceclofenac, Ketorolac, and Sulindac in Human Urine Using the Microemulsion Electrokinetic Chromatography Method

Abstract

A method to determine aceclofenac, ketorolac, and sulindac in human urine samples using microemulsion electrokinetic chromatography (MEEKC) has been developed in this study. The optimization of MEEKC conditions was carried out by changing the microemulsion compositions including the buffer pH, borate salt concentration, surfactant concentration, co-surfactant concentration, organic modifier concentration, and oil droplet concentration. The optimum separation of selected drugs was obtained with a composition of microemulsion containing 10 mM borate buffer pH 9, 0.5% sodium dodecyl sulphate (SDS), 6.6% n-butanol, 6.0% acetonitrile, and 0.8% ethyl acetate. Excellent linearity was obtained in the range concentration of 25 to 200 ppm with r² > 0.999. Limits of detection (LOD, S/N = 3) and limits of quantification (LOQ, S/N = 10) were 2.72 to 4.75 and 9.08 to 15.85 ppm, respectively. The solid-phase extraction (SPE) method using C-18 as an adsorbent and the solid phase micro-tip extraction (SPMTE) method using multiwalled carbon nanotubes (MWCNTs) as an adsorbent were used to clean-up and pre-concentrate the urine samples prior to the MEEKC analysis. The best recoveries of the selected drugs in the spiked urine sample were 91 to 103% with RSD of 1.26 to 4.03% (n = 3) using the SPE-MEEKC method.

1. Introduction

The COVID-19 pandemic has increased the demand for pharmaceuticals, such as non-steroidal anti-inflammatory drugs (NSAIDs). These drugs have been employed to treat COVID-19-associated fever, pain, and inflammation symptoms [1,2]. The analysis of NSAIDs in biological samples such as urine and plasma can be used to monitor the level of the ingestion of the drugs. Several analytical methods for the determination of NSAIDs have been reported, including gas chromatography [3], liquid chromatography [4,5], and capillary electrophoresis (CE) [6,7,8] methods. The advantages of CE for drugs analysis compared to chromatographic techniques include high separation power, low sample consumption, and less organic solvent [9,10].

Microemulsion electrokinetic chromatography (MEEKC), a mode of the CE method, has been demonstrated to have a reliable separation of both neutral and charged analytes. It involves microemulsion where water and oil were mixed, providing a larger partitioning area of the neutral and charged analytes compared to micelles. Commonly, microemulsion in MEEKC is composed of a surfactant, co-surfactant, organic modifier, and immiscible nanometer oil droplet in a buffer solution [11,12,13,14,15].

Biological samples, such as urine and plasma, often acquire a clean-up method that provides high selectivity and the usage of small-volume samples. The extraction methods for NSAIDs have been performed using liquid–liquid extraction (LLE) and solid phase extraction (SPE) on various biological and water samples [3,4,5,6,16]. Solid phase membrane tip extraction (SPMTE) was used as a sample preparation method [17,18,19]. SPMTE could reduce the extraction time and solvent usage. Multiwalled carbon nanotubes (MWCNTs) have been used as the adsorbent in SPMTE to determine triazine herbicides prior to micro-liquid chromatography [17]. MWCNTs were selected as adsorbents in this research due to their thermal stability and unique electrical properties. This extraction method was proven to give comparable LODs to the commercial SPE [17]. Mesoporous crystalline material-41 (MCM-41) was also used as an adsorbent in SPMTE for NSAIDs analysis, namely diclofenac, ketoprofen, naproxen, and mefenamic acid, in urine samples. This method has showed excellent recovery, repeatability, and reproducibility [18].

The present study developed and optimized the MEEKC method for the simultaneous determination of three NSAIDs, aceclofenac, ketorolac, and sulindac (Figure 1), in human urine samples. NSAIDs are acidic compounds with pKa values in the range of 3–6, and exist in the anionic form in a medium with pH > 7. As of the writing of this manuscript, publications on the analysis of these three drugs using SPE-MEEKC and SPMTE-MEEKC methods range from limited to none. Several parameters in the microemulsion background electrolyte (BGE), and also CE conditions, have been optimized in this research. The optimized MEEKC combined with SPE using C-18 as an adsorbent and SPMTE using MWCNTs as an adsorbent were then applied to determine the selected drugs in human urine samples. A comparison between the SPE-MEEKC and the SPMTE-MEEKC in terms of recoveries of the selected drugs is presented.

2. Materials and Methods

2.1. Materials

The analytes obtained from Sigma Aldrich (St. Louis, MO, USA) were aceclofenac, ketorolac, and sulindac. Sodium dodecyl sulphate (SDS) was obtained from Fischer Chemicals (Loughborough, Leics, UK), the SPE cartridge (LiChrolut^® RP-18 sorbent, 500 mg) was from Merck (Darmstadt, Germany), and multiwalled carbon nanotubes (MWCNTs) were purchased from Sun Nanotech (Nanchang, China). The polypropylene (PP) sheet (157 µm thickness; 0.2 µm pore size) was obtained from Membrana (Wuppertal, Germany), and purified water (18 MΩ cm) was collected from Millipore Water Purification System (Molsheim, France). All organic solvents were HPLC grade. Stock solutions of individual NSAIDs (2000 ppm in acetonitrile) were stored in the refrigerator at 5 °C. Final dilutions were prepared by diluting the stock solution with acetonitrile. All of the reagents and materials were used as purchased, without prior modifications.

2.2. Methods

2.2.1. Buffer and Microemulsion Preparation

Borate buffer was prepared by dissolving appropriate amounts of sodium tetraborate anhydrous using purified water, and the pH solution was adjusted using boric acid or NaOH solution (the changes in borate concentration due to the addition of boric acid are negligible in this experiment). The microemulsions (oil-in-water, O/W) were prepared by weighing the right amounts of SDS, n-butanol, acetonitrile, and ethyl acetate in borate buffer solution (w/v) and then sonicating them for 30 min until they became homogenous. The MEEKC conditions were optimized in this research, whereas the initial MEEKC conditions (microemulsion content and CE condition) were adapted from the previous study [19].

2.2.2. CE Instrumentation

A CE instrument from Agilent HP3D System (Agilent Technologies, Wald-Bronn, Germany) equipped with photo-diode array detector and a temperature control was used. An untreated fused-silica capillary (50 µm i.d., 375 µm o.d., and 54.5 cm of total length, 23 cm of effective length) was used for all of the separation process. The conditioning of a new capillary was performed by subsequently rinsing with 1 M NaOH solution for 10 min, with purified water for 30 min, and finally with microemulsion BGE solution for 15 min. Between runs, the capillary was rinsed with 0.1 M NaOH solution for 3 min, purified water for 3 min, and BGE for 3 min. Sample injection was performed hydrodynamically at 50 mbar for 7 s, and UV detection was 200 nm. The applied voltage and temperature were optimized in this research.

2.2.3. Sample Collection and Pretreatment

Human urine samples were obtained from a drug-free healthy volunteer and performed in accordance with the approval from the Research Ethics Committee of our institution. The urine samples were diluted to 1:1 (v/v) with purified water, and the pH sample was adjusted to 2 with 0.1 M hydrochloric acid solution to facilitate the extraction process by increasing the solubility of the NSAIDs. The spiked urine samples were prepared with spiking analytes at 1 ppm, and then the pH solution was adjusted.

2.2.4. Solid Phase Extraction (SPE)

This SPE procedure was slightly modified from a previous study [20]. SPE C-18 cartridge was preconditioned with a series of 6 mL of solvents, including hexane, acetone, and acidified purified water (pH 2) at 2 mL/min flow rate. Hexane was used to remove non-polar impurities, while acetone was used to remove polar impurities. The acidified water (pH 2) prepared the stationary phase to capture the acidic analytes. After preconditioning, a 10 mL acidified urine sample (pH 2) spiked at 1 ppm of each selected drug was loaded onto the cartridge at a 2 mL/min flow rate. The retained analytes were sequentially eluted with acetone (1 mL), methanol (2 mL), and then with acetone (2 mL). The eluted analytes were concentrated by evaporating the solvent using a gentle flow of nitrogen gas to dryness. It was reconstituted with 200 µL of acetonitrile.

2.2.5. Solid Phase Membrane Tip Extraction (SPMTE)

The preparation of the SPMTE device and the extraction procedure was similar to that of a previous experiment [17]. The extraction of selected NSAIDs from samples was performed using SPMTE with MWCNTs as a sorbent, isopropanol as a conditioning solvent, 10 mL of sample volume, 30 min of extraction time, 15 min of desorption time, sample pH 2, and 2.5% NaCl as salt addition concentration. The solid phase membrane tip was inserted into both the blank and spiked urine samples. Finally, the extracted NSAIDs were then desorbed with a 200 µL acetonitrile using ultrasonication.

3. Results and Discussion

3.1. Optimization of the MEEKC

3.1.1. Borate Buffer pH

The effect of the buffer pH was first explored with a pH ranging from 7 to 10.

Table 1. Optimization of borate buffer pH.

Peak *	Buffer pH	Peak Area *	Rs **	N **
1	7	81.14	-	33,157
	8	103.21	-	44,168
	9	101.78	-	88,576
	10	77.85	-	55,300
2	7	42.99	1.51	39,996
	8	53.68	2.06	54,699
	9	56.98	4.40	97,422
	10	61.04	2.79	66,220
3	7	103.90	6.90	25,785
	8	146.64	8.94	33,289
	9	137.24	8.18	69,081
	10	158.79	11.13	42,349

* Peak identification: (1) sulindac; (2) ketorolac; and (3) aceclofenac. Unit of peak area: (mAU × min). ** The number of theoretical plates (N) and the resolution (R_s) were calculated according to Equations (1) and (2), as follows [6]:

N = 16 (t/w)²

(1)

R_s = 2 (t₂ − t₁)/(w₁ + w₂)

(2)

where t is the migration time and w is the baseline width of the peak.

shows the results for the effect of the buffer pH on the peak area, resolution, and plate number of the analytes. Based on the results, the resolution of three analytes were greater than 1.50 for all of the buffer pHs. The best separation efficiencies for all of the analytes (N > 69,000) were obtained at buffer pH 9. This buffer pH was selected as the optimal pH and was employed in all subsequent investigations.

3.1.2. Borate Concentration

Borate concentrations were also evaluated from 2.5 mM to 12.5 mM.

Table 2. Optimization of borate concentration *.

Peak	Borate Concentration (mM)	Peak Area	Rs	N
1	2.5	103.15470	-	87,656
	7.5	106.29944	-	73,044
	10.0	117.34855	-	64,902
	12.5	112.99209	-	58,637
2	2.5	55.11003	3.74	96,642
	7.5	60.55586	3.23	81,250
	10.0	65.75339	3.22	72,622
	12.5	61.11577	3.30	70,557
3	2.5	136.20381	9.86	68,994
	7.5	149.40866	9.95	55,371
	10.0	157.86875	9.35	45,543
	12.5	150.25440	8.40	44,674

* Peak identification and notation, as in Table 1.

shows the results for the effect of borate concentrations on the resolution, separation efficiency, and peak area. From this experiment, the resolution of three analytes was greater than 1.50 for all of the borate concentrations. An amount of 10 mM of borate was chosen as the optimal borate concentration since it resulted in the highest peak area with good resolution and separation efficiency. This borate concentration was employed in all subsequent investigations.

3.1.3. Surfactant Concentration

In this study, SDS was used as surfactant, varying from 0.25% to 1.25% (w/v). As illustrated in

Table 3. Optimization of SDS concentration *.

Peak	SDS (%)	Peak Area	Rs	N
1	0.25	98.32646	-	81,842
	0.50	110.29612	-	74,069
	1.00	111.29242	-	58,456
	1.25	90.98324	-	41,461
2	0.25	35.99672	5.19	94,659
	0.50	40.22963	4.74	85,577
	1.00	59.73791	3.15	68,264
	1.25	30.02828	1.59	52,886
3	0.25	122.89176	3.37	76,173
	0.50	140.46258	4.78	64,713
	1.00	150.52068	8.54	42,739
	1.25	125.62084	10.98	30,540

* Peak identification and notation, as in Table 1.

, the resolution of three analytes were greater than 1.50 for all of the SDS concentrations. A 0.5% SDS was chosen as the optimal SDS concentration, since it resulted in a better separation efficiency than 1.00% and 1.25% SDS, with peak area greater than 0.25% SDS. This SDS concentration was used in all subsequent investigations.

3.1.4. Co-Surfactant Concentration

The n-butanol was used as the co-surfactant in this research. A series of n-butanol concentrations ranging from 3.3% to 13.2% (w/v) were investigated. As shown in Figure S1, the baseline resolution of three analytes (R_s > 1.50) was obtained using 3.3% and 6.6% n-butanol. In addition, a poor resolution was observed using 9.9% n-butanol (R_s < 1.50 between aceclofenac and ketorolac) and using 13.2% n-butanol (R_s < 1.0 between aceclofenac and sulindac). A 6.6% n-butanol was chosen as the optimal concentration, since it rendered a better resolution than 3.3% n-butanol. This concentration was employed in all subsequent investigations.

3.1.5. Organic Modifier Concentration

Acetonitrile was used as the organic modifier in this study, ranging from 3.0% to 12.0% (w/v). As presented in Figure S2, the baseline resolution of three analytes (R_s > 1.50) was obtained using 6% acetonitrile. Poor resolutions (R_s < 1.50) between aceclofenac and ketorolac were observed using 9% and 12% acetonitrile. At 3% acetonitrile, no peaks were observed in the electropherogram. Therefore, a 6% acetonitrile was selected as the optimal concentration and was employed in the further experiment.

3.1.6. Oil Droplet Concentration

The ethyl acetate was used as the oil droplet in this research. The effects of different ethyl acetate concentrations (0.2% to 0.8% w/v) were then explored in this experiment. The baseline resolution of three analytes (R_s > 1.50) was obtained using 0.2%, 0.4%, and 0.8% ethyl acetate. Further increment of ethyl acetate concentration would reduce the peak resolution of three analytes. A 0.8% ethyl acetate was selected as the optimal condition, since it gave the shorter analysis time (Figure S3). This concentration was employed in all subsequent investigations.

3.1.7. Temperature

The effect of temperature on the separation of three selected drugs was evaluated in this study from 20 °C to 30 °C. The baseline resolution of three analytes (R_s > 1.50) was obtained using all the separation temperatures (see

Table 4. Optimization of temperature *.

Peak	Temperature (°C)	Migration Time (min)	Peak Area	Rs	N
1	20	11.888	94.05359	-	112,363
	25	10.411	97.88517	-	97,228
	30	8.451	90.45343	-	48,154
2	20	12.777	35.32412	6.25	140,632
	25	11.165	37.61095	5.63	114,177
	30	8.781	33.54822	3.45	45,076
3	20	13.647	131.10137	5.71	103,746
	25	11.811	135.42228	4.42	88,639
	30	9.596	126.35067	3.62	45,076

* Peak identification and notation, as in Table 1.

). A 25 °C was selected as the optimal separation temperature since it gave the highest peak area of three analytes.

3.1.8. Applied Voltage

The effect of applied voltage on the separation of three selected drugs was also investigated in this study from 20 kV to 30 kV. The baseline resolution of three analytes (R_s > 1.50) was obtained using all of the applied voltages. At 30 kV, the migration time of the analytes was less than 8 min (see Figure S4). From this experiment, 30 kV was selected as the optimal applied voltage, since it gave the shorter analysis time.

3.2. Analytical Performance of the MEEKC Method

A typical electropherogram of the NSAIDs in the optimized MEEKC method is presented in Figure 2. Migration order of the analytes is sulindac (peak 1), ketorolac (peak 2), and aceclofenac (peak 3). The analytical performance of the optimized MEEKC method is shown in

Table 5. Analytical performance of the optimized MEEKC method *.

Parameter	Peak 1	Peak 2	Peak 3
Linearity range (ppm)	25–200	25–200	25–200
r2	0.9999	0.9996	0.9998
RSD (%, n = 3)
Migration time	0.70	1.65	0.97
Peak Area	2.03	2.09	2.30
LOD	3.51	4.75	2.72
LOQ	11.72	15.85	9.08

* Peak identifications and MEEKC conditions, as in Figure 2.

. Good repeatability in the migration time and peak area was obtained, ranging from 0.70 to 1.65% and 2.03 to 2.30%, respectively. All calibration curves were linear for all of the NSAIDs over the concentration range of 25 to 200 ppm of standards with r² higher than 0.999. The LOD (S/N = 3) and LOQ (S/N = 10) of the three analytes ranged from 2.72 to 4.75 ppm and 9.08 to 15.85 ppm, respectively.

3.3. Analysis of Urine Samples

To validate the optimized MEEKC method, the SPE using C-18 as an adsorbent was first applied for the analysis of three drugs in the blank and spiked urine samples prior to the MEEKC method. A typical electropherogram of blank and spiked urine samples using the SPE-MEEKC method are presented in Figure 3a. The recoveries of selected NSAIDs were calculated with the calibration graph of NSAIDs standards with known concentrations (see Table S1). From this experiment, excellent recoveries of NSAIDs in spiked urine samples ranged from 91 to 103% with RSD between 1.26 and 4.03%, as presented in

Table 6. Comparison between the SPE-MEEKC and the SPMTE-MEEKC methods *.

Method	Parameter	Peak 1	Peak 2	Peak 3
SPE-MEEKC	Recovery (%)	91	98	103
	RSD (%, n = 3); (intra-day)	1.33	2.33	1.26
	RSD (%, n = 9); (inter-day)	3.26	4.03	2.73
SPMTE-MEEKC	Recovery (%)	87	79	96
	RSD (%, n = 3); (intra-day)	2.04	3.15	3.66
	RSD (%, n = 9); (inter-day)	5.33	5.80	4.93

* Peak identifications and conditions, as in Figure 3.

.

In addition, the SPMTE using MWCNTs as an adsorbent was also applied and compared with the SPE method for the analysis of three drugs in the blank and spiked urine samples prior to the MEEKC method. Recoveries of selected NSAIDs from human urine samples were also calculated with the calibration graph of NSAIDs standards with known concentrations (see Table S1). A typical electropherogram of blank and spiked urine samples using SPMTE-MEEKC is shown in Figure 3b. From this experiment, recoveries of three drugs in the spiked urine samples using SPMTE-MEEKC were good for all analytes, ranging from 87% to 96% with RSD between 2.04% and 5.80% (see Table 6).

Simultaneous separation of the selected NSAIDs has been successfully achieved by the optimized MEEKC with resolutions greater than 2.0 for all of the analytes and with a migration time within 8 min. The migration time of sulindac in this result (less than 7 min) was faster than that of a previous study (greater than 9 min) [7]. The optimized MEEKC combined with SPE using C-18 as an adsorbent and SPMTE using MWCNTs as an adsorbent has been successfully applied to determine the selected drugs in the spiked human urine samples. Results showed that the SPE method gave better recoveries compared to the SPMTE method. However, the SPMTE method offers lower solvent and adsorbent consumptions than the SPE method. The SPMTE has a less-organic solvent (about 400 times lower than SPE) and a less-adsorbent mass (about 170 times lower than SPE) usage. It can reduce costs and function in environmentally friendly way. Overall, the SPMTE-MEEKC method has been demonstrated to be a promising alternative for the analysis of selected NSAIDs in biological samples.

4. Conclusions

The presented method is a proper eco-friendly alternative analysis which can be used to determine NSAIDs in human urine samples. It requires only a small amount of samples and reagents instead of hazardous organic solvents. The method was validated using SPE using C-18 as an adsorbent and SPMTE using MWCNTs as an adsorbent. The results show excellent recoveries (79–103%) for all of the analytes in the spiked urine samples.