Morphine In Urine Samples Research Articles

Electrochemical Determination of Morphine in Urine Samples by Tailoring FeWO4/CPE Sensor.

Morphine (MORPH) is natural alkaloid and mainly used as a pain reliever. Its monitoring in human body fluids is crucial for modern medicine. In this paper, we have developed an electrochemical sensor for submicromolar detection of MORPH. The sensor is based on modified carbon paste electrode (CPE) by investigating the FexW1-xO4 ratio in iron tungstate (FeWO4), as well as the ratio of this material in CPE. For the first time, the effect of the iron-tungsten ratio in terms of achieving the best possible electrochemical characteristics for the detection of an important molecule for humans was examined. Morphological and electrochemical characteristics of materials were studied. The best results were obtained using Fe1W3 and 7.5% of modifier in CPE. For MORPH detection, square wave voltammetry (SWV) was optimized. Under the optimized conditions, Fe1W3@CPE resulted in limit of detection (LOD) of the method of 0.58 µM and limit of quantification (LOQ) of 1.94 µM. The linear operating range between 5 and 85 µM of MORPH in the Britton-Robinson buffer solution (BRBS) at pH 8 as supporting electrolyte was obtained. The Fe1W3@CPE sensor resulted in good selectivity and excellent repeatability with relative standard deviation (RSD) and was applied in real-world samples of human urine. Application for direct MORPH detection, without tedious sample pretreatment procedures, suggests that developed electrochemical sensor has appeared to be a suitable competitor for efficient, precise, and accurate monitoring of the MORPH in biological fluids.

New method of identifying morphine in urine samples using nanoparticle-dendrimer-enzyme hybrid system

The incorporation of morphine (MOR) into the nanoparticle structure is a viable alternative to traditional enzyme usage. It has good biological potential to separate MOR from real urine samples. In this study, a new method of MOR identification in real urine samples was synthesized using the β-glucuronidase-dendrimer poly amidoamine (PAMAM) enzyme hybrid system. Replacing MOR in dendrimer cavities significantly reduces enzyme consumption. The replacement technique is done in dendrimer cavities in two stages as an alternative to β-glucuronidase enzyme and even MOR. In this paper, firstly, PAMAM dendrimer G2 was synthesized based on silica. The β-glucuronidase enzyme was replaced inside its dendrimer cavities and the compound was released into a real urine sample containing MOR. The enzyme was extracted from dendrimer cavities. The MOR- β-glucuronidase enzyme bond broke. In the next stage of the process, free MOR entered the PAMAM dendrimer G2 cavities. MOR was detected in real urine samples.

Plant Extract and Herbal Products as Potential Source of Sorbent for Analytical Purpose: An Experimental Study of Morphine and Codeine Determination Using HPLC and LC-MSMS.

Solid-phase microextraction (SPME) is an analytical method for microextraction of analytes, in which the analytes bind to the sorbent on the surface of the SPME fiber. Many types of chemical agents are used as sorbent; however, many of these sorbents cause secondary contamination or are not cost-effective. Here, aqueous extract of Ferula gummosa was evaluated as potential source of sorbent for simultaneous microextraction of morphine and codeine. For this purpose, multiwalled carbon nanotubes were carboxylated with H2SO4/HNO3 (3:1) and then functionalized with aqueous extract of F. gummosa. Functionalization was confirmed by Fourier transform infrared and Raman spectroscopy measurements as well as scanning electron microscopy analysis. Porous polypropylene hollow fibers were filled with the functionalized carbon nanotubes (CNTs) and used for analyte extraction in urine sample at 40°C and pH6 for 2min. Reversed-phase high-performance liquid chromatography (RP-HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that the fiber could preconcentrate 1ng/mL of morphine and 0.75ng/mL codeine in urine sample and was successfully used for 30 times with no significant loss in the extraction efficiency. Limit of detection (LOD) and limit of quantification (LOQ) for morphine were 1 and 3.3ng/mL, respectively. LOD and LOQ for codeine were determined 0.75 and 2.47ng/mL, respectively. Recovery of the fiber was 80% and 93% for morphine and codeine, respectively. SPME fiber using extract of F. gummosa plant was used for the detection of a small amount of morphine in urine sample. Therefore, plants can be considered as abundant and cheap sources of sorbent for various analytical purposes.

Detection of morphine in urine based on a surface plasmon resonance imaging immunoassay.

Based on the surface plasmon resonance imaging (SPRi) technique, a new detection method for morphine in urine samples was developed. Sample labelling was not required, and qualitative and quantitative analysis could be completed in 20 minutes. According to an indirect competitive immunoassay, the mixture of morphine at different concentrations and morphine antibody at a certain concentration as the mobile phase was reacted with morphine BSA fixed on a chip surface in a competitive way. A calibration curve was obtained by correlating the signals generated from SPRi with the concentrations of morphine. By the addition of morphine to a blank urine sample, this method was confirmed to be feasible for the detection of morphine in actual urine. The limit of detection was as low as 9.59 ng mL-1. This method is fast and sensitive and can be applied in many fields.

Molecular modeling and experimental study of a new peptide-based microextraction fiber for preconcentrating morphine in urine samples.

Antimicrobial peptides (AMPs) are best known for their bactericidal properties; however, due to their unique and flexible structures, they have also been proposed as potential selective sorbents for specific molecules. In the present study, we aimed to design and produce a new peptide-based microextraction fiber for preconcentrating morphine in urine samples. The binding of morphine to the peptide was first evaluated by computational simulation using the Molecular Operating Environment (MOE) 2015.10 software. A similar study was then performed using DS BIOVIA Materials Studio 2017 v17.1.0.48, which confirmed the results of the simulation carried out with MOE. Afterwards, those results were also confirmed by experimental research. In the experimental evaluation, carbon nanotubes (CNTs) were initially carboxylated with H2SO4/HNO3 (3:1) and then functionalized with the peptide. FTIR analysis, Raman measurements, and SEM imaging were used to confirm that CNT functionalization was successful as well as to check the nanostructure of the fiber. To evaluate the functionality of the fiber, it was inserted into a microtube containing a urine sample that included morphine and then sonicated for 5min at 40°C. Afterwards, the fiber was washed with methanol 20% (H2O/methanol) and the resulting sample was analyzed by HPLC. This procedure was repeated for different concentrations of morphine in the urine sample. The computational and experimental results showed that a morphine concentration as low as 0.25ppb in urine could be adsorbed and detected using the peptide fiber. Therefore, given its semi-selective binding affinity for morphine, this peptide-based fiber can be considered a new approach to the detection of small amounts of morphine in biological samples.

Preconcentration of morphine in urine sample using a green and solvent-free microextraction method

Abstract The ability of extraction and preconcentration of small amounts of substances from biological samples is very important in medical toxicology. On the other hand, minimal use of organic solvents is an important issue to prevent environmental damage. In the present study, we developed a new solid phase microextraction fiber using plant extracts as sorbent for extraction and preconcentration of morphine in urine sample. For this purpose, raw carbon nanotubes (CNTs) were functionalized with tobacco extracts. Functionalization was confirmed by Fourier transform infrared (FTIR) and Raman spectroscopy in addition to scanning electron microscope (SEM) images. The functionalized CNTs were coated on polypropylene hollow fiber. The results of HPLC analysis showed that the produced fiber could preconcentrate a very low concentration of morphine (0.25 ng/ml) in small volume of urine samples. Limit of detection (LOD) and limit of quantification (LOQ) for the produced fiber were determined 0.25 ng/ml and 0.825 ng/ml, respectively, and recovery of the fiber was determined 89% at LOQ. The produced fiber provided a recyclable and solvent free method for extraction of a trace amount of morphine, which can be successfully used for up to 30 times with no significant loss in the extraction efficiency.

Potential application of amino acids in analytical toxicology

The ability of extraction and preconcentration of small quantities of substances from biological samples is important in analytical sciences, particularly forensic medicine. In the present study, we evaluated the binding potential of amino acids to produce a new solid phase microextraction fiber based on carbon nanotube (CNTs) for extraction and preconcentration of small amount of morphine in urine sample. Raw CNTs were first carboxylated and then functionalized with 3 amino acids including glutamate, arginine, and cysteine. Functionalization was confirmed by FTIR analysis, Raman spectroscopy and SEM imaging. The functionalized CNTs were coated on polypropylene hollow fiber and used for preconcentration. The results of HPLC analysis in isocratic elution mode using acetonitrile-sodium acetate (10:90, v/v; pH 4; 0.01 M) as the mobile phase showed that amino acids are able to adsorb morphine and the prepared fiber could preconcentrate a very low concentration of morphine (0.25 ppb) in urine matrix. In addition, the fiber was successfully used for up to 30 times with no significant loss in the extraction efficiency. Lowest limit of detection (LOD) and limit of quantitation (LOQ) was 0.07 and 0.25, respectively. Also, the lowest and best recovery of the fiber was 87.8% and 139% at LOQ, which belonged to glutamate and arginine, respectively. The fibers based on amino acids can be used for the detection of a small amount of morphine in biological samples, which are not detectable by conventional methods. Simple mechanism of these fibers in preconcentrating morphine makes them a novel candidate for detection of other opiates and drugs of abuses in crime scene investigations and postmortem examinations several days after exposure.

Highly sensitive amperometric sensor for morphine detection based on electrochemically exfoliated graphene oxide. Application in screening tests of urine samples

Graphene oxide modified screen-printed electrodes have been tested as amperometric sensors for morphine determination. The results demonstrate that the arising of electrocatalytic processes ascribable to the graphene coating, combined with the use of a suitable cleaning procedure, allow the sensor to achieve higher sensitivity (2.61 nA ppb−1) and lower limit of detection (2.5 ppb) with respect to those reported in the literature for similar devices.Due to very low detection limit found, the device is suitable to detect the presence of morphine in urine samples after a very simple and rapid pre-treatment of the matrix, allowing the removal of interfering species affecting the voltammetric responses. Tests performed in synthetic urine samples demonstrate that the presence of the electrocatalytic coating is mandatory in resolving the peak due to morphine oxidation in respect to uric acid. The sensor proposed is, thus, suitable to detect this drug even at concentration values below the cut-off levels defined by European and American regulations. These results allow us to propose the sensor for screening tests in portable devices, to be applied in systematic controls of drug abuses, e.g. in drivers and in men at work.

Developing a new sensitive solid-phase microextraction fiber based on carbon nanotubes for preconcentration of morphine

The ability of extraction and preconcentration of small amounts of substances from biological samples is important in forensic medicine. In the present study, we produced a new solid-phase microextraction fiber based on carbon nanotube (CNTs) for extraction and preconcentration of small amount of morphine in urine sample. Raw nanofibers were first carboxylated with H2SO4/HNO3 (3:1) and then functionalized with ethylenediamine. Functionalization was confirmed by FTIR and Raman spectroscopy as well as SEM analysis. The functionalized CNTs were coated on a porous polypropylene hollow fiber. A new electrical device was designed and manufactured for adsorption–desorption of the analyte, in which the voltage could be reversed and the analytes could desorb into the washing solution for HPLC analysis. The results showed that the prepared fiber could adsorb a very low concentration of morphine (0.5 ppb) in urine matrix, and was successfully used for up to 50 times with no significant loss in the extraction efficiency. Recovery of the fiber was 77% at 0.5 ppb. The optimum condition for the fiber was 2 min at 1 V of the manufactured electrical catalyst controller. This fiber can be used for the detection of a small amount of morphine in biological samples, which are not detectable by conventional methods. Simple mechanism of this fiber in preconcentrating morphine makes it a novel candidate for detection of other opiates and drugs of abuses in crime scene investigations and postmortem examinations several days after exposure. The prepared electric microextraction device could also efficiently concentrate the morphine in the urine matrix.

Computational-aided design of magnetic ultra-thin dummy molecularly imprinted polymer for selective extraction and determination of morphine from urine by high-performance liquid chromatography

In this work, a novel magnetic ultra-thin dummy molecularly imprinted polymer (MMIP) for morphine (MO) was prepared. In order to obtain highly selective recognition cavities, the MMIP has been designed using semi-flexible docking to screen the optimal monomer and its ratio to morphine from six representative monomers. Furthermore, the dummy template was creatively screened by semi-flexible docking method from opioid drugs. The system of dihydrocodeine (DI) as dummy template, methacrylamide (MAC) as founctional monomer, ethyleneglycol dimethacrylate (EGDMA) as crosslinker was chosen for MO imprinting. The morphological and magnetic properties of MMIP were characterized by FT-IR, TEM and VSM. The results suggested that molecularly imprinted polymer (MIP) was synthesized evenly on Fe3O4 surface. The adsorption experiments revealed that MMIP showed better extraction capacity and selectivity toward MO and its analogues than the non-imprinted polymer (NIP). The MMIP possessed adsorption capacity of 14.71mg/g for MO and the imprinting factor was 2.10 at separate adsorption and 1.87 at competitive adsorption. A magnetic molecularly imprinted solid phase extraction coupled with HPLC method (M-MISPE-HPLC) has been established for the analysis of MO in urine sample. The developed method was validated for its linearity (0.038–100mgL−1 R2=0.9937), precision (1.07%–3.72%) and accuracy (83.62%–100.37%).

Ultrasonic-assisted magnetic solid phase extraction of morphine in urine samples by new imprinted polymer-supported on MWCNT-Fe3O4-NPs: Central composite design optimization

Multiwalled carbon nanotubes (MWCNTs) were magnetized with Fe3O4 nanoparticles (MWCNTs-Fe3O4-NPs) and subsequently coated by vinyl end groups (Vinyltrimethoxysilane). MWCNT-Fe3O4-NPs were used as support for a new morphine (MO) molecularly imprinted polymer (MWCNT-Fe3O4-NPs@MO-MIP) by surface imprinting polymerization method. The MWCNT-Fe3O4-NPs@MO-MIP was characterized by FTIR, VSM and SEM techniques and successfully used for determination of MO. Ultrasonic-assisted magnetic solid phase extraction followed by UV–vis spectrophotometer (UAMSPE-UV–vis) was investigated for MWCNT-Fe3O4-NPs@MO-MIP and compared with non-imprinted polymer (NIP) using batch method. Central composite design under response surface methodology was used for the evaluation of the effect of variables, individually, as well as their possible interaction effects on the adsorption process. The variables such as sonication time, MWCNT-Fe3O4-NPs@MO-MIP mass, initial concentration of MO and pH were investigated in this study. At optimum experimental conditions, UAMSPE-UV–vis method was exhibited a linear range of 0.8–8.7mgL−1 of the MO concentration with a detection limit of 0.18mgL−1. The relative standard deviation for the analyte was found to be lower than 2.32%. The MWCNT-Fe3O4-NPs@MO-MIP adsorption capacity was found to be 37.01mgg−1. The enrichment and preconcentration factors were found to be 107.01 and 98.21, respectively. The developed method was finally applied successfully to the determination of MO in urine and wastewater samples with the recoveries ranged from 96.40 to 105.6%.

A new platform for sensing urinary morphine based on carrier assisted electromembrane extraction followed by adsorptive stripping voltammetric detection on screen-printed electrode

Electromembrane extraction (EME) coupled with electrochemical detection on screen-printed carbon electrode has been developed for the quantification of morphine in urine samples. Charged morphine molecules were extracted from an aqueous sample by applying an electrical potential through a thin supported liquid membrane (SLM) into an acidic aqueous acceptor solution (20µL) placed inside the lumen of a hollow fiber. Then, the acceptor solution was mixed with 20µL of NaOH solution (0.1M) and analyzed using screen printed electrochemical strip. Differential pulse voltammetry (DPV) peak current at 0.18V was selected as the signal and the influences of experimental parameters were investigated and optimized using Box–behnken design and also one-variable-at-a-time methodology as follows: adsorptive accumulation time, 40s; SLM, 2-nitrophenyl octyl ether+10% tris-(2-ethylhexyl) phosphate+10% di-(2-ethylhexyl) phosphate; pH of the sample solution, 6.0; pH of the acceptor solution, 1.0; EME time, 24min; EME potential, 90V and stirring rate, 1000rpm. The calibration curve which was plotted by the variation of DPV currents as a function of morphine concentration was linear within the range of 0.005–2.0µgmL−1. The limit of detection and the limit of quantification were 0.0015 (S/N=3) and 0.005µgmL−1, respectively. Finally, the proposed method was able to determine morphine simply and effectively at concentration levels encountered in toxicology and doping.

Development of a Nanogold-Based Immunochromatographic Assay for Detection of Morphine in Urine Using the Amor-HK16 Monoclonal Antibody

A simple, rapid competitive immunochromatography (ICG) strip test was developed to detect morphine in urine samples using a monoclonal antibody produced in-house and conjugated to gold nanoparticles. Hybridoma cells were cultured and the Amor-HK16 monoclonal antibody against morphine was obtained from the supernatant after purification by salting out and passing through a Protein G-Agarose affinity column. Morphine was obtained from morphine sulfate and a C6-hemisuccinate derivative of morphine was prepared, conjugated to bovine serum albumin, and immobilized to a nitrocellulose membrane as the test line. Goat anti-mouse antibody was used as a binder in the control line in the detection zone of the strip. Colloidal gold particles of diameter approximately 20 nm were prepared and conjugated to the monoclonal antibody. The detection limit of the test strip was found to be 2000 ng/mL of morphine in urine samples. Reliability was determined by performing the ICG test on 103 urine samples and comparing the results with those obtained by thin-layer chromatography. The sensitivity of the test was 100%, and the analysis time for the assay was approximately 5 min. The new ICG method was adequately sensitive and accurate for the rapid screening of morphine in urine.

Direct and Simple Electrochemical Determination of Morphine at PEDOT Modified Pt Electrode

AbstractA simple and rapid method for morphine detection is described based on PEDOT electrode in the presence of SDS. The electrochemistry of morphine is investigated by CV, LSV and SWV. The effect of common interferences on the current response of morphine namely AA and UA is studied. The electrode is applied to the selective determination of morphine in urine samples in the linear ranges 0.3–8 µmol L−1 and 10–60 µmol L−1, with low detection limits of 50 and 68 nmol L−1, respectively and recovery of 96.4 %. The application of PEDOT is realized in determination of morphine in tablets successfully.

Electrochemical determination of morphine at ordered mesoporous carbon modified glassy carbon electrode

A sensitive electrochemical detection scheme for morphine has been proposed by utilizing the ordered mesoporous carbon modified glassy carbon electrode (OMC/GCE). The electrochemical behaviour of morphine at the OMC/GCE was investigated by cyclic voltammetry. The modified electrode displayed a decrease in the overpotential (ca. 82 mV) and an obvious increase in the peak current (80 times) compared to the bare glassy carbon electrode (GCE). The results indicated that OMC/GCE remarkably enhanced electrocatalytic activity towards the oxidation of morphine. Linear response was found over the range from 0.1 μM to 20 μM with a high sensitivity of 1.74 μA/μM and low detection limit of 10 nM (S/N = 3). And the relative standard deviation was 0.86% ( n = 5). Moreover, combining with a medium-exchange procedure, the OMC/GCE has also been successfully applied to the selective determination of morphine in urine samples with a low detection limit of 50 nM and satisfied recovery of 96.4%. The good results indicate that the OMC/GCE holds great promise in practical application.

Strip-based immunochromatographic assay using specific egg yolk antibodies for rapid detection of morphine in urine samples

Using specific egg yolk antibodies (IgY), a strip-based immunochromatographic assay was developed for rapid detection of morphine in urine samples. IgY type antibody against morphine was generated by immunizing chickens with well-characterized monoacetyl morphine–protein conjugate. The antibody was labeled with gold nanoparticles and used as an immunoprobe in the dipstick format for the visual detection of morphine in urine samples. The dipstick was developed using three membranes: an application pad made of glass fiber membrane to hold the tracer, a signal generation test line on nitrocellulose membrane (detection zone) and a cellulose membrane used as an absorption pad. Analytes of interest (morphine and its analogues) added to the sample well, dissolved the labeled antibody (tracer), and the antigen–antibody complex formed was transported by the flow caused by capillary action to the test line. The color signal of test line in proportion to the morphine concentration in urine samples was measured using a detector. The developed dipstick assay format was optimized, showing the average IC50 values of morphine as low as 9.45ng/mL, the detection range of 1–1000ng/mL and the lowest detection limit 2.5ng/mL under optimal conditions of analysis. The correlation between the developed dipstick and ELISA was 0.948 in the analysis of urine samples spiked with morphine. The developed dipstick could be a highly sensitive and convenient tool for rapid detection of opiate drugs in samples with high degree of stability.

A novel metabolic pathway of morphine: formation of morphine glucosides in cancer patients.

To characterize directly the conjugated metabolites of morphine in urine samples of cancer patients. Urine samples from the patients were treated by solid-phase extraction method and chromatographed using three high-performance liquid chromatography systems. Conjugated metabolites were directly detected with liquid chromatographic/ion trap mass spectrometric (LC/MSn) technique by selected ion monitoring, full scan MS/MS and MS3 modes. Six conjugated metabolites including two new metabolites M5 and M6 were found. Morphine-3-glucuronide (M-3-G) and morphine-6-glucuronide (M-6-G) were identified by comparing their l.c. retention times and multistage mass spectra with those of the reference substances. Two novel metabolites, morphine-3-glucoside and morphine-6-glucoside, as well as normorphine glucuronides were identified by comparing their mass fragment patterns and l.c. retention times with those of M-3-G and M-6-G. Hydrolysis of urine samples with beta-glucosidase and beta-glucuronidase provided further evidence of the metabolites M5 and M6 as morphine glucosides. The excretion amounts of morphine conjugates in urines were in the order of morphine glucuronides, morphine glucosides and normorphine glucuronides. In the present study, the applications of l.c. separation and multistage mass spectra have permitted the direct identification of conjugated metabolites of morphine. To our knowledge, this is the first report about O-linked glucosides of morphine at 3-aromatic and 6-aliphatic hydroxyl groups.

Qualitative determination of urinary morphine by capillary zone electrophoresis and ion trap mass spectrometry.

A rapid, sensitive method for the determination of morphine and amphetamine was developed using capillary zone electrophoresis coupled with electrospray interface (ESI), ion-trap tandem mass spectrometry (CE-ESI-MS2). Morphine and amphetamine were separated in 20 mM ammonium acetate buffer (pH 6.6) and detected by ion-trap mass detector in different analytical time segments (0-6.25 min for amphetamine and 6.25-12.0 min for morphine) in which the tune file for each compound was used separately. Molecular ions of morphine (m/z 286) and amphetamine (m/z 136) were detected at 5.77 and 6.83 min, respectively, while product ions of MS2 for each compound (m/z 229, 201 for morphine and m/z 119 for amphetamine) were detected almost exactly at the same time with their parent compounds. The limits of detection (LOD) for MS2 determination were 30 and 50 ng/mL for amphetamine and morphine, respectively, with an S/N ratio of 3. For more sensitive detection of morphine, the sample was injected for a longer time (i.e., 80 s) and hydrodynamically transported into a CE capillary for MS detection. Morphine and its product ion appear at 0.36 and 0.39 min on the ion chromatogram, respectively, with a five-fold increase of detection sensitivity (LOD, 10 ng/mL). The CE-MS system thus established was further applied for forensic urine samples screened as morphine-positive by fluorescence polarization immunoassay (FPIA). These results indicated the feasibility of CE-ESI-MS2 for confirmative testing of morphine in urine sample.

Automated kinetic-spectrofluorimetric method for the determination of morphine in urine.

The continuous addition of reagent technique was used with spectrofluorimetric detection for the kinetic determination of morphine in urine samples. The method thus developed is based on the oxidative dimerization of morphine to fluorescent pseudomorphine by potassium hexacyanoferrate(III) in a basic medium. The optimum pH, oxidant concentration and instrumental variables were determined. The method permits the sensitive determination of morphine over a wide concentration range (15-925 ng ml-1) with high precision (relative standard deviation approximately 2%), selectivity and sample throughput (48 samples h-1). A novel sorption--desorption procedure was used to isolate morphine from whole urine, which provided recoveries of between 85 and 100%. The results showed the usefulness of the proposed method for controlling a wide variety of medicinal problems (e.g., therapeutic, overdose and doping analysis).

Determination of morphine and codeine in urine by gas chromatography—mass spectrometry

GC-MS is one of the recommended analytical techniques for the identification and confirmation of opiates in urine. A method for the qualitative detection and quantitation of codeine and morphine in urine samples by this technique has been developed. This method is also suitable for the detection of their main metabolites in urine: norcodeine and normorphine. It also allows the identification of 6-monoacetylmorphine in urine, which can be used as a confirmatory marker of heroine abuse. The derivatized compounds are separated by capillary gas chromatography (GC) and identified by mass spectrometry (MS) in the selective ion monitoring acquisition mode (SIM). The recoveries from urine at concentrations of 1000 ng ml-1 are 72% for codeine and 80% for morphine. The method is linear in the range studied (0-1000 ng ml-1) for codeine and morphine.