1. Introduction
In an effort to increase the selectivity and sensitivity of electrochemical sensors, sensors have been developed based on molecularly imprinted polymers and nanoparticles. Two natural polymers have been used as functional molecules in the development of molecularly imprinted polymer (MIP)-based sensors, namely chitosan and starch [
1,
2,
3]. Cassava starch, which is a natural polymer, has the potential to be a functional polymer in the development of MIP-based sensors. Polymer molecules in cassava starch can be crosslinked by the addition of sodium tripolyphosphate (STPP) in a basic NaOH solution [
4,
5,
6]. Based on these results, cassava starch can be used as a basic polymer for membranes in the development of electrochemical sensors for the detection of acetaminophen and caffeine, simultaneously. Increasing the sensitivity of a sensor can be done by adding a conductivity material, such as Fe
3O
4 nanoparticles.
Fe
3O
4 nanoparticles were chosen as additives in membranes because they can increase the electrical conductivity of polymers [
7]. Fe
3O
4 nanoparticles are known to increase the electrical conductivity of polyindole/polyvinyl alcohol (PIN/PVA) composites. The electrical conductivity of PIN/PVA composites is 1.25 mS/cm, while the electrical conductivity of PIN/PVA composites containing Fe
3O
4 nanoparticles is 100 S/cm [
8]. Adding 0.1% Fe
3O
4 nanoparticles in the nata de coco membrane for potentiometric detection of phenol compounds can increase sensitivity up to 30% [
9]. Fe
3O
4 nanoparticles have been used for the development of diazinon sensors using nata de coco membranes [
10] and polyvinyl alcohol-based MIP membranes for the detection of chlorpyrifos [
11] and monosodium glutamate [
12].
Acetaminophen is one of the antipyretic and analgesic compounds that is most widely used as a safe and effective painkiller. Caffeine is an alkaloid which is a derivative of N-methyl from xanthine and is widely used to treat asthma, nasal congestion, and headaches. Headache medicines on the market generally contain a combination of acetaminophen and caffeine in amounts of 500–600 mg and 35–65 mg per tablet, respectively. Ensuring the quality of medicines circulating in the market, especially over-the-counter medicines, such as headache medications, is urgent. This is because there are several cases of the circulation of counterfeit medicines and repackaging expired medicines. The most important activity is determining the amount of active substances in these medicines. Determination of caffeine and paracetamol content in multicomponent medicines can be done by the titrimetric and the HPLC methods. The titrimetric method requires a long analysis time and is less sensitive, whereas the HPLC method has a high analytical sensitivity but it is considerably expensive [
13,
14,
15]. Another method of analysis, such as spectrophotometry, without separation is less accurate because there are other substances in the medicines which might interfere the measurement. Therefore, this study seeks to develop a simultaneous, easier, quicker, and more accurate determination of acetaminophen and caffeine in headache medicine by using selective membranes based on cassava starch.
Acetaminophen is 4-N-acetamino phenol or commonly called paracetamol. It can be oxidized in an acidic environment [
16] following the reaction as shown in
Figure 1. Several electrochemical detection methods for acetaminophen have been developed, including the use of electrodes from Screen Printed Graphene Electrodes. Acetaminophen in biological samples can be determined using this sensor in the range of 0.1–50 µM at pH 7 [
17].
An acetaminophen detection method in medicines has also been developed using Multi Walled Carbon Nanotubes (MWCNT) nano material and chitosan–Cu complex compounds as sensors/working electrodes. These sensors can detect acetaminophen in the concentration range of 0.1–200 µmol/L at pH 7. To increase its sensitivity, ascorbic acid and dopamine are added [
18]. The use of nano material with CuO–CuFe
2O
4 for the detection of acetaminophen and codeine, simultaneously, has resulted in a system with a high sensitivity where acetaminophen can be detected in the concentration range of 0.01–1.5 µmol/L. The detection was applied to biological fluid samples [
19]. The ability to detect compounds, with performance at very low concentrations is not applicable for pharmaceutical samples. Detection of acetaminophen is best obtained by the voltammetry method both individually and simultaneously with the detection of other compounds [
20,
21,
22,
23,
24].
Caffeine can be oxidized in an acidic environment by the mechanism shown in
Figure 2. A study to develop a detection method has been carried out for electrochemical detection of caffeine using carbon paste as a working electrode and applied to pharmaceutical samples. In that experiment caffeine was detected in the concentration range of 1–80 µM [
25]. The same detection method for caffeine has also been developed but it uses carbon electrodes, in the concentration range of 20–100 µM, and is applied to beverage samples [
26]. The development of electrochemical sensors for the detection of caffeine, both individually and simultaneously with other compounds, has been widely developed but is generally applied in beverage samples [
27,
28,
29,
30,
31].
Several electrochemical sensors have been developed based on MIP to detect caffeine and acetaminophen individually [
32,
33]. Utilizing natural polymers from renewable materials as functional molecules in the preparation of MIP is an effort to produce low-cost and environmentally friendly sensors. Research on the development of MIP-based electrochemical sensors from cassava starch—Fe
3O
4—is a new breakthrough to produce sensors that are inexpensive, selective and sensitive and environmentally friendly.
2. Materials and Method
2.1. Materials
Chemicals used in the experiments were acetaminophen standard (Sigma Aldrich, Surabaya, Indonesia), caffeine standard (Sigma Aldrich), Britton Robinson (BR) buffer solution (pH 2–7), phosphate buffer (PB) solution (pH 7), cassava starch (local product), sodium tripolyphosphate for synthesis, sodium tripolyposphate STPP (Sigma Aldrich), glycerol for synthesis (Sigma Aldrich), sodium hydroxide (Merck), ethanol (Merck), Fe3O4 50–100 nm (Sigma Aldrich).
The tools used in this study were glassy carbon working electrodes (GCE) with a disk diameter of 5 mm (Metrohm RDE.GC50) with electrode shafts, galvanostat potentiostat (Autolab PGSTAT204), Shimadzu 8400S fourier transform infrared spectroscopy (FTIR) scanning electron microscopy (SEM) FEI Inspect S50, pH meter Senz TI-13MO597, Yenaco YNC-OV-30L oven, shakers and glassware.
2.2. Preparation of MIP Membrane
The procedure for making sensors was adapted from experiments conducted by da Silva, N., Sechi, M., Teixeira, P.M. (2017) [
6]. We added 2 g of cassava starch to boiling water, and stirred until the volume reached 100 mL (2% w/v), then a few drops of 0.1 M NaOH solution were added to the pH value of 10. Next, 22 mL STPP was added along with 11 mL acetaminophen and 11 mL caffeine (concentrations of acetaminophen and caffeine as in
Table 1). The mixture was stirred at 80 °C for eight hours, then placed in a petri dish and dried at 80 °C overnight. The formed membrane was removed and washed with 3 × 30 mL of ethanol until it is free of acetaminophen and caffeine, confirmed spectrophotometry. The length of time for each washing step was one hour while shaken at 200 rpm. The washed membrane is then dried in the oven for two hours at 50 °C. The membranes were made in three different compositions, shown in
Table 1.
2.3. Electrode Modifications
A 0.05 g of dry membrane, 2 mL of hot water (70 °C), and 15 µL glycerol was mixed and continuously stirred for 30 min to form a homogeneous solution (viscous solution). Meanwhile, the glassy carbon electrode (GCE) surface was rinsed with ethanol and dried at 50 °C for 30 min. Next, the GCE surface was sprayed with a membrane suspension, then dried at 50 °C for 1 hour 30 min. An electrode modification was also made by adding 11 µL suspension of Fe3O4 nanoparticles 0.1% (w/v).
2.4. Cyclic Voltammetry (CV) and Differential Pulse Voltammetry (DPV) Measurements
The measurement was carried out on acetaminophen (1 mM), caffeine (1 mM), and acetaminophen-caffeine solutions (1:1 mM), in a BR buffer of pH 7. The electrodes used as a reference in the measurements were Ag/AgCl (3 M KCl) and Pt wires used as auxiliary electrodes. The potential applied to the CV is −0.1–1.8 volts vs Ag/AgCl (3 M KCl), with a scan rate of 0.1 V/s. The potential applied to DVP is −0.3–1.6 volts with a scan rate of 0.01 V/s, amplitude modulation 0.025 V, modulation time 0.05 s, and interval time 0.5 s.