Advances in Physics Research, volume 5

Soedirman International Conference on Mathematics and Applied Sciences (SICOMAS 2021)

Development of Ketorolac Analysis in Water Samples

using Micellar Electrokinetic Chromatography

Dadan Hermawan1,* Cacu Cacu1, Amin Fatoni1, Suwandri1, Wan Aini Wan Ibrahim2,
Hassan Y Aboul-Enein3

1
Department of Chemistry, Faculty of Mathematics and Natural Science, Jenderal Soedirman University, Indonesia
2
Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia, 81310 Johor Bahru, Malaysia
3
Department of Pharmaceutical and Medical Chemistry, Pharmaceutical and Drug Industries Research Division, Na-
tional Research Centre (NRC), Dokki, Cairo, Egypt
*
Corresponding author. Email: [email protected]

ABSTRACT
Quantitative determination of ketorolac, a nonsteroidal anti-inflammatory drug (NSAID) in water samples was reported
using the micellar electrokinetic chromatography (MEKC) method. The on-line preconcentration technique at MEKC
was then investigated to increase the detection sensitivity of ketorolac. A 6-fold increase in sensitivity factor was
achieved using normal stacking mode (NSM)-MEKC. The average recovery of ketorolac in tap water samples was
102.16% (RSD = 0.06%). The current NSM-MEKC method is simple, has short analysis time, high accuracy, and uses
fewer organic solvents (environmentally friendly).

Keywords: Ketorolac; Micellar electrokinetic chromatography; Water sample

1. INTRODUCTION and was the first to be approved for parenteral use and
postoperative therapy [2-3]. At physiological pH,
Ketorolac, a nonsteroidal anti-inflammatory drug ketorolac tromethamine dissociates to form the anion
(NSAID), is a drug that has been shown to have opioid- ketorolac. It can also be used for the treatment of pain
sparing effects and reduces opioid-related side effects after cesarean delivery, cancer-related pain, and in the
in the treatment of postoperative pain [1]. Ketorolac emergency department for the treatment of renal colic
was developed as a safer alternative to opioid pain, sickle cell crisis, migraine headaches, and
analgesics and is now available as a generic drug. musculoskeletal pain. Ketorolac has been used safely
Ketorolac or known as (RS)-5-benzoyl-2,3-dihydro- and effectively in certain pediatric populations but is
1H-pyrrolizine-1 carbox -ylic acid is a synthetic not currently recommended for use in children under 17
pyrrolizine carboxylic acid derivative. Ketorolac has a years of age [4].
different shape from other NSAIDs in that it has a
stereogenic center in the pyrrole ring and has an Terabe in 1984 [5], introduced hybrid
asymmetric carbon in the 2-arylpropionic acid side electrophoresis and a chromatographic method called
chain. Ketorolac is a non-steroidal anti-inflammatory micellar electrokinetic chromatography (MEKC) and
drug that includes a non-narcotic analgesic drug that has become reliable in the high-efficiency separation of
has antipyretic properties 20 times stronger than aspirin charged and neutral analytes using capillary
and was patented in 1976 and in 1989 was approved by electrophoresis instruments. (CE) without any changes
the USFDA for medical use. Ketorolac tromethamine is [6-11]. Capillary electrophoresis (CE) is one of the
an NSAID that is used for several clinical indications most reliable analytical liquid phase separation

Copyright © 2022 The Authors. Published by Atlantis Press International B.V.

This is an open access article distributed under the CC BY-NC 4.0 license -http://creativecommons.org/licenses/by-nc/4.0/. 12
 Advances in Physics Research, volume 5

techniques due to its small sample requirements, low simple, fast analysis, high accuracy and requires
operating costs, and high separation efficiency. CE has minimal use of organic solvents (environmentally
the availability of a CE mode which allows separation friendly).
of almost all analytes. This technique uses a voltage
that is applied to a glass capillary tube containing a
buffer solution that produces the driving force of the
electroosmotic flow [13]. The injected sample is
separated based on the electrophoretic mobility
between the ions and molecules through the capillary,
which is a result of the object's charge and the frictional
force it experiences. MEKC belongs to the category of
separation methods and is a variant of capillary
electrophoresis (CE).
Figure 1. Chemical structure of ketorolac
In MEKC, the stationary phase is the anionic
surfactant as the pseudo stationary phase and the
surrounding aqueous phase becomes the mobile phase. 2. MATERIALS AND METHODS
Sodium dodecyl sulfate (SDS) is the most widely used
2.1 Materials
surfactant in the MEKC method [14-16]. The
separation of analytes is based on the principle of Ketorolac as an analyte was purchased in St. Louis,
partition difference between the micellar phase and the MO, USA at the Sigma-Aldrich site. The chemical
aqueous phase. Micelles in the MEKC method are structure of ketorolac is shown in Figure 1. Sodium
added to the CE buffer solution and the stationary phase hydroxide and anhydrous sodium tetraborate as
to assist in separating neutral molecules. The micelle solvents under the brand name “Darmstadt”, Germany
system is chosen in such a way that the charge of the purchased from Merck. Sodium dodecyl sulfate (SDS)
micelles results in their opposite electrophoretic as a chiral selector was purchased from Fischer
mobility as opposed to electroosmotic [17-18]. Chemicals located in Loughborough, Leics, UK. JT
Baker in Pennsylvania, USA, provided the organic
The poor CE concentration sensitivity is due to the
solvent (HPLC grade). Deionized water (DI) was
small sample size (1–10 L) and the short optical path
purified with Millipore Simplicity (Simpak®2)
length and capillary diameter for the absorbance
(Barnstead, USA). The standard stock solution was
detector [19-23]. Electrochemical measurements and
prepared by dilution with methanol which was used as
highly sensitive detectors for laser-induced
the working standard solution. Standard stock is stored
fluorescence are one possible solution. Another
in the refrigerator until needed.
solution is to expand the optical path such as bubble
cells or Z-type cells, while minimally lowering the 2.2 Instrumentation
resolution, which can increase sensitivity. However, all
A CE system from Agilent Technology (7100,
these methods take a long time and the hardware is a bit
Waldbronn, Germany) equipped with a DAD and
expensive and a bit complicated. Online sample
operating at a wavelength of 320 nm was used for the
preconcentration is one of the most reliable solutions
analysis. The separation process uses uncoated fused
for increasing the level of concentration sensitivity,
silica capillaries, which have a total length of 64.5 cm
where a longer-than-usual plug sample is injected and
and 56 cm for the detector size, and the inside diameter
focused on the capillary prior to separation. The use of
(ID) is 50 µm. The CE system (Chromatography
pressurized injection, also known as hydrodynamics or
Station CSW 1.7) was used to collect analytical data.
electrokinetic, can be used. Sample stacking and
1.0 M NaOH was used for conditioning. DI water was
sweeping are recognized as two online sample
used for new capillary equalization for 30 minutes,
preconcentration techniques in providing increased
followed by 0.1 M NaOH solution for 15 minutes, DI
concentration sensitivity in MEKC [24-26].
water for 15 minutes, and finally with BGE solution for
This work aims to develop the MEKC method, 10 minutes. Hydrodynamic injection in all cases was
which is very potent and can be used for the analysis of used to optimize separation at 50 mbar for 5 s (5 µL
NSAIDs, namely ketorolac. This method was then used sample volume) at the capillary inlet.
to evaluate the application of normal stacking mode
(NSM) [27], as an online preconcentration technique to
increase the concentration sensitivity of MEKC. 3. RESULTS AND DISCUSSIONS
method for the analysis of ketorolac in water samples.
It is known that the low sensitivity of MEKC
The MEKC method proposed for ketorolac analysis is
concentration is caused by several factors, including the

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small number of samples injected (1-10 L sample seconds. The decrease in peak height occurred when
volume) [28-33]. This study uses the normal stacking there was an increase in sample injection time (more
mode applied to the optimized MEKC system to than 80 seconds), while the peak area did not increase
increase the detection sensitivity of ketorolac. The significantly (data not shown). After confirming that no
stacking process is performed when the conductivity of ketorolac was found in the tap water sample, the NSM-
the sample is significantly lower than that of the MEKC method was used to analyze the ketorolac in the
running buffer. The sampling procedure in the normal spiked tap water sample. Figure 2 shows an
mode under MEKC conditions is referred to as normal electropherogram of 0.75 g/mL of ketorolac in a spiked
stacking mode (NSM). The application of the NSM tap water sample and a tap water sample using the
method on the MEKC system uses a lower conductivity optimized NSM-MEKC method. There is no
of the sample solution compared to the interference matrix peak with a standard rejection peak.
micellar/separation solution. Pure water is used to
dilute the sample stock solution [34-36].
The NSM method is used to optimize the sample
injection time and the best sample injection time is 80

Figure 2. Electropherogram (A) 0.75 µg/mL ketorolac in spiked tap water samples; and (B) tap water samples; with
the optimized NSM-MEKC method.

The average recovery of ketorolac in spiked tap on the application of other online preconcentration
water samples was 102.16% (RSD = 0.06%, n=3). The techniques at MEKC for ketorolac analysis.
NSM-MEKC method increased the detection
sensitivity of ketorolac 6-fold when compared to the AUTHORS’ CONTRIBUTIONS
conventional MEKC injection method. These results
indicate that NSM-MEKC tends to improve the DH performed CONCEPTUALIZATION,
detection of ketorolac. METHODOLOGY, SUPERVISION, and REVIEW. C
did INVESTIGATION, DATA CURATION,
4. CONCLUSIONS WRITING and EDITING. AF made VALIDATION,
Analysis of ketorolac in water samples has been SOFTWARE, PROJECT ADMINISTRATION, and
reported using the micelle electrokinetic SUPERVISION. S performed SUPERVISION,
chromatography (MEKC) method. The on-line PROJECT ADMINISTRATION, and VALIDATION.
preconcentration technique at MEKC using the normal WAWI did CONCEPTUALIZATION, SUPERVISION,
stacking mode suggests an increased possibility of FORMAL ANALYSIS, and VALIDATION. HYA
detection of ketorolac. The results obtained can be used contributed to CONCEPTUALIZATION,
as a reference in a preliminary study for further studies SUPERVISION, FORMAL ANALYSIS, AND
VALIDATION.

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ACKNOWLEDGMENTS [10] M.E. El-Kommos, N.A Mohamed, A.F. Abdel

Hakiem. Selective micellar electrokinetic
This work was supported by the Ministry of chromatographic method for simultaneous
Education, Culture, Research, and Technology determination of some pharmaceutical binary
Indonesia and Universitas Jenderal Soedirman mixtures containing non-steroidal anti-
Indonesia through the Basic Research Grant 2021. inflammatory drugs, J. Pharm. Anal, 2013, pp. 53–
60.
[11] M. Silva, Micellar electrokinetic chromatography:
REFERENCES
a practical overview of current methodological
and instrumental advances, Electrophoresis, 2011,
[1] R.R. Sawkar, V.B. Patil, M.M. Shanbhag, N.P. pp. 149–165.
Shetti, N.M. Tuwar, T.M. Aminabhavi. Detection [12] Y. Zhao, L. Xu. Chiral separation of d,l-p-
of ketorolac drug using pencil graphite electrode, hydroxyphenylglycine by ligand exchange
Biomed. Eng. Adv, 2021, pp. 100009. micellar electrokinetic capillary chromatography,
[2] R. Bhushan. Liquid chromatographic Chromatographia, 2015, pp. 717–721.
enantioseparation, determination, bioassay and [13] Q. Zhu, G.K.E. Scriba. Advances in the use of
isolation of enantiomers of Ketorolac: A review, cyclodextrins as chiral selectors in capillary
Acta Chromatogr, 2021, pp. 1-17 electrokinetic chromatography: fundamentals and
[3] A. Macià, F. Borrull, M. Calull, C. Aguilar. applications, Chromatographia, 2016, pp. 1403–
Capillary electrophoresis for the analysis of non- 1435.
steroidal anti-inflammatory drugs, TrAC - Trends [14] L.Q. Li, J.T. Baibado, Q. Shen, H.Y. Cheung.
Anal. Chem, 2008, pp. 133–153. Determination of the authenticity of plastron-
[4] N. Vadivelu, D. Chang, E.M. Helander, G.J. derived functional foods based on amino acid
Bordelon, A. Kai, A.D. Kaye, D. Hsu, D. Bang, I. profiles analysed by MEKC, J. Chromatogr. B
Julka. Ketorolac, oxymorphone, tapentadol, and Anal. Technol. Biomed. Life Sci, 2017, pp. 23–30.
tramadol: a comprehensive review, anesthesiol, [15] Q. Liang, H. Chen, F. Li, X. Du. Simultaneous
Anesthesiol. Clin, 2017, pp. 1–20, sensitive mekc–lif determination of homocysteine,
[5] S. Terabe, K. Otsuka, K. Ichikawa, A. Tsuchiya, T. homoarginine, and six arginine metabolic
Ando. Electrokinetic separations with micellar derivatives in fluids from type 2 diabetics with
solutions and open-tubular capillaries, Anal. peptic ulcer bleeding, Chromatographia, 2015, pp.
Chem, 1984, pp. 111–113, 1049–1056.
[6] K.C.A. Rezende, N.M. Martins, M. Talhavini, [16] P.C. Lin, Y.H. Hsieh, F.F. Liao, S.H. Chen.
Wendell, K.T. Coltro, Determination of the Determination of free and total levels of phenytoin
alcoholic content in whiskeys using micellar in human plasma from patients with epilepsy by
electrokinetic chromatography on microchips, MEKC: An adequate alternative to HPLC,
Food Chem, 2020, pp. 127175. Electrophoresis, 2010, pp. 1572–1582.
[7] K. Ciura, H. Kapica, S. Dziomba, P. Kawczak, M. [17] W. Xiao, C. Chen, Q. Zhang, Q.H. Zhang, Y.J. Hu,
Belka, T. Bączek. Biopartitioning micellar Z.N. Xia, F.Q. Yang, Separation Study of eight
electrokinetic chromatography–concept study of isoflavones by mekc with different surfactants,
cationic analytes, Microchem. J, 2020, pp. 104518. Chromatographia, 2015, pp. 1385–1393.
[8] H.Y. Ko, C.J. Shih, Y.H. Lin, Y.L. Chen [18] A. Dervishian, J. Goodwin, C. King, C. Copper.
Determination of phenylenediamines in hair Micellar electrokinetic chromatographic analysis
colors derivatizated with 5-(4, 6-dichlorotriazinyl) of historic dyes and their photofading products,
aminofluorescein via micellar electrokinetic Chromatographia, 2021, pp. 979–983.
chromatography, J. Food Drug Anal, 2019, pp. [19] L.Q. Peng, S.L. Dong, W. Xin, X.T. Zhen, J. Yang,
825–831. Y. Chen, J.C. Tian Xie. Simultaneous separation
[9] H.L. Minglei Wu, F. Gao, Y. Zhang, G. Wang, Q. and concentration of neutral analytes by
Wanga. Sensitive analysis of antibiotics via cyclodextrin assisted sweeping-micellar
hyphenation of field-amplified sample stacking electrokinetic chromatography, Anal. Chim. Acta,
with reversed-field stacking in microchip micellar 2020, pp. 224–230.
electrokinetic chromatography, J. Pharm. Biomed.
Anal, 2015, pp. 91–98.

15
 Advances in Physics Research, volume 5

[20] G.D. Roberto Gotti, J. Fiori, S. Bosi. Field- [30] X. Peng, H. Wang, B. Yang, X. Zhan, Y. Wu.
amplified sample injection and sweeping micellar Field-Amplified sample injection-capillary
electrokinetic chromatography in analysis of electrophoresis for the determination of bisphenol
glyphosate and aminomethylphosphonic acid in a, α-naphthol and β-naphthol in drinks and lake
wheat, J. Chromatogr. A, 2019, pp. 357–364. water, Chromatographia, 2016, pp. 327–333.
[21] P.K. Marta Gładysz, M. Król, M. Woźniakiewicz. [31] L. Kartsova, D. Moskvichev, E. Bessonova, M.
The increase of detection sensitivity of micellar Peshkova. Imidazolium Ionic liquids in
electrokinetic capillary chromatography method microemulsion electrokinetic chromatography for
of stamp pad inks components by applying a separation of polyphenol antioxidants,
sample stacking mode for the purpose of Chromatographia, 2020, pp. 1001–1008.
questioned document examination, Talanta, 2018,
[32] G. Sunil, M. Jambulingam, S. Ananda
pp. 287–295.
Thangadurai, D. Kamalakannan, R.
[22] D. Hermawan, I.M. Yatim, K. Ab Rahim, M.M. Sundaraganapathy, C. Jothimanivannan.
Sanagi, W.A. Wan Ibrahim, H.Y. Aboul-Enein. Development and validation of ketorolac
Comparison of HPLC and MEEKC for tromethamine in eye drop formulation by RP-
miconazole nitrate determination in HPLC method, 2017, Arab. J. Chem. pp. S928–
pharmaceutical formulation, Chromatographia, S935.
2013, pp. 1527–1536.
[33] D. Hermawan, W.A. Wan Ibrahim, M.M. Sanagi,
[23] H.Y. Aboul.-Enein, D. Hermawan, W.A. Wan H.Y. Aboul-Enein. Chiral separation of econazole
Ibrahim, M.M. Sanagi. Chiral separation of using micellar electrokinetic chromatography with
econazole using micellar electrokinetic hydroxypropyl-γ-cyclodextrin, J. Pharm. Biomed.
chromatography with hydroxypropyl-γ- Anal. 2010, pp. 1244–1249.
cyclodextrin, J. Pharm. Biomed. Anal, 2010, pp.
[34] M. Sertić, A. Mornar, B. Nigović (2020) Simple
1244–1249.
and rapid micellar electrokinetic chromatography
[24] H.J. Lin, K.P. Hsieh, S.S. Chiou, H.S. Kou, S.M. method for simultaneous determination of
Wu, Determination of deferasirox in human febuxostat and its related impurities,
plasma by short-end injection and sweeping with Chromatographia, 2020, pp. 993–1000.
a field-amplified sample stacking and micellar
[35] M. Rucins, D. Baron, A. Plotniece, J. Petr.
electrokinetic chromatography, J. Pharm. Biomed.
Determination of hormone antagonists in waste-
Anal, 2016, pp. 497–502.
water samples by micellar electrokinetic
[25] Z. Mal, P. Gebauer, P.B. Ek. Contemporary sample chromatography, Chromatographia, 2018, pp.
stacking in analytical electrophoresis, 1607–1612.
Electrophoresis, 2011, pp. 116–126.
[36] J. de Jesús Olmos-Espejel, I. Ocaña-Rios, A.
[26] W.A. Wan Ibrahim, D. Hermawan, M.M. Sanagi, Peña-Alvarez, C.J. Catenza, K.K. Donkor.
H.Y. Aboul-Enein. Stacking and sweeping in Micellar electrokinetic chromatography method
cyclodextrin-modified MEKC for chiral development for sensitive monitoring of rotenone
separation of hexaconazole, penconazole and in lake waters, Chromatographia, 2020, pp. 241–
myclobutanil, Chromatographia, 2020, pp. 305– 247.
309.
[27] J.P. Quirino. Modern injection modes (stacking)
for CE, Anal. Sep. Sci, 2015, pp. 531–554.
[28] D. Hermawan, W.A. Wan Ibrahim, S.M.A. Wahib,
H.Y. Aboul.-Enein, M.M. Sanagi. Chiral
separation of vinpocetine using
cyclodextrin_modified micellar electrokinetic
chromatography, Chirality, 2012, pp. 252–254.
[29] P.M. Nowak, E. Sekuła, P. Kościelniak.
Assessment and comparison of the overall
analytical potential of capillary electrophoresis
and high-performance liquid chromatography
using the rgb model: how much can we find out?,
Chromatographia, 2020, pp. 1133–1144.

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