Recent Advances in Electroanalysis Techniques Used for Morphine in Managing Postoperative Pain

Abstract

Determination of a particular drug in pharmaceutical preparations and biological fluids is critically important in pharmaceutical and medical sciences to avoid its overdose. Effective analysis requires sensitivities at ppb level or even less in the biological fluids with high selectivity. Morphine is a potent analgesic drug that is used to relieve severe pains like postoperative pain, labor pain, and cardiac pain. It is a μ-opioid agonist which acts directly on the central nervous system to relieve pain. It is very important to monitor the doses of morphine in the patient’s body under examination since the overdose may cause disruption to the central nervous system. As the applications of analytical instruments are progressing, modern electrochemical methods are attracting interest for the analysis of therapeutic agents or their metabolites in medical samples since these methods are economic and can detect extremely low concentrations approximately 10 ng/ml. A review of the principles and application of modern electroanalytical techniques, namely, cyclic voltammetry, differential pulse voltammetry, square wave voltammetry, and amperometry, is presented. The use and advantages of these techniques at different electrodes for the detection of morphine have been discussed. The analytical applications of these techniques to pharmaceutical compounds in dosage forms and biological media are also discussed.

1. Introduction

Morphine is a narcotic analgesic drug mainly used for the relief of postoperative pain, cardiac pain, pain of childbirth, and terminal cancers. It is one among the 50 different alkaloids present in opium and poppy derivatives. It is the active metabolite derived from heroin (3,6-diacetylmorphine). Morphine is a μ-opioid agonist which acts directly on the central nervous system to relieve pain [1]. Consequently, it can cause disruption to the central nervous system if not used properly. The minimum lethal dose is 200 mg but in case of hypersensitivity 60 mg can bring sudden death. In the case of drug addiction, 2–3 g/day can be tolerated [2]. Therefore, the determination of concentration of morphine in the patient’s body is a very important issue.

Morphine is a benzylisoquinoline alkaloid with two additional ring closures. It has a rigid pentacyclic structure consisting of a benzene ring, two partially unsaturated cyclohexane rings, a piperidine ring and a tetrahydrofuran ring. The structure of morphine is given in Figure 1. The first three rings make the phenanthrene ring system which has little conformational flexibility. There are two hydroxyl functional groups (a phenolic –OH and an allylic –OH), an ether linkage, a basic 3o-amine function, and 5 centers of chirality with morphine exhibiting a high degree of stereoselectivity of analgesic action.

Figure 1.

Structure of morphine.

Morphine is metabolized into morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G). The metabolism of morphine occurs not only in the liver but may also take place in the brain and the kidneys. The glucuronides are mainly eliminated via bile and urine. A highly polar metabolite that is unable to pass the blood-brain barrier is glucuronides. Although morphine glucuronidation in human brain tissue has been shown, the capability is much lower than in the liver. This shows that morphine glucuronides, despite their high polarity, can enter the brain and that the M3G and M6G concentrations detected in the cerebrospinal fluid (CSF) following systemic injection represent hepatic metabolism of morphine [3].

Moreover, it has been found that there has been a significant increase in morphine-related crimes in recent years. More and more people have started taking this drug including youngsters. Drug-related crime has been a serious worldwide problem. Therefore, there is an urgent demand to develop a rapid and sensitive analytical method for prohibiting the overuse of morphine as well as its determination in pharmaceutical and clinical samples. Traditional analytical methods like gas chromatography (GC), high-performance liquid chromatography (HPLC), mass spectrometry (MS), etc. have been developed for the determination of morphine. This concentration of drug can be detected by HPLC [4, 5, 6] as well as GC-MS in urine samples [7]. However, these methods need expensive equipment and professional operators, which may not satisfy the needs of modern drug prohibition programs. The development of simple, convenient, highly sensitive, versatile, fast, and economic techniques is very necessary to control the overuses of this drug causing many side effects. Recently, electroanalytical methods have been widely used for the analysis of morphine. Different methods in electroanalytical techniques have been discussed in this chapter for the detection of morphine in clinical samples.

2. Postoperative pain relievers

The effective relief of pain to a patient undergoing surgery is of extreme importance as it has substantial physiological benefits. Reducing pain with a minimum amount of side effects is the main goal of postoperative pain relievers. Various agents (opioid vs. nonopioid), routes (oral, intravenous, neuraxial, regional), and modes (patient controlled vs. “as needed”) are available for the treatment of postoperative pain. Despite years of advances in pain management, the mainstay of postoperative pain therapy in many settings is still opioids. Opioids bind to receptors in the central nervous system and peripheral tissues and modulate the effect of the nociceptors [8].

Moderate to severe pain, either acute or chronic, is among the conditions for which morphine sulfate is FDA approved. Morphine, which is most frequently used in pain management, significantly reduces pain in individuals [9]. The management of palliative/end-of-life care, ongoing cancer treatment, and vaso-occlusive pain during sickle cell crises are clinical scenarios that benefit greatly from morphine medication [10]. The off-label use of morphine is common for practically any painful disease. When patients in the emergency room do not respond to first- and second-line medications for musculoskeletal pain, stomach discomfort, chest pain, arthritis, and even migraines, morphine is administered [11].

In a certain case study, 280 individuals undergoing various types of surgery, including thoracic, upper, and lower abdominal, perineal, obstetric, and orthopedic procedures, were evaluated for postoperative pain alleviation. Through an indwelling epidural catheter, morphine (2/4 mg) was administered following the procedure. Only 3.5% of patients reported being unsatisfied, compared to 87% who had excellent analgesia. In 30% of cases, a single injection provided total pain relief for the whole postoperative time. The remaining patients had a mean analgesic duration of 10.7 hours (SD ± 4.3). Although the immediate effect after 4-mg doses may well involve systemic reactions due to rapid vascular uptake of morphine from the spinal fluid, plasma morphine concentrations obtained after 2-mg doses suggest a localized spinal action as the basis for the lengthy duration of analgesia [12].

3. Electroanalysis of morphine

Few electroanalytical methods have been identified by researchers for the determination of morphine. The mechanism of electrochemical oxidation of morphine has been shown in Figure 2. The electrochemical oxidative comportment of morphine in aqueous solution was very helpful to predict the oxidation peaks of this drug. The anodic peaks obtained in the voltammogram are due to the oxidation of phenolic and tertiary amine groups present. It has also been verified that a weak peak obtained is due to the formation of a dimer in phenolic group oxidation but not due to further oxidation of pseudomorphine [13].

Figure 2.

Electrochemical oxidation of morphine.

The electroanalytical methods include amperometry, differential pulse voltammetry (DPV), square wave voltammetry, and cyclic voltammetry (CV). These methods have been critically reviewed in this chapter.

3.1 Amperometry

In this electroanalytical technique, an indicator (working electrode) is subjected to a constant reducing or oxidizing potential, and the resulting steady-state current is recorded. This method can be used to detect electroactive chemicals in the solution because the measured current’s amplitude frequently varies on the concentration of the reduced or oxidized component. A small number of researchers have used it to find morphine in biological samples that were spiked or real.

A relatively stable and highly sensitive cobalt hexacyanoferrate-modified electrode was employed for the first time to study the pharmacokinetics of morphine in rat brain after an intravenous administration of morphine (25 mg/kg). The peak current was linearly related to the morphine concentration in the range of 1.0 × 10⁻⁶ M–5.0 × 10⁻⁴ M at +0.60 V (vs. Ag/AgCl) with a detection limit of 5.0 × 10⁻⁷ M [14]. Another highly sensitive and durable sensor for amperometric determination of morphine has been reported using CNT [15]. A glassy carbon electrode (preheated at 50°C for 5 min) was modified with multiwalled carbon nanotubes (MWCNTs) by simply rubbing electrode surface on filter paper powdered with CNT. This modified electrode showed its potential as a selective and sensitive electrocatalyst for the determination of morphine with a detection limit of 0.2 μM in concentration range 0.5–150 μM and sensitivity of 10 nA/μM. The amperometric response of the modified electrode was found to be incredibly stable over a continuous operation of 30 min. This proves the electrocatalytic potential of CNT-modified GC electrode for sensing morphine.

Molecularly imprinted polymers (MIP) have also been employed for morphine detection [16, 17]. For synthesizing molecular imprinted polymer, monomer with specific functional group is made to interact with a template and then polymerized with thermally/UV stimulated initiators. An electrode modified with MIP particles within the conducting poly(3,4-ethylenedioxythiophene) polymer, PEDOT (MIP/PEDOT-modified electrode), which immobilizes particles onto an indium tin oxide (ITO) glass has been prepared as presented in Figure 3. This modified electrode showed good capability for amperometric detection of morphine in terms of sensitivity, operating potential, and reproducibility of the MIP/PEDOT-modified electrode [16]. On the other hand, PEDOT has been applied as electroactive film onto the surface of electrode and the intended amperometric sensor showed a sensitivity of 91.86 μA/cm² per mM with a detection limit of 0.2 mM for morphine detection [17].

Figure 3.

Schematic for the preparation of the MIP/PEDOT-modified electrode [16].

In another unique method, the electrooxidation pathway for morphine was first investigated in neutral medium (pH = 6), then hydrodynamic amperometry was used for the determination of morphine using Prussian blue film-modified palladized aluminum electrode. Result showed that the detection limit of this method was about 0.8 μM with a linear concentration range 2–50 μM which was less efficient than the above reported one [18].

3.2 Differential pulse voltammetry

In DPV, short pulses with limited amplitude are superimposed upon a staircase waveform. This method can provide improved selectivity for observing different redox processes than other voltammetric methods. Various results have shown good sensitivity for the detection of morphine when the DPV method was used for electroanalysis [19, 20]. In few cases, typical electrochemical cells can be replaced with contracted screen-printed electrochemical strips for sensors that have proven to be of great potential in application areas like pharmaceutical, environmental, and food analysis applications. These screen-printed electrodes provide a simple, inexpensive, and user-friendly path of electrochemical measurements. However, for the electroanalysis of drug residuals in biological fluids, particularly in the case of drug abuse, low concentration of drug residuals, interferences of other electroactive biological species, fouling the electrode’s surface, and sample amount limitations are the major difficulties. These challenges can be overcome by using electromembrane extraction (EME) before electroanalysis on screen printed strips. This method has been developed for morphine to quantify its concentration in urine samples. DPV peak current at 0.18 V was selected as the signal and the calibration curve which was plotted by the variation of DPV currents as a function of morphine concentration was linear within the range of 0.005–2.0 μg/mL. The limit of detection and the limit of quantification were 0.0015 (S/N = 3) and 0.005 μg/mL, respectively [21]. Another type of screen-printed electrode containing metal sulfide nanosheets modified graphite has been found able to determine morphine simply and effectively at concentration levels encountered in toxicology and doping. Results indicated linear response in a concentration range between 0.05 and 600.0 μM of morphine with 0.03 μM limit of detection [22].

A screen-printed electrode modified by electrochemically exfoliated graphene oxide has shown good potential for detecting morphine in real samples [23]. Synthetic and real urine samples with a maintained pH of 6.6 and 7.0, respectively, were taken with added concentration of morphine. The modified electrode exhibited a high-performance sensing ability with sensitivity of 2.61 nA/μM and a detection limit of 2.5 μM. The voltammetric response of modified electrode with different concentrations of morphine has been shown in Figure 4.

Figure 4.

Differential pulse voltammogram for different morphine concentrations [23].

Real sample analysis has been performed for measuring the potential of synthesized material for the detection of morphine [24]. Urine samples of drug-addicted patients were taken for this study where the sample was collected after 5 h from the last abuse. This sample was firstly centrifuged and then the supernatant was filtered and hydrolyzed before electrochemical analysis. Currently, urine samples are frequently used to assess drug usage. Diacetylmorphine, which is heroin, is converted to morphine in urine samples. Heroin quickly deacetylates to 6-monoacetylmorphine (6-MAM), which has a potency of about six times that of morphine. More deacetylation of 6-MAM results in the formation of morphine. Concentration of morphine to codeine in urine of heroin abusers is therefore analyzed. MWCNTs/SnO₂–Zn₂SnO₄ modified carbon paste electrodes have shown promising potential for detection of morphine and codeine in urine samples. Under optimum conditions, the fabricated electrode showed wide linear ranges from 0.1 to 310.0 μM for morphine oxidation and from 0.1 to 600.0 μM for codeine oxidation. The electrochemical performance was better with a detection limit of 0.009 μM in real samples than other working electrodes with satisfactory recoveries [24].

Under optimized conditions, a sensitive and selective voltammetric sensor has been developed for the detection of morphine. This method used MWCNTs and polydopamine to modify the GC electrode. Electrocatalytic efficiency was evaluated using DPV that showed the potential of modified electrode for the determination of morphine in human plasma and urine samples with a liner dynamic range of 0.075–75.0 μM and detection limit of 0.06 μM [25]. A portable device containing such a sensible and disposable sensor needs to be developed to control drug abuse by persons at work and during driving.

3.3 Square wave voltammetry

Square wave voltammetry is a form of linear potential sweep voltammetry that uses a combined square wave and staircase potential applied to a stationary electrode. Studies on electroanalytical methods for detection of morphine have also been carried out using square wave voltammetry. Many materials, individually and as composites, have been investigated by researchers that can be used for sensing morphine concentration. In a particular study, MWCNTs, MIP, and gold nanoparticles have been employed for modification of pencil graphite electrodes. Under optimized conditions of several effective parameters, the calibration curve by square wave voltammetry was linear in two linear domains, over the range of 0.008–5 μM vs. Ag/AgCl, and the detection limit was 2.9 nM [26]. The aforesaid electrochemical sensor was successfully applied for MO determination in real samples such as human urine and plasma.

As discussed in the previous subsection 2.2, screen-printed electrodes have shown great potential as sensors. An immunosensor based on graphene screen-printed electrode modified with gold nanoparticles has been reported for morphine detection [27]. After modification of electrode with gold nanoparticles, cysteamine was also self-assembled on electrode surface via thiol interaction to introduce terminal amino groups to the electrode surface. The electrodes were then used to fabricate the immunosensor by covalent immobilization of antibodies against morphine. This sensor worked because of a competition between morphine and the morphine-bovine serum albumin conjugate for the immobilized antibodies on the sensor surface and the resulting change in the square wave voltammetry reduction current using the hexacyanoferrate system as an electrochemical probe. A sensitive and selective detection of morphine in the concentration range 0.1–100 ng/mL with a detection limit of 90 pg/mL was obtained. This method was also applied for the determination of morphine in spiked saliva samples and showed high recoveries. Such a sensitive determination method for morphine is of great interest for public health.

Recently, iron tungstate (FeWO₄) has been used for developing an electrochemical sensor for sub micromolar detection of morphine [28]. The effect of iron-tungsten ratio has been extensively studied for achieving the best possible characteristics and Fe₁W₃ with 7.5% of modifier in carbon paste electrode showed the best results. The limit of detection of this modified electrode was 0.58 μM and limit of quantification was 1.94 μM. The linear operating range was between 5 and 85 μM of morphine in the Britton-Robinson buffer solution at pH 8. The developed electrode resulted in good selectivity and excellent repeatability when applied in real biological samples like human urine. Selectivity of the method is a crucial parameter for the application in real-word sample analysis. To investigate this, the possibility for the detection of 50 μM of morphine was investigated in the presence of ascorbic acid, uric acid, citric acid, dopamine, and glucose under optimized conditions. The results of square wave voltammetric studies in the presence and absence of the above-mentioned interferents have been shown in Figure 5. The oxidation potential of morphine and other alkaloids, that can be present in urine samples were obtained at different potentials. As found in the literature, morphine and alkaloids are simultaneously determined. The developed electrochemical sensor has appeared to be a suitable competitor for efficient, precise, and accurate monitoring of morphine in biological fluids [28]. There are studies that show the detection of morphine with codeine by the researchers [29, 30].

Figure 5.

Square wave voltammograms of morphine at Fe1W3@CPE in absence (green line) and presence (red line) of (A) ascorbic acid (AA); (B) uric acid (UA); (C) citric acid (CA); (D) dopamine (DOP); (E) glucose (GLU); and (F) peak current signal (%) before and after addition of interferents [28].

3.4 Cyclic voltammetry

To get both qualitative and quantitative data regarding electrochemical reactions, CV, a potentiodynamic electrochemical technique, is frequently used. During a CV measurement, the working electrode’s potential in relation to the reference electrode is determined in the system under investigation, and the potential is scanned back and forth between predetermined higher and lower limits. The current flowing between the working electrode and the counter electrode is monitored concurrently. CV has been used by several researchers for determination of morphine using modified electrodes [31, 32, 33, 34].

Due to their excellent redox mediator properties, some metal hexacyanoferrates have been used for the development of electroanalytical methods for detection of morphine [14, 16]. In the above cases, the cyclic voltammograms of the modified electrode showed the presence of well-defined redox peaks. We have also studied the electrocatalytic potential of synthesized cadmium hexacyanoferrate-CNT nanocomposite for oxidation of morphine [35]. The synthesized material can be drop cast over the surface of glassy carbon electrode which remains stable with no significant loss in electrocatalytic activity up to 10 days. This voltammetric sensor worked well in spiked urine samples with a detection limit of 0.21 μM that confirms that this modified electrode will show good performance for the determination of morphine in human urine fluid samples. Few electrochemical sensors have also been developed that can measure the concentration of morphine in urine samples but due to high detection limit they will show less sensitivity than others [36]. A ZnO/CNT nanocomposite modified carbon paste electrode showed linear range for morphine determination from 0.1 to 700 μmol/L and the detection limit was calculated as 0.06 μmol/L [37].

A simple and sensitive voltammetric sensor has been reported for simultaneous determination of Morphine (Mp) and Buprenorphine (Bp) [38]. The complete method has been described graphically in Figure 6. It involved embedment of rhodium nanoparticles in a carbon matrix followed by its carbonization. Then after the composite was cast over the surface of GCE for its voltammetric characterization. The modified glassy carbon electrode with rhodium nanoparticles-mesoporous carbon composite showed high potential for simultaneous determination of Morphine (Mp) and Buprenorphine (Bp), with a linear range and limit of detection of 0.1–20 μM and 40 nM, respectively for morphine, and these data were obtained about 0.1–14 μM and 45 nM, respectively, for buprenorphine. This method involved easy and fast preparation with high efficiency as a sensor [38].

Figure 6.

Cyclic voltammetric determination of morphine (Mp) and buprenorphine (Bp) [38].

4. Conclusion

Diversity of electrochemical sensors developed for the detection of morphine is presented in this chapter. Efforts are being made by researchers to develop a simple, sensitive, economical, and accurate electroanalytical method for the determination of morphine which can be used for pharmaceutical and clinical applications. One of the major challenges is the selection of electrode material that is responsible for limit of detection and sensitivity. For designing a potential sensor, molecular-level understanding of the correlation between the surface structure and reactivity is very important factor which governs the selectivity and sensitivity. Future studies in this area should be more focused on understanding interfacial reaction kinetics to design novel sensors suitable for use in all practical applications.

Acknowledgments

The University of Petroleum and Energy Studies, Dehradun is gratefully acknowledged for supporting the present work. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Conflict of interest

The authors declare no conflict of interest.