High‑Performance Liquid Chromatography for Determining a Mixture of Nonsteroidal Anti‑inflammatory Drugs

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https://doi.org/10.1007/s13391-021-00306-8

ORIGINAL ARTICLE - ELECTRONICS, MAGNETICS AND PHOTONICS

High‑Performance Liquid Chromatography for Determining a Mixture of Nonsteroidal Anti‑inflammatory Drugs

Md. Mynul Hassan¹ · Sung‑Wook Nam¹

Received: 16 July 2021 / Accepted: 23 July 2021 / Published online: 13 August 2021

Abstract

Nonsteroidal anti-inflammatory drugs (NSAIDs), which block the activity of cyclooxygenase (COX) isoenzymes and inhibit the synthesis of prostaglandin, have been used for pain relief. For the enhancement of the potency or the decrease of side- effects, NSAIDs are normally prescribed as a mixture with other chemical components including caffeine and proton pump inhibitor. Here, we developed a method to separate a mixture of three NSAIDs, such as aspirin, paracetamol, and naproxen, using reverse-phase high-performance liquid chromatography (RP-HPLC). An isocratic mobile phase consisting of acidic water and acetonitrile was selected to run at a low flow rate, such as 0.8 mL/min. The mixture of three NSAIDs was injected at a low volume into a C18 column having 150 mm in length and characterized using a UV detector at 230 nm. We identified three peaks in the chromatogram indicating three compounds. The elution time of the peaks was less than 10 min. The method proposed here can be used for identification of the combination of NSAIDs.

Graphic Abstract

Keywords High-performance liquid chromatography · Nonsteroidal anti-inflammatory drugs · Drug mixture · Separation · Short column

1 Introduction

Nonsteroidal anti-inflammatory drugs (NSAIDs) are regarded as cyclooxygenase (COX) inhibitors and gener- ally prescribed as pain relievers for headaches, rheumatoid arthritis, postoperative pain, and orthopedic fractures [1–3]. NSAIDs constitute one of the most widely used

* Sung-Wook Nam nams@knu.ac.kr

1 Department of Molecular Medicine, School of Medicine, Kyungpook National University, Daegu 41405,

Republic of Korea

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classes of drugs, with more than 70 million prescrip- tions and more than 30 billion tablets sold annually in the United States [4]. Despite NSAIDs’ beneficial effects, their use has been limited due to their side effects, such as mucosal injury to the gastrointestinal tract and the risk of thrombotic cardiovascular events [5, 6]. To reduce the gastrointestinal side effects, NASIDs are frequently treated as a mixture form with proton pump inhibitors such as omeprazole, esomeprazole, and pantoprazole [7–9]. In addi- tion, for the enhancement of the drug potency, NSAIDs have been treated with caffeine as a fixed-dose combination [10, 11]. Therefore, there are growing researches on the simultaneous analysis of the mixture forms regarding NSAIDs [1–6].

Aspirin (2-acetoxybenzoic acid), paracetamol (N-acetyl- 4-aminophenol), and naproxen [(S)-(+)-2-(6-methoxy- 2-naphthyl)propionic acid] are classic NSAIDs even available in local drug stores. Hence, it is meaningful to understand the separating principle among aspirin, paracetamol, and naproxen, especially by high-performance liquid chromatography (HPLC), which is one of the most widely used tools for separating drug mixtures [12, 13]. To separate chemicals via HPLC, the fine control of the affinity between stationary phase and analyte is important [14, 15].

For reverse phase (RP)-HPLC method, the chromatography column is filled with non-polar stationary phase like C18 coated silica beads which have a high affinity with non-polar chemical components. Therefore, the non-polar ones in a mixture are likely to migrate more slowly than the polar ones [14, 15].

HPLC has been used for the quantitative and qualitative analysis of NSAIDs [16–20]. However, it is a challenge to separate the multiple components of NSAIDs with an isocratic method using a short column [16]. In this regard, we present an isocratic RP-HPLC method that determines aspirin, paracetamol, and naproxen in a combined solution to study the effect of molecular structures on the separating process. In contrast to normal phase-HPLC where stationary phase is highly polar, RP-HPLC is based on the non- polar stationary phase [14, 15]. In particular, we focus on the effect of molecular polarity of the NSAIDs on the elution time in HPLC chromatogram.

The molecular structures of aspirin, paracetamol, and naproxen are shown in Fig. 1. Interestingly, aspirin, paracetamol, and naproxen have both phenyl ring and carbonyl group in common, yet they are known to be separated by HPLC owing to the difference of charge status, polarity, and atomic coordination. By analyzing the retention time of the chromatogram with the molecular structures, we propose a mechanism to separate aspirin, paracetamol, and naproxen. In our belief, the separating mechanism of NSAIDs described here would be applicable for the analysis of the

2 Experimental

2.1 Chemicals and Solvents

HPLC grade acetonitrile (Honeywell Burdick & Jackson, Ulsan, Korea), and analytical reagent grade orthophosphoric acid 85%, potassium dihydrogen phosphate, and triethyl- amine (Merck, Darmstadt, Germany) were purchased. Water was purified using a Smart2Pure (Thermo Scientific) system. The mobile phase was filtered using PTFE membrane filter of 0.45 μm pore size. Each aspirin (Bayer Bitterfeld GmbH), paracetamol (Janssen Korea Ltd.), and naproxen (Chong Kun Dang Pharma, Korea) tablet contains 500 mg of active pharmaceutical ingredient (API).

2.2 First Dilution

The first dilution procedures to prepare stock solutions are described in Fig. 2a. For aspirin, paracetamol, and naproxen tablets, 10 tablets of each drug were ground to a fine powder using a glass mortar and pestle. Aspirin (60.0 mg), paracetamol (30.3 mg), and naproxen (52.0 mg) were transferred to 100 mL volumetric flask each. A mixture of acidic water and acetonitrile at a ratio of 70:30 was used as a diluent.

Approximately, 75 mL of diluent was added to each volumetric flask. The solutions were shaken and sonicated for 15 min. After cooling down the solution, the 100 mL volumetric flasks were completely filled with the diluent. Each solution was stored as a stock solution of aspirin, paracetamol, and naproxen.

2.3 Second Dilution and Drug Mixture Preparation The second dilution procedure was conducted to prepare individual drug solution as shown in the left panel of Fig. 2b.

From each stock solution of aspirin, paracetamol, and naproxen, 2 mL was transferred to each 50 mL volumetric flask. Then, the volumetric flasks were completely filled with mobile phases. The mobile phase solutions consisted of acidic water and acetonitrile with the ratio of 70:30 with the pH between 2.5 and 3.0. By considering the pKa values of aspirin, paracetamol, and naproxen, pH ~ 2.5–3.0 of the

Fig. 1 Molecular structures of a 2-acetoxybenzoic acid (aspirin), b N-acetyl-4-aminophenol (paracetamol), c (S)-(+)-2-(6-methoxy- 2-naphthyl)propionic acid (naproxen)

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mobile phases was adjusted to make the drug compounds have acidic forms. The second-diluted solutions of the individual drugs were transferred to the HPLC vial via 0.25 μm PTFE filters.

For preparing the drug mixture, 2 mL stock solutions of aspirin, paracetamol, and naproxen are combined in a 50 mL volumetric flask, as shown in the right panel in Fig. 2b. The flask was completely filled with a mobile phase solution.

For the drug mixture solution, the mobile phase consisted of acidic water (pH 2.5) and acetonitrile at the ratio of 85:15.

The drug mixture solution was transferred to the HPLC vial through a 0.25 μm PTFE filter.

2.4 Chromatographic Conditions

HPLC makes use of high-pressure pumps that speed the move- ment of analytes down the column [14]. HPLC technology needs a sophisticated coordination of electronic and optical

devices with silica bead-based column system combined with novel surface chemistry and high purity reagents [21–24].

As described in Fig. 2c, we used the Waters company HPLC system that includes a gradient multi-solvent delivery system (Waters Delta 600 pump), helium degasser, autosam- pler (Waters 717 plus), and a UV/VIS detector (Waters 2489 lamp). A Waters Empower 2 software was used to control the system. HPLC analysis was conducted isocratically at room temperature using a Waters Atlantis C18 (100 Å, 3 µm, 4.6 mm × 150 mm) column.

The flow rate was varied from 0.8 to 1.0 mL/ min and the eluent was monitored by an UV detector with wavelength ranging from 230 to 260 nm. The injection volume was 10–20 µL. The run time is 10–15 min.

Fig. 2 a First dilution of aspirin, paracetamol, and naproxen to pre- pare stock solutions, b Second dilution and drug mixture preparation.

c A schematic of high-performance liquid chromatography (HPLC).

A reservoir contains the solvent, and a high-pressure pump is used to

generate a specified flow rate of the mobile phase. An injector intro- duces the sample into the mobile phase for the sample to pass through the HPLC column. The UV detector identifies the separated compounds as they are eluted from the HPLC column

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Fig. 3 High-performance liquid chromatography (HPLC) chromatograms of the analysis of the nonsteroidal anti-inflammatory drugs showing

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3 Results

Figure 3a–c show chromatograms of aspirin, paracetamol, and naproxen, respectively. The retention times of aspirin, paracetamol, and naproxen are 3.31, 2.55, and 4.94 min, respectively. We identified that the retention time of paracetamol is shortest among them. Since the stationary phase of RP-HPLC is a hydrophobic material such as C18, the greater the polarity of analyte molecule, the less the affinity with stationary phase, thereby reducing the retention time [15]. Based on the principle, we interpret that paracetamol behaves as the most polar compound among them. On the other hand, the retention time of naproxen is longest, implying that naproxen behaves as the least polar compound among them.

Figure 3d shows chromatogram of drug mixture of aspirin, paracetamol, and naproxen. The sequence of the retention time of the compounds is as following: Paracetamol, asprin, and naproxen. The result is consistent with those of single compounds shown in Fig. 3a, b, and c. The retention time of aspirin, paracetamol, and naproxen is summarized in Table 1.

The pKa value influences the solubility and the ionized form of the compounds [25–27]. In particular, pKa value determines the charge state depending on pH of the solution. The pKa values of aspirin, paracetamol, and naproxen are 3.41, 9.46, and 4.19, respectively. Since pH of the mobile phase that we used is 2–3, less than the pKa values of aspirin, paracetamol, and naproxen, it is expected that the mobile phase induces all the compounds to have acidic forms [25, 26]. Note that aspirin and naproxen are acidic compounds due to carboxylic acid group (–COOH), whereas paracetamol is a basic compound due to amide group (–NH–). Therefore, as the acidic forms, aspirin and naproxen have no charges, that is, the net charge is 0, whereas paracetamol have one positive charge, thus having the +1 net charge as an ionized form [25]. Based on the charge status of each compound, it is expected that paracetamol has the highest solubility among the compounds, which was reflected in our experiment.

4 Discussions

It is fairly common for drugs to be classified as acids, bases, neutral or zwitterionic [26]. Aspirin and naproxen are acids (–COOH), whereas paracetamol is base (–NH–).

By considering the functional group of each compound, we interpret the chromatogram. Since pH of the mobile phase in our experiment is 2–3, which is an acidic environ- ment, only paracetamol has positive charge status whereas aspirin and naproxen have neutral forms.

Besides, molecular polarity is another contributor to the retention time of HPLC chromatogram [26]. In the analysis of the dipole moment based on the difference of the electronegativity of the atoms, it is noticed that paracetamol has the functional groups of –OH, –NH–, C=O, in which O and N have relatively large electronegativity values, as shown in the chemical structures of Fig. 1.

Interestingly, all O and N atoms are directed downwards, thus paracetamol is expected to be the most polar molecule among them. On the other hand, naproxen has two conju- gated phenyl rings, whereas aspirin and paracetamol have one phenyl ring each. Therefore, it is expected that naproxen has the lowest polarity among them. Based on the analysis of molecular structure, we predict the molecular polarity as followings: Paracetamol > Aspirin > Naproxen.

The prediction is well established in the experimental results. In the RP-HPLC, the stationary phase is C18 which is hydrophobic. Therefore, the polar compound has less affinity with the stationary phase, thus having shorter retention time [12–15]. As shown in the chromatogram in Fig. 3, the retention time of paracetamol is shortest, followed by aspirin and naproxen. We found that the ten- dency of the retention time of aspirin, paracetamol, and naproxen are consistent with the results reported in other studies [16–20].

5 Conclusions

In conclusion, we have developed an isocratic RP-HPLC method to analyze a mixture of NSAIDs, including aspirin, paracetamol, and naproxen. We have identified an optimal isocratic condition for analyzing the mixture of aspirin, paracetamol, and naproxen by adjusting the mobile phase and pH parameters. In particular, we interpret the charge status of the NSAIDs compounds by comparing the pKa of drugs and the pH of mobile phase. Also, we analyze the molecular structure to predict the polarity. In terms of both charge status and molecular polarity, paracetamol is expected to have the shortest retention time, which is well matched with our experiments. The parameter adjusting

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principle can be used for the identification of combination of NSAIDs with other drugs.

Acknowledgements This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Ministry of Science

& ICT (2017M3A9G8083382). This research was supported by NRF-2018M3A9H3023077/2021M3A9H3016063.

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summary of aspirin, paracetamol, naproxen, and their mixture

Aspirin Paracetamol Naproxen Mixture

Aspirin Paracetamol Naproxen

Retention time (min) 3.31 2.55 4.94 3.15 2.82 5.52

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