Xiaoyue Shan
Bingsheng Yang
Yanna Le- Department of Public Health and Clinical Services, Hangzhou Occupational Disease Prevention and Treatment Hospital, Hangzhou, China
Morphine (MOR) is a commonly utilized opioid analgesic in the field of pain management. However, its potential for abuse presents substantial public health concerns. This suggests the necessity for developing an accurate and sensitive analytical method for the determination of MOR and its metabolites in biological specimens. In this review, various analytical techniques, including HPLC, LC-MS, UHPLC-MS and electrochemical sensors, have been applied for the measurement of MOR and its metabolites in biological samples. LC-MS is the gold standard due to its high sensitivity and specificity. In addition, electrochemical methods are also highlighted as a primary analytical approach in this research.
1 Introduction
Illicit drug abuse has long been a threat to public health and social stability (Wood et al., 2009). As reported by the World Drug Report 2021, around 275 million individuals worldwide engaged in the use of illicit drugs in 2020. Illicit drugs such as morphine (MOR) are used in large scale and the addiction is a major public health challenge (Ripanda et al., 2022). MOR is a strong opioid analgesic compound that comes from opium and it is employed to manage moderate-to-severe acute and chronic pain, especially those undergoing surgical procedures (Jordan and Hart, 1991). MOR is relatively water-soluble and its solubility in lipids is poor (Andersen et al., 2003). Approximately 90% of MOR that is taken orally is eventually eliminated via urine within 24 h and nearly 10% of the eliminated MOR remaining in its unmetabolized form (McQuay et al., 1997). The majority of MOR is absorbed by the liver via the organic cation transporter (OCT) and undergoes rapid metabolism predominantly mediated by UGT2B7, generating various metabolites. Significantly, morphine 3-glucuronide (M3G) and morphine 6-glucuronide (M6G) exhibit distinct biological activities (Tzvetkov et al., 2013; Morrish et al., 2006; Klepstad et al., 2000). Clinical Researches have shown that M6G possesses potent analgesic properties and contributes to the effects produced by MOR (Portenoy et al., 1992). M6G had 50 times greater potency than MOR in its antinociceptive function (Holmquist, 2009). In contrast to both MOR and M6G, M3G has no analgesic potency, but may have anti-analgesic and (neuro-)excitatory effects (Lötsch, 2005). The structures of MOR and M6G, M3G are shown in Figure 1.
Figure 1. The structures of (a) MOR; (b) M3G and (c) M6G.
Despite the widespread use of MOR, it may produce physical and psychological reliance, presenting a considerable danger of addiction and abuse (Volkow et al., 2019). Indeed, overdoses of MOR may lead to serious opioid-related side effects including respiratory problems, nausea, vomiting, diarrhea, central nervous system disorders and even death (Clavijo et al., 2011). Consequently, monitoring MOR and its transformation products in different biological specimens is highly significant for overseeing drug addicts and tracking drug abuse (Zhao et al., 2022). To date, there are numerous analytical methods available today, such as gas chromatography-mass spectrometry (GC-MS) (Tiago Rosado et al., 2019; Ferreira et al., 2020; Magalhães et al., 2018; Shang et al., 2020; Norouzi et al., 2020; Fernandez-Lopez et al., 2019; Prata et al., 2019; Özbunar et al., 2019; Jain et al., 2020), liquid chromatography (Ghorani-Azam et al., 2021; Derakhshanrad et al., 2021; Bazargan et al., 2022; Abdolmohammad-Zadeh et al., 2019; Soltani et al., 2018; Riahi-Zanjani et al., 2019; Alahyari et al., 2018; Ebrahimi Rahmani et al., 2017; Riahi-Zanjani et al., 2018), liquid chromatography-mass spectrometry (LC-MS) (Ghorani-Azam et al., 2021; Truver and Swortwood, 2018; Wang et al., 2019; Tang et al., 2019; Grabenauer et al., 2018; Grabe et al., 2018; Alcántara-Durán et al., 2018; Edmund Rab et al., 2023; Al-Asmari et al., 2022; Al-Asmari, 2020; Amvrosios Orfanidis et al., 2021; Orfanidis et al., 2020; Orfanidis et al., 2018; Henkel et al., 2018; Kristoffersen et al., 2018; Sadler Simões et al., 2018; Lu et al., 2020; Yuancheng Wang et al., 2024; Fernández et al., 2018; Jin et al., 2021; Bassotti et al., 2020; Fa et al., 2019; Hernandez et al., 2022; Bakke et al., 2019; Nedahl et al., 2019; Scendoni et al., 2022; Di Fazio et al., 2018; Danso et al., 2019), capillary electrophoresis (Emara et al., 2019), electrochemical methods (Zare et al., 2021; Verrinder et al., 2021; Ren et al., 2021; Rasitanon et al., 2024; Akbarian et al., 2018; Rajaei et al., 2019; Habibi et al., 2022; Wester et al., 2019; Wester et al., 2018; Wang et al., 2023; Maccaferri et al., 2019; Kumary et al., 2019; Zahra Nazari, 2022; Hadi and Fariba Garkani, 2020; Jahani et al., 2020; Ognjanović et al., 2022; Sedigheh Akbari et al., 2020; Jahanbakhshi, 2019; Eissa et al., 2019; Mohsen Saeidi et al., 2023; Zhang et al., 2021; Abraham et al., 2019; Salajegheh et al., 2018; Bahrami et al., 2020; Aliabadi and Rounaghi, 2019; Yousefi et al., 2020; Rahimi et al., 2020; Barthwal et al., 2018), optical sensors (Alhaddad and Sheta, 2020; Nebu et al., 2018; Cao et al., 2019; Singh et al., 2017; Karimzadeh et al., 2022; Masteri-Farahani and Mosleh, 2019; Masteri-Farahani and Askari, 2019; Masteri-Farahani and Mollatayefeh, 2019; Abnous et al., 2018; Feng et al., 2022; Zhang et al., 2020; Rohani Bastami et al., 2022) and Raman spectroscopy (Li et al., 2020; Yu et al., 2018; Mao et al., 2018; AkÇAn et al., 2020; Yu et al., 2019) have been recognized as powerful tools for identifying MOR and its metabolic products in diverse biological materials, including serum, urine, and saliva. Among these, MS is considered the gold standard due to its high sensitivity and specificity. However, electrochemical methods are particularly promising, offering distinct advantages of high sensitivity, low cost, portability, and the ability to analyze complex samples with minimal pretreatment (Gowda et al., 2023; Gowda et al., 2025; Gowda et al., 2021). During the past 10 years, measurements of MOR and its metabolic products in various biological specimens was a challenging field. As the biological matrix’s complexity and the drugs' trace quantities can interfere with the analytical procedures of identification and quantification, some of the researches employ diverse sample pretreatments to achieve the extraction and preconcentration of these drugs (Ansari and Karimi, 2017; Chen et al., 2021).
This review aims to critically discuss liquid chromatographic and electrochemical methods for determining MOR and its metabolic derivatives in diverse biological matrixes published from 2018 to 2024. Various sample preparation procedures that researchers have employed to extract MOR and its metabolites from biological specimens are discussed. The review provides an overview of analytical procedures that have been established to monitor MOR and its metabolic derivatives in samples with different matrix compositions, mainly focusing on LC-MS and electrochemical methods, since they are the most frequently applied for quantitative detection of MOR and its metabolites. Furthermore, the merits and limitations of these analytical procedures have been addressed.
2 Methods
Literature search was conducted using Web of Science, Science Direct, PubMed and Google Scholar to identify relevant English publications published between 2018 and 2024. keywords including “morphine”, “quantification”, “chromatographic”, and “electrochemical determination” were utilized. The reviewed publications were selected considering chromatographic and electrochemical methods for the analysis of MOR and its metabolites in biological samples.
3 MOR detection methods
3.1 Samples pretreatment
The determination of MOR and its metabolic derivatives in biological matrices is challenging due to complex matrix effects. Endogenous compounds such as organic matter, fats, proteins, and other interferents would significantly affect the reproducibility, sensitivity, and selectivity of the measurements. Therefore, an efficient sample preparation to increase the MOR level and eliminate the interfering compounds is generally required. In recent studies, centrifugation and filtration are commonly used methods in the processing of urine or blood to minimize the matrix effects. Dilution (Alcántara-Durán et al., 2018; Edmund Rab et al., 2023; Bassotti et al., 2020; Danso et al., 2019) is also a very simple sample preparation procedure among the reported techniques to minimize the sample matrix effects. In one study, Bassotti et al. (2020) designed a LC-MS/MS analytical protocol to analyze 17 various abused substances in oral fluid matrices. The analytical procedure including a sample dilution and centrifugation: 40 µL of the oral fluid was diluted with 160 µL of water (1:4 dilution) and 200 µL of 15 different internal standards were added. After the centrifugation process, the supernatant was immediately detected using LC-MS/MS. This simple sample pretreatment may reduce the sample loss and matrix interferences.
However, among the sample preparation techniques used to detect MOR and its metabolic derivatives from biological fluids, liquid-liquid extraction (LLE) (Alahyari et al., 2018; Amvrosios Orfanidis et al., 2021; Orfanidis et al., 2020; Orfanidis et al., 2018; Henkel et al., 2018; Kristoffersen et al., 2018; Sadler Simões et al., 2018; Bakke et al., 2019; Scendoni et al., 2022) or solid-phase extraction (SPE) (Ghorani-Azam et al., 2021; Derakhshanrad et al., 2021; Bazargan et al., 2022; Abdolmohammad-Zadeh et al., 2019; Soltani et al., 2018; Riahi-Zanjani et al., 2019; Ebrahimi Rahmani et al., 2017; Riahi-Zanjani et al., 2018; Truver and Swortwood, 2018; Wang et al., 2019; Tang et al., 2019; Grabenauer et al., 2018; Grabe et al., 2018; Al-Asmari et al., 2022; Al-Asmari, 2020; Lu et al., 2020; Fernández et al., 2018; Fa et al., 2019; Hernandez et al., 2022; Nedahl et al., 2019; Di Fazio et al., 2018) is the most frequently practiced approach. LLE has been shown to be a highly effective option due to its main advantages including simplicity and high extraction coefficient. Despite these advantages, the LLE procedure consumes large quantities of sample and organic solvents that might be limited by labor-intensive procedures and relatively expensive cost. Therefore, selecting a suitable organic solvent is an important factor. Methanol (Orfanidis et al., 2018), chloroform (Alahyari et al., 2018; Fernández et al., 2018), acetonitrile (Amvrosios Orfanidis et al., 2021; Orfanidis et al., 2020; Henkel et al., 2018) or as a mixtrue of solvents including methanol/acetonitrile (3:1) (Sadler Simões et al., 2018), ethyl acetate/heptane (8:2) (Kristoffersen et al., 2018), chloroform/2-propanol (Magalhães et al., 2018) or choline chloride-menthol-phenylacetic acid (Norouzi et al., 2020) have been utilized as organic solvents in the LLE procedure to extract MOR and its metabolites in biological matrices. Recent literature report that liquid microextraction techniques have been widely applied for MOR and its metabolites extraction from their complicated biological samples as an eco-friendly approach that decreases environmental pollution and reduces pretreatment costs (Dmitrienko et al., 2020). For instance, Jain and colleagues et al. (Jain et al., 2020) designed a GC-MS approach to determine MOR in illicit opium in which samples were extracted by dispersive LLE (DLLME). In DLLME, first, a cloudy solution was initially generated by using 1.1 mL of a chloroform/acetone mixture (1:10) to the opium sample. Then, the mixture was exposed to ultrasonication and centrifuged, resulting in the extraction solvent droplet settling at the tube’s base. After drying the residue under a nitrogen stream, it was redissolved in ACN. Then, the solution was treated with N,O-Bis(trimethylsilyl)acetamide (BSA) to prepare it for the subsequent GC-MS analysis. With extraction recoveries of 89.7%–92.3%, the DLLME methodology successfully achieved simultaneous extractions.
SPE has become a commonly used sample preparation technique for the preconcentration of MOR and its metabolic derivatives. The selection of a suitable adsorbent greatly affects the analytical parameters and extraction efficiency (Cai et al., 2003). Numerous adsorbents have been reported. These encompass polyoxometalate (POM)-based frameworks (Derakhshanrad et al., 2021; Bazargan et al., 2022), carbon nanotubes (CNTs) (Ghorani-Azam et al., 2021; Soltani et al., 2018; Riahi-Zanjani et al., 2019; Riahi-Zanjani et al., 2018), graphene oxide (GO) (Abdolmohammad-Zadeh et al., 2019; Lu et al., 2020), and molecularly imprinted polymer (MIP) (Ebrahimi Rahmani et al., 2017). Derakhshanrad et al. (2021) employed a micro-solid-phase extraction (D-µSPE) based on POM frameworks to extract abused drugs in hair specimens. The unique modular design of the POM-based frameworks could exhibit strong affinity for opioid drugs. This material was efficient for the enrichment of MOR and its metabolic derivatives. Despite the aforementioned advantages of SPE, it remains labor-intensive and expensive. Thus, environmentally friendly strategies, such as magnetic solid phase extraction (MSPE), have been created. In a recent investigation conducted by Lu et al. (2020), a synthesized GO-Fe3O4 nanocomposite for simultaneous preconcentration of 8 drugs, including MOR, from urine samples prior to UHPLC-MS/MS analysis. Owing to its potent magnetic characteristics, and suitable functional groups, the adsorbent showed exceptional selectivity to the drugs. The recoveries were rangd from 80.4% to 105.5%. Additionally, the efficiency of their GO-Fe3O4-based MSPE strategy was compared with other pretreatment methods, such as MSPE using magnetic C18 as the adsorbent and DLLME using chloroform/MeOH (1:2.5) as the solvent. The GO-Fe3O4 MSPE methodology demonstrated lower costs and easier handling, providing better reusability. Thus, this approach shows promising potential for monitoring psychoactive drugs in clinical and forensic areas.
3.2 HPLC
Currently, LC remains the dominant analytical platform for isolating and purifying compounds in complicated biological specimens. The instrumental and analytical properties from recent literatures are summarized in Table 1. Various LC methods coupled with ultraviolet (UV) (Ghorani-Azam et al., 2021; Derakhshanrad et al., 2021; Bazargan et al., 2022; Abdolmohammad-Zadeh et al., 2019; Soltani et al., 2018; Riahi-Zanjani et al., 2019; Riahi-Zanjani et al., 2018) and photodiode array detector (PDA) (Alahyari et al., 2018; Ebrahimi Rahmani et al., 2017) have been extensively used for the sensitive quantification of MOR and its metabolic derivatives due to their easy operation and relatively low cost. For example, Abdolmohammad-Zadeh et al. (2019) reported an easy and rapid HPLC-UV technique to simultaneously determine MOR and COD in numerous biological matrices. A Fe3O4/rGO/Ag nanocomposite was developed for the MSPE of MOR and COD. After that, determination was conducted using C18 column (250 × 4.6 mm, 5 µm) with the mobile phase of phosphate buffer and ACN (60:40, v/v), delivered at 0.8 mL/min. The LOD and enrichment factor (EF) for MOR were 1.8 ng L−1 and 1000. For both MOR and COD, relative recoveries in human samples ranged from 97.0% to 102.5%. This MSPE-HPLC-UV approach presented acceptable precision, excellent EF and a short analysis time. In another research, Ebrahimi Rahmani et al. (2017) introduced an HPLC-PDA method for estimating MOR in biological samples. A magnetic MIP was designed to extract MOR from blood and urine samples. Separation was performed on an XDB-C18 column (4.6 × 50 mm, 1.8 µm) with acetate buffer-ACN (60:40, v/v) mobile phase at 1.0 mL/min. The method demonstrated linear quantification over 0.1–30.0 μg/mL. Method sensitivity was established with an LOD of 0.03 μg/mL and LOQ of 0.08 μg/mL. In plasma samples, recovery rates fluctuated between 84.9% and 105.5%, whereas in urine samples, they ranged from 94.9% to 102.8%. However, some of these methods exhibited low sensitivity, with the lowest LOD obtained using UV detection being 0.1 ng/mL for MOR (Riahi-Zanjani et al., 2019).
Table 1. LC detection methodologies for MOR and its transformation products in biological matrixes.
3.3 LC-MS
LC is coupled with MS/MS to identify and quantify MOR. The selectivity and sensitivity of LC-MS/MS methods are greatly influenced by analytes ionization efficiency. Thus, selecting a proper ionization source is the key concern. Recently, various ionization sources including electrospray ionization (ESI), its enhanced variant heated electrospray ionization (HESI), and atmospheric pressure chemical ionization (APCI) have been routinely utilized. Among these, ESI has emerged as the predominant ion source for the determination of MOR and its metabolites (Ghorani-Azam et al., 2021; Truver and Swortwood, 2018; Wang et al., 2019; Tang et al., 2019; Grabenauer et al., 2018; Grabe et al., 2018; Alcántara-Durán et al., 2018; Edmund Rab et al., 2023; Al-Asmari et al., 2022; Al-Asmari, 2020; Amvrosios Orfanidis et al., 2021; Orfanidis et al., 2020; Orfanidis et al., 2018; Henkel et al., 2018; Kristoffersen et al., 2018; Sadler Simões et al., 2018; Lu et al., 2020; Fernández et al., 2018; Jin et al., 2021; Bassotti et al., 2020; Fa et al., 2019; Bakke et al., 2019; Nedahl et al., 2019; Scendoni et al., 2022; Di Fazio et al., 2018). LC-MS analyses of MOR usually employ an ESI source, with detection performed in either positive or negative switching mode. Although APCI and HESI are applied less frequently for monitoring MOR, two LC-HRMS studies have successfully used HESI for the ionization of opioid compounds, including MOR (Hernandez et al., 2022; Danso et al., 2019). Moreover, both multiple reaction monitoring (MRM) and selected reaction monitoring (SRM) modes are utilized to achieve greater sensitivity and selectivity. In most of the methods described here, positive-mode ESI is the preferred ionization method, coupled with triple quadrupole mass analyzer for its superior performance in MOR and its metabolites analysis. The mode of MRM has often been performed due to its great sensitivity and selectivity in analyzing MOR and its metabolic derivatives.
Truver and Swortwood (2018) suggested a SPE-LC-MS/MS approachemploying positive-ion ESI for the concurrent quantification of novel synthetic opioids (NSOs), MOR, and buprenorphine with LODs of 5.0 ng/mL. Separation was conducted using a Poroshell 120 EC-C18 column (100 × 3.0 mm, 2.7 μm) with a gradient system of: (A) aqueous 0.05% formic acid/5 mM ammonium formate and (B) 0.1% formic acid in ACN, delivered at 0.5 mL/min. MS detection employed MRM mode, utilizing the following mass transitions: m/z 286.3 → 165.0 for MOR; 328.3 → 165.0 for 6-acetylmorphine (6-AC); 468.6 → 396.2 for buprenorphine. This method could be employed to analyze drug trends across various demographic groups in the market. In a similar study, four heroin metabolites in human urine, including MOR, 6-monoacetylmorphine (6-MAM), M3G and M6G were quantitatively analyzed using an SPE-LC-MS/MS approach (Wang et al., 2019). Following the SPE procedure, the separation of analytes was achieved by using a Waters XBridge® C18 column (150 × 3.0 mm, 35 μm). MS analysis employed MRM mode, monitoring the characteristic fragment transition m/z 286.3 → 165.1, 328.2 → 165.2, 462.2 → 201.2, 462.2 → 286.3 for MOR, 6-MAM, M3G and M6G, respectively. Tang and co-workers (Tang et al., 2019) proposed a robust SPE-LC-MS/MS method (ESI+) for the detection of MOR, M3G, M6G, and clonidine in plasma. The validated method showed linear responses within the ranges of 1.0–1000.0 ng/mL for MOR and 0.25–100.0 ng/mL for clonidine, with LODs established between 0.05 and 0.2 ng/mL. The method exhibited good selectivity, stability, and negligible matrix effect (close to 100%) for MOR, M3G, M6G. In addition, it was successfully validated through the analysis of clinical plasma samples. Grabenauer’s group (Grabenauer et al., 2018) introduced an LC-MS/MS approach (ESI+) for quantifying MOR and other analytes in oral fluid. Separation employed an Agilent poroshell 120 SB C18 column (100 × 2.1 mm, 2.7 µm) with a methanol/formate gradient (0.5 mL/min). The transitions were as follow: 300.2 → 165.0 for COD; m/z 286.2 → 165.2 for MOR; 328.2 → 211.1 for 6-AC; and 289.2 → 165.0 for MOR-d3. The LODs reached 0.02 ng/mL for 6-AC and 0.04 ng/mL for COD and MOR. In another study, the same research group performed a LC-MS/MS approach to analyze MOR, M3G, M6G and other drugs in human hair, with LODs of 0.2–1.0 pg/mg (Grabe et al., 2018). The described methodology detected opioid biomarkers in all 46 hair samples from users, demonstrating its applicability in forensic and clinical settings. Alcántara-Durán et al. (2018) reported a nanoflow LC-HRMS methodology, based on direct urine dilution, for determing abused and performance-enhancing compounds in urine. According to the authors, this method was simple and sensitive, with LOQ below 5 μg/L for most target analytes. The results of matrix effects revealed that a dilution 1:50 of the samples showed a negligible matrix effect for all target analytes. In contrast to SPE, this method demonstrated superiority in high-throughput capability, simplicity, and reproducibility. This method could be recommended for the preliminary application of anti-doping and drug abuse in the future. Edmund Rab et al. (2023) proposed an LC-HRMS approach to simultaneously screen for and determine abused drugs in postmortem human samples. Two hundred postmortem blood and one hundred urine specimens were determined. The authors conducted a comparison between the performance of two techniques: a two-step screening/quantitation process and the LC-HRMS methodology. LC-HRMS represents the preferred choice for analyzing MOR and its metabolic derivatives in diverse biological matrices.
3.4 UHPLC-MS
UHPLC (or UPLC) offers many advantages including improved resolution, higher precision, and reduced analysis time. These characteristics have led to the wide application of UHPLC-based methods for analyzing MOR and its metabolites in biological specimens (Al-Asmari et al., 2022; Al-Asmari, 2020; Amvrosios Orfanidis et al., 2021; Orfanidis et al., 2020; Orfanidis et al., 2018; Henkel et al., 2018; Kristoffersen et al., 2018; Sadler Simões et al., 2018; Lu et al., 2020; Yuancheng Wang et al., 2024; Fernández et al., 2018; Jin et al., 2021; Bassotti et al., 2020; Fa et al., 2019; Hernandez et al., 2022). Al-Asmari and colleagues (Al-Asmari et al., 2022) reported a SPE-UHPLC-MS/MS approach for quantifying 6-MAM, 6-AC, MOR, and COD in postmortem stomach tissue. A Raptor Biophenyl column (50 × 3.0 mm, 2.7 µm) was employed for the separations. A mobile phase composed of 10 mM ammonium formate and MeOH was utilized. The flow rate was 0.3 mL/min. The LODs were 0.3 ng/g for 6-MAM, 0.4 ng/g for 6-AC, 0.3 ng/g for MOR, and 0.2 ng/g for COD. MOR exhibited a retention time (RT) of 4.7 min. An SPE-UHPLC-MS/MS approach for detecting 60 substances in around 1,000 postmortem blood samples was also reported by the same research team. This technique achieved outstanding sensitivity (LOD: 0.4 ng/mL) and enabled rapid analysis for MOR. The RT was 4.5 min (Al-Asmari, 2020). Orfanidis’s research group (Amvrosios Orfanidis et al., 2021) detailed a UHPLC-MS/MS approach for measurement of 84 drugs in postmortem blood specimens. This method employed ESI in dual-polarity mode (switching between positive and negative polaritie). The RT was shortened to 1.24 min and the LOD for MOR reached 1.23 ng/mL. The matrix effect for drugs ranging from 70.6% to 97.4% was within the accepted range. The same group published another UHPLC-MS/MS method to quantify 84 abused drugs in human liver samples (Orfanidis et al., 2020). The matrix effect was in the range of 73.3%–119.0% and no significant matrix effects was observed for the drug measurement. Following a QuEChERS procedure, the extracted samples underwent detection via UHPLC-MS/MS operating in MRM mode with separation performed on an Acquity BEH C18 column. The duration of the analysis was under 1.5 min (RT: 1.24 min). This study marked the first application of UHPLC-MS-MS combined with QuEChERS to simultaneously determine 84 drugs in human liver tissue. Orfanidis et al. (2018) also studied a UHPLC-ESI-MS/MS method to quantitatively analyze 27 drugs in human skeletal tissue. LLE with MeOH was used for analyte extraction, and the method achieved baseline resolution within 89 s (RT: 1.28 min) with an LOD of 1.28 ng/g. According to the authors, this represented the first methodology to detect and quantify 27 drugs in human bones. In a similar study, Henkel et al. (2018) presented a rapid (RT: 3.2 min) and sensitive (LOD: 2.5 ng/g) UHPLC-ESI-MS/MS method utilizing a Luna PFP column (150 × 2.0 mm, 5.0 µm) with gradient elution of the mobile phase (A: 2% formic acid containing 2.0 mmol/L ammonium formate, and B: MeOH modified with 0.1% formic acid) to quantify 10 abused drugs in non-mineralized dental biofilm. The analytes were extracted from the plaque through ultrasonication using ACN and the resulting extracts were subjected to UHPLC-MS/MS detection n MRM mode. The method demonstrated successful application in 3 real postmortem plaque cases. Kristoffersen et al. (2018) developed a novel ultra-fast (analysis time <1.5 min) high-throughput UHPLC-ESI-MS/MS methodology capable of determining 12 drugs in whole blood. The authors utilized a supported liquid extraction (SLE) plate, using an optimized binary solvent system of ethylacetate and heptane (80:20, V/V) for the extraction of analyte. The methodology demonstrated robustness and reliability, having been applied to approximately 9,900 antemortem and 1,000 postmortem whole blood samples. Simões’s team (Sadler Simões et al., 2018) presented a UHPLC-ESI-MS/MS technology to determine 11 illicit drugs in dried blood spot (DBS) specimens. A combination of MeOH and ACN at a volume ratio of 3:1 was utilized as the extraction solvent. The linearity was ranged from 1.0 to 500.0 ng/mL, while the LOD for MOR was 1.0 ng/mL. Notably, the separation process required only 0.83 min and this innovative technology was applied to analyze 64 real samples and demonstrated an acceptable correlation between the data from DBS and those derived from laboratory’s conventional whole blood analysis procedures. In 2020, Lu et al. (2020) conducted a UHPLC-ESI-MS/MS methodology to determine 8 psychoactive drugs in urine samples. They applied MSPE using GO-Fe3O4 as the adsorbent for analyte extraction. The LOD was established at 0.2 ng/mL, and the analysis of MOR took only 1.37 min. Wang’s group (Yuancheng Wang et al., 2024) reported a UHPLC-ESI(+)-MS/MS method for detecting MOR and its metabolites in mouse urine. TpBD-COF-functionalized magnetic nanospheres (Fe3O4@SiO2@TpBD) were used as the adsorbent for analyte extraction. The RTs for MOR, M3G, and M6G were 3.06 min, 4.61 min, and 4.10 min, respectively, with LODs of 0.53 pg/mL, 0.33 pg/mL, and 0.16 pg/mL. The advantages of the method were its simplicity, speed, and sensitivity. Fernández’s team (Fernández et al., 2018) utilized a BEH Shield RP18 column (100 × 2.1 mm, 1.7 µm) and ESI(+)-MS/MS in MRM mode for determining 20 illicit drugs in oral fluid specimens. DLLME procedure was utilized for analyte extraction and the MRM transitions were configured at 286.00 > 201.04 (m/z) for MOR. This work demonstrated an ultra-fast run time (RT: 0.67 min) and the primary limitation of this method was its high LOD at 25.0 ng/mL. Jin et al. (2021) conducted a sensitive UHPLC-ESI(+)-MS/MS methodology to analyze MOR and ralated opioids in urine samples. The RTs for MOR, M3G, M6G were 2.5 min, 2.42 min and 1.51 min, respectively, with LODs of 0.11 ng/mL, 0.07 ng/mL and 0.07 ng/mL. The proposed approach shows great promise for application in opioid-related pharmacokinetics, bioequivalence studies, and clinical research. MRM mode is the primary mass spectrometric technique for analyzing MOR and its metabolites and SRM mode has also been utilized (Bassotti et al., 2020; Fa et al., 2019). A UHPLC-ESI(+)-MS/MS technique utilizing SRM mode to analyze 17 abused drugs in oral fluid was conducted by Bassotti et al. (2020). The RT was 1.8 min. The minimal sample volume and simple sample handling effectively reduced analyte loss and matrix effects. The matrix effect for MOR was 98.3% and no significant matrix interference was observed. Fabresse’s group (Fa et al., 2019) established a UHPLC-ESI(+)-MS/MS method with SRM mode to quantify 10 drugs in oral fluid. This method had low sensitivity. The LOD was 10.0 ng/mL. Hernandez and colleagues (Hernandez et al., 2022) presented a UHPLC-HRMS technique utilizing HESI in positive mode for detecting 15 compounds in meconium. Two distinct SPE procedures were employed. The LOD was 0.5 pg/mg and analysis time was 3.22 min. The methodology has been applied for routine toxicological screening in high-risk pregnancies. The specifics of these methodologies are presented in Table 1. In summary, LC is simple and cost-effective for the measurement of MOR and its metabolites. It is a challenge to distinguish MOR from other analogs due to the method’s low sensitivity and poor selectivity. In contrast, MS, particularly when combined with UHPLC, has revolutionized the quantification of these compounds in biological fluids. UHPLC-MS/MS technique utilizing MRM in positive ESI mode has considered as the most frequently applied approach for MOR analysis. These methods offer excellent sensitivity and specificity, but the relatively high cost of equipment and maintenance makes MS-based methods not commonly applied in clinical laboratories.
3.5 Electrochemical sensors
Electrochemical sensing have gained significant attention in recent literature as a promising approach for MOR and its metabolites quantification. The advantages including sensitivity, selectivity, operational simplicity, cost-effectiveness, rapid response times, and potential for miniaturization (Pushpanjali et al., 2020; Bhimaraya et al., 2023; Manjunatha, 2020a; Prinith et al., 2021; Hareesha et al., 2019; Manjunatha, 2018). Achieving accurate detection of MOR in complex biological matrices is challenging. The possible interferents include dopamine (DA), ascorbic acid (AA) and uric acid (UA) (Manjunatha and Deraman, 2017). To address this, functional materials have been developed to coat the electrodes. The role of modified electrodes is crucial in electrochemical sensing. The application of functional materials on the electrode surface can significantly enhance signal transduction (Manjunatha, 2020b; Edwin et al., 2021). Currently, various nanomaterials have been employed to modify working electrodes for sensitive and selective determining MOR and its metabolites in different matrices (Table 2).
Table 2. Electrochemical detection methodologies for MOR and its transformation products in biological matrixes.
3.5.1 Carbon nanotube-based electrodes
In 2021, Zare’s team (Zare et al., 2021) developed a novel MOR sensor by modifying a carbon paste electrode (CPE) with a MgO-decorated single-walled carbon nanotube (MgO/SWCNTs) nanocomposite and a 1-methyl-3-octylimidazolium tetrafluoroborate (MOCITFB) for drug analysis. The authors established a linear range spanning from 0.003 to 320.0 µM. The LOD was calculated as 0.8 nM. Verrinder et al. (2021) presented a sensor modified with Nafion/SWCNT. Its aim was to determine MOR in capillary whole blood. The combination of SWCNT networks and a Nafion coating allowed for the fabrication of flexible, reproducible sensor strips capable of direct electrochemical determination in untreated capillary whole blood. The authors managed to achieve a linear response in the range of 0.5–10.0 μM, and the LOD was 0.48 μM. The sensor showed strong resistance to interference from other opioids and potential interferents. Furthermore, single-determination measurements were conducted with capillary samples from 3 volunteers. An average recovery of 60% was obtained (lower than results of LC-MS methods), demonstrating that the sensor only determined the free fraction of MOR. With a total assay time of only several minutes and a sample volume of 40 μL, the Nafion/SWCNT sensor strips show real potential for point-of-care rapid test of MOR in whole blood. Ren et al. (2021) prepared Ti3C2TX and carbon nanomaterial (Ti3C2TX/MWCNTs) to construct an electrochemical sensor platform aimed at detecting MOR in physiological conditions. The sensor exhibited a linear detection range from 0.01 to 100.0 µM with a low LOD of 9.2 nM. This sensor provided a favorable recovery rate ranging from 94.0% to 100.5% in serum and urine samples. Rasitanon et al. (2024) established wearable sensors utilizing a novel ink composed of CNTs, graphite, and styrene ethylene butylene styrene block copolymer (SEBS) (“FLEX-CNT” ink) for real-time electrochemical detection of urinary potassium ions and MOR. The MOR sensor exhibited a linear response range of 0.1–1000.0 µM. The LOD was 0.024 µM. The potassium sensor showed a linear response range of 0.1–1000.0 mM. It was able to detect low concentration downs to 1.0 µM. Additionally, these wearable sensors are impervious to common interfering agents, making them highly suitable for noninvasive urine analysis applications. Akbarian’s team (Akbarian et al., 2018) designed a sophisticated sensor system. This system combined transition metal oxide-carbon nanomaterial composites with organic molecular recognition elements. As a result, the sensor resolved the overlapping signals, it could distinguish and simultaneously quantify diclofenac, MOR, and mefenamic acid under conditions relevant to physiology. The results revealed the LODs were as low as 0.008 µM for diclofenac, 0.4 µM for MOR, and 0.5 µM for mefenamic acid, using SWV technique. Moreover, the sensor showed good selectivity with recoveries of 98.8%–102.2% for real samples. Rajaei’s group (Rajaei et al., 2019) showed a high sensitive electrochemical approach for quantifying MOR using a MWCNT/La3+-CuO nanoleaf-modified CPE (La3+-CuO/MWCNTs/CPE). Using CV technique, they achieved a linear detection range from 0.05 to 325.0 µM, with a LOD of 8.0 nM. Furthermore, the sensor possessed long-term stability (over 3 weeks) and good selectivity for MOR against its analogs. The sensor exhibited reliable performance in real urine samples, with recoveries of 97.5%–102.3%. Habibi et al. (2022) studied a CMK-5 mesoporous carbon modified GCE to simultaneously analyze MOR and methadone using fourier transform SWV. The presence of CMK-5 led to fast electron transfer for both compounds owing to its large surface area and numerous edge plane defect sites. The authors reported that both analytes exhibited linear ranges of 0.1–4.0 µM. Specifically, the LOD was 0.027 µM for MOR and 0.029 µM for methadone. The sensor demonstrated no interference in the presence of matrix components. The performance of the as-fabricated sensor was successfully explored in real urine samples with recoveries of 97.0%–105.6%. Wester et al. (2019) developed a disposable dual-layer Nafion-coated SWCNT modified CPE for the voltammetric detection of MOR and COD using DPV. The Nafion/SWCNT electrode produced better peak separation compared to the uncoated electrode. They achieved two linear ranges for MOR: 0.05–1.0 µM and 1.0–10.0 µM. Additionally, the LOD was 0.071 µM. The sensor showed no interference in the presence of AA and UA and was utilized to analyze MOR and COD in human fluids. The same group (Wester et al., 2018) previously used an anodically treated titanium/tetrahedral amorphous carbon (Ti/ta-C) electrode to detect MOR and paracetamol in PBS at pH 7.4. According the authors, this sensor demonstrated a linear correlation for MOR within the range of 0.1–10.0 µM. The anodic treatment of Ti/ta-C electrodes achieved selective detection of MOR with a LOD of 9.8 nM in the presence of paracetamol.
3.5.2 Graphene-based electrodes
In 2023, Wang et al. (2023) developed a graphene/Co3O4 (Gr/Co3O4) composite modified graphite rod electrode (GRE) to determine MOR by means of cyclic voltammetry (CV) and differential pulse voltammetry (DPV) techniques. The peak current of MOR on the Gr/Co3O4-GRE was 12.5 times in comparison with the bare electrode. The Gr/Co3O4-GRE demonstrated excellent electrocatalytic activity. This sensor indicated that the Gr/Co3O4-GRE exhibited an excellent sensitivity to MOR within 0.5–100.0 µM, with its LOD reaching 80.0 nM, showcasing remarkable anti-interferent capacity and long-term stability, making it a potentially valuable means for clinical, environmental, and forensic measurements of MOR. Maccaferri et al. (2019) fabricated screen-printed electrodes (SPEs) functionalized with exfoliated GO (EGO) for amperometric detection of MOR in phosphate buffer (0.1 M PBS, neutral pH). Through DPV measurements, the authors obtained a linear calibration curve within the range of 30.0–275 ppb, a remarkable sensitivity of 2.61 nA/μM and a LOD as low as 2.5 ppb. In addition, the sensor demonstrated high reproducibility and sensitivity, enabling its use in rapid drug abuse screening. Kumary et al. (2019) prepared a reduced GO supported copper-amino acid (RGO/Cu-poly (Ala)) composite modified GCE to determine MOR in neutral PBS. The RGO/Cu-poly (Ala) exhibited excellent electrocatalytic activity towards MOR. This phenomenon was attributed to its large surface area and abundant edge plane defects. They obtained a linear range of 50.0–80.0 µM, and the LOD was found to be 47.0 nM through DPV measurements. The sensor displayed good repeatability, reproducibility, and long-term stability, maintaining 95% of its initial response over a month. Furthermore, it was successfully applied to real blood serum with recoveries of 101.1%–103.0%, which validates its practical utility. Zahra Nazari (2022) described an innovative electrochemical sensor featuring thioglycolic acid-functionalized cadmium selenide quantum dots embedded in GO (TGA-capped CdSe QDs/GO) to simultaneously detect MOR and methadone. The multi-layers GO coating exhibited a higher electrochemical capacitance than the bare electrode. Furthermore, the high specific capacitance and efficient electron transfer capabilities of CdSe QDs contributed to the enhanced sensitivity in electrochemical detecting MOR and methadone. The sensor exhibited linear responses from 0.05 to 323.0 µM for MOR and 0.1–350.0 µM for methadone by CV and DPV. The LODs were 0.038 µM and 0.027 µM, respectively. The sensor showed high reproducibility and anti-interface capability. The sensor’s performance in quantifying MOR in real serum samples was assessed, with acceptable recoveries of 98.4%–102.7%. Hadi’s group (Hadi and Fariba Garkani, 2020) fabricated a new type of magnetic core-shell GO/magnetite@silica (GO/Fe3O4@SiO2) nanocomposite-modified graphite SPE for quantifying MOR in urine samples. The sensor exhibed a linear range from 1.0 to 100.0 µM with a LOD of 0.75 µM. The satisfactory recovery was 98.0% and 103.1% in the determination of MOR in urine specimens. Jahani’s team (Jahani et al., 2020) developed a graphene nanoribbon modified screen printed electrode (G/SPE) to detect MOR and diclofenac. The sensor offered a linear range from 0.07 to 600.0 µM. The LOD for MOR was determined to be 20.0 nM by DPV, CV and chronoamperometry.
3.5.3 Metal/metal oxide-based electrodes
Ognjanovi’c’s group (Ognjanović et al., 2022) reported a CPE-based sensor modified with iron tungstate (FeWO4). The electrochemical characteristics of the FeWO4/CPE were greatly influenced by the tungsten concentration. In addition, the FeWO4/CPE showed electrocatalytic activity toward the MOR determination. The LOD and LOQ were measured at 0.58 µM and 1.94 µM by square wave voltammetry (SWV) in Britton-Robinson buffer (BRBS). The sensor also exhibited selectivity for MOR against interferents including glucose (GLU), AA, DA, CA, and UA. This sensor proved accurate determination of MOR in human urine, with recovery rates of 99.0%–101.0% and negligible matrix effects. Sedigheh Akbari et al. (2020) designed a GCE modified with β-MnO2 nanoflowers (β-MnO2-NF) to monitor MOR and methadone. EIS revealed that the Rct of the β-MnO2-NF/GCE was about 215 Ω and the Rct of bare GCE was about 710 Ω. The decrease in Rct demonstrated the enhanced electron transfer kinetics promoted by the β-MnO2-NF modification. The DPV measurements revealed linear response ranges of 0.1–250.0 µM for MOR and 0.1–200.0 µM for methadone. The LODs were 8.3 nM and 5.6 nM, respectively. Moreover, the sensor exhibited excellent repeatability, reproducibility, and strong resistance to interference capability for MOR detection, demonstrating its successful quantification in actual samples. Jahanbakhshi (2019) fabricated a rhodium nanoparticles-decorated mesoporous carbon (RhNPs-MC)-modified GCE for MOR and buprenorphine detection. The linear ranges of MOR and buprenorphine were 0.1–20.0 µM and 0.1–14.0 µM, respectively, by DPV. LODs were 40.0 nM for MOR and 45.0 nM for buprenorphine, respectively. Finally, the RhNPs-MC/GCE sensor was satisfactorily applied to human serum and pharmaceutical matrices with recoveries in the range of 95%–108.5%. Eissa and colleagues (Eissa et al., 2019) introduced a multiplex electrochemical immunosensor based on an AuNP functionalized disposable electrode array. This innovative sensor enabled the detection of MOR, tetrahydrocannabinol (THC), and cocaine’s main metabolite benzoylecgonine (BZC). The LODs were 1.2 pg/mL for MOR, 7.0 pg/mL for THC, and 8.0 pg/mL for BZC by SWV. The selectivity test showed no significant cross-reactivity. Moreover, the recovery rates of spiked urine sample were 88.0%–115.1%. Saeidi’s group (Mohsen Saeidi et al., 2023) reported an ultrasensitive electrochemical sensor based on Au-Ag nanoparticle-modified ZIF-67 on a screen printed carbon electrode (Au-Ag@ZIF-67/SPCE) for the determination of MOR in urine samples. Using DPV and amperometry (AMP) techniques, they achieved a broad linear dynamic range of 0.05–600 μM, with a low LOD of (3 ± 0.2 nM). The biosensor demonstrated good selectivity for MOR against various analogs. Additionally, the biosensor was further evaluated in urine samples from healthy donors. Zhang’s group (Zhang et al., 2021) designed a voltammetric sensor based on modified CNHs-CHI@PtNPs/GCE to detect MOR and 3,4-methylenedioxymethamphetamine (MDMA) in 0.04 M BRBS. The CNHs@PtNPs demonstrated a good electrocatalytic activity toward MOR and MDMA. The sensor exhibited a linear range of concentrations of 0.05–25.4 µM, and LODs of 0.020 µM for MOR and 0.018 µM for MDMA.
3.5.4 Polymer-based electrodes
Abraham et al. (2019) developed a novel electrochemical sensor based on a poly (cetyltrimethylammoniumbromide)/graphene oxide (poly (CTAB)/GO) modified GCE for MOR quantification. The poly (CTAB)/GO greatly enhanced the electrochemical response of MOR compared to that of bare electrode. The sensor achieved a linear detection range for MOR between 50.0 and 60.0 µM, with a sensitivity as low as 0.36 µM by DPV. The sensor showed good repeatability, stability (over 2 weeks), and resistance to interference from AA and UA. Additionally, the team evaluated the sensor’s performance in measuring spiked MOR in human biofluids, obtaining recovery rates of 96.0%–103.3%. Recently, Salajegheh’s team (Salajegheh et al., 2018) engineered a high selective MIP sensor by modifying a GCE with L-lysine functionalized sodium alginate-activated carbon (SA-AC) for MOR detection. The as-prepared MIP possessed unique selectivity towards MOR, which was attributed to the electrostatic interactions between L-lysine and MOR. The selectivity of the MIP/SA-AC/GCE was conducted to investigate the effect of other analogs on this MIP sensor. The results indicated that a 10-fold excess of COD, heroin, naltrexone and buprenorphine caused no significant influence on the peak current of the MIP/SA-AC/GCE. Using DPV technique, a linear range spanning from 0.1 to 1000.0 µM was achieved, accompanied by a LOD of 48.0 nM. Furthermore, this sensor exhibited satisfactory selectivity, stability, repeatability, and recognition capability in real human urine samples.
3.5.5 Other materials-based electrodes
Bahrami et al. (2020) fabricated an alternative sensing platform by modifing a CPE with electrospun magnetic nanofibers (MNFs) for MOR detection. According to the results of the electro chemical impedance spectroscopy (EIS), the bare CPE indicated electron transfer resistance (Rct) of ∼8818 Ω while the Rct of MNFs/CPE declined to 4479 Ω, indicating that MNFs modification effectively promoted electron transfer. This method achieved a linear range of 0.0033–245.0 µM, and an LOD of 1.9 nM by CV and DPV. In addition, the sensor’s good stability, reproducibility, and anti-interference capability enabled the reliable detection of MOR in actual samples. Aliabadi’s team (Aliabadi and Rounaghi, 2019) fabricated a hydrogel-based CPE sensor for MOR quantification under physiological conditions. The sensor presented a linear range from 5.0 to 200.0 µM, with a LOD of 1.0 µM by DPV. The fabricated sensor demonstrated good repeatability, satisfactory reproducibility, interference-free operation, and long-term stability (for over 35 days). The sensor was also utilized to find MOR in a drop of urine sample. Yousefi’s team (Yousefi et al., 2020) introduced a modified magnetic CPE (MMCPE) for the simultaneous SWV detection of both MOR and methadone. Owing to the high surface area and electrode sensitivity of the magnetic nanoparticles, the MMCPE demonstrated high responsiveness to various the electro-active compounds. In 0.04 M BRBS (pH 8.0), linear responses from 0.005 to 1.8 µM for MOR and 0.01–8.0 µM for methadone were obtained. The LODs were found to be 1.6 nM for MOR and 3 nM for methadone. Moreover, the sensor demonstrated reliable reproducibility and stability. The sensor also displayed good selectivity in the presence of common interferents, such as AA, DA, Na+, K+, Mg2+, and Cl−. Eventually, it was successfully utilized to quantify MOR and methadone in urine samples with satisfactory accuracy.
Electrochemical sensors offer many advantages, such as fast response, low cost, and the ability to be miniaturized. Nevertheless, the matrix effects in real samples cannot be ignored. Most of the 26 studies assessed selectivity. However, five of them did not investigate interference. Common interfering substances (AA, UA, and DA) were chosen for evaluation due to their similar chemical structure or oxidation potential to MOR. However, there are no reports on using electrochemical methods to distinguish MOR from its major metabolites (M3G and M6G). This is still a big challenge.
3.6 Chromatographic versus electrochemical methods for MOR detection
MS is the golden standard for quantifying MOR. It offers high sensitivity and specificity. However, it is frequently time-consuming, costly, and large. Electrochemical sensors offer advantages including rapid response, cost-effectiveness, and portability. However, matrix effects can significantly affect sensors’ sensitivity and recovery. These issues prevent the commercial development of many sensors and limit their use to academic research. Overall, LC-MS is high accurate and electrochemical sensing is low-cost and portable. Future studies should aim to make LC-MS less expensive while creating precise electrochemical sensors.
4 Conclusion
Accurate measurement of MOR and its metabolites in complex biological systems is a challenge. This review focuses on the pretreatment techniques, LC, and electrochemical methods for detecting MOR and its metabolic derivatives in various biological specimens. The analytical processes uses simple pretreatment step like LLE or SPE. These methods aim to reduce or eliminate matrix interferences while facilitating the preconcentration of the target analytes. Subsequently, LC and electrochemical methods are commonly utilized to monitor trace levels of MOR and its metabolites in biological system. Future studies should focus on enhancing the sensitivity and selectivity of electrochemical and MS methods for MOR quantification. Additionally, future research should aim to the miniaturization of electrochemical and MS systems for MOR quantification. Such portable devices facilitate fast on-site analysis, minimizing the need for laboratory-based testing. Moreover, further optimization should focus on improving the cost-effectiveness and simplifying the procedures of these methods to facilitate large-scale application.
Author contributions
XS: Writing – original draft. BY: Investigation, Writing – review and editing. LZ: Formal Analysis, Writing – review and editing. JS: Formal Analysis, Writing – review and editing. YJ: Formal Analysis, Writing – review and editing. JQ: Formal Analysis, Writing – review and editing. ST: Formal Analysis, Writing – review and editing. HY: Formal Analysis, Writing – review and editing. YL: Writing – review and editing.
Funding
The authors declare that financial support was received for the research and/or publication of this article. The Hangzhou Medical and Health Science and Technology Project (Grant No. A20220302, A20220757, A20230954, and A20221009) and the Hangzhou Foundation for Development of Science and Technology (Grant No. 20241029Y084, 2024KY234 and 2025HZFKY7).
Acknowledgements
The authors extend their sincere gratitude to the Hangzhou Medical and Health Science and Technology Project (Grant No. A20220302, A20220757, A20230954, and A20221009) and the Hangzhou Foundation for Development of Science and Technology (Grant No. 20241029Y084, 2024KY234 and 2025HZFKY7) for their generous financial support. The authors would like to thank Wei Zhang from Shiyanjia Lab (www.shiyanjia.com) for the paper review service. The authors hereby declare that there are no additional financial or non-financial conflicts of interest associated with the content of this manuscript, beyond those explicitly stated above.
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Abbreviations
AMPH, Amphetamine; METH, Methamphetamine; EAHF-SPME, Electrical-accelerated hollow fiber solid-phase microextraction; D-µSPE, Micro-solid-phase extraction; M3G, Morphine-3-glucuronide; MTD, Methadone; 6-MAM, 6-Acetylmorphine; M6G, Morphine-6-glucuronide; 6-AC, 6-Acetylcodeine; COC, Cocaine; BZE, Benzoylecgonine; 6-MAM, 6-Monoacetylmorphine; COD, Codeine; US-DLLME, Ultrasound-assisted dispersive liquid-liquid microextraction; IHC, Immunohistochemistry; NSO, Novel synthetic opioids; DLLME, Dispersive liquid-liquid microextraction; Graphene oxide-Fe3O4, GO-Fe3O4; SLE, Supported liquid extraction; PB-SPME, Peptide-based solid-phase microextraction; DBS, Dried blood spot; PP, Protein precipitation; C6G, Codeine 6b-glucuronide; MCNTs, Magnetic carbon nanotubes; MMIP, Magnetic molecularly imprinted polymer.
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Keywords: analytical procedures, biological samples, morphine, metabolites, electrochemistry, chromatography
Citation: Shan X, Yang B, Zhang L, Shao J, Jin Y, Qiu J, Tan S, Ye H and Le Y (2026) Current methodologies to the analysis of morphine and its metabolites in biological matrices. Front. Anal. Sci. 5:1680414. doi: 10.3389/frans.2025.1680414
Received: 06 August 2025; Accepted: 20 November 2025;
Published: 07 January 2026.
Edited by:
Jayant Gowda, BLDEA’s Commerce BHS Arts and TGP Science College, IndiaReviewed by:
J. G. Manjunatha, Mangalore University Constituent College, IndiaSzymon Świątek, Adam Mickiewicz University, Poland
Copyright © 2026 Shan, Yang, Zhang, Shao, Jin, Qiu, Tan, Ye and Le. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Yanna Le, bGV5YW5uYXpmeUAxMjYuY29t