Determination of Morphine in Human Urine by the Novel Competitive Fluorescence Immunoassay

A competitive fluorescence immunoassay for the identification and quantification of morphine has been developed on the basis of hapten-coated plate format. Hapten was prepared through covalent conjugating a morphine derivative with albumin bovine. In the immunoassay, the hapten was inoculated on a 96-well plate and then bound with monoclonal antibodies labeled with a signal indicating dye, fluorescein isothiocyanate (FITC). Unbound FITC-antibodies were rinsed off from the plate. The fluorescein intensity decreases in the presence of morphine molecules due to the competitively binding to antibodies against hapten. The intensity is inversely correlated with the concentration of morphine. In quantitative analysis for urine samples, we obtained a linearity range of 0.2 μg/mL∼2.5 μg/mL, along with a detection limit of c.a. 1 ng/mL. The fluorescence immunoassay shows low cross-reactivity (below 10%) to 6-acetylmorphine, 3-acetylmorphine, and heroine. The developed method produced comparable results to the standard GC-MS/MS method. In conclusion, a rapid and efficient screening tool for morphine in clinical human urine has been established.


Introduction
Morphine (MOR), which is one of the main components in opium, is widely used in relieving severe pain for patients, especially those undergoing a surgical operation, and it is recommended for mildly alleviating cancer-related pain by the World Health Organization (WHO) [1]. MOR is a precursor of narcotic analgesics, among which the most representative drug are heroin, codeine, pethidine, etc. MOR analysis in biological fluids has been applied in forensic cases as an indicator of heroin usage [2] and in routine pharmacokinetic studies [3]. Abused and overdosed of MOR drug is not only dangerous to human health but also injury to the central nervous system, especially the fatal ingested dose is 120 mg. It has been reported that almost 10% of discharged MOR remains unmetabolized, and 90% of orally administrated MOR is passed in the urine within 24-hour period [4]. erefore, to prevent overdose-induced toxicity, it is very necessary to adopt precise examination means for sensitively monitoring the concentration of MOR in patients' blood and urine sample [5,6].
In this work, a competitive immunofluorescence method was used to qualitatively and quantitatively analyze MOR in human urine. For the competitive immunofluorescence method, fluorescein isothiocyanate (FITC) was used as the fluorescent dye to label antibodies. MOR in sample solution and the hapten coated on microporous plate competed with each other to bind with FITC-labeled MOR antibody. e fluorescence intensity is associated with the concentration of MOR, and the correlation index of the affine function for MOR was greater than 0.99%. e relative standard deviation (RSD) of the results obtained from the new method was lower than GC-MS/MS analysis results, while detection limit of our new method was two orders of magnitude lower.
e results show that we have successful established a new competitive immunofluorescence method to detect MOR in human urine. Most importantly, the new method does not require sample preparation as other methods do. Unless special instructions, the experimental data were obtained by averaging five groups parallel experiments.

Materials and Methods
roughout the experiment, all chemicals were analytical grade and redistilled water was used.

MOR Hapten
Synthesis. An amine-MOR hapten was synthesized according to the literature procedures (see Figure 1) [31]. In order to be immunogenic, the target MOR molecule with small relative molecular weight (MW 285) was conjugated with a carrier protein, forming complex biological macromolecules.
Step 1: synthesis of normorphine, as shown as Compound No. 2 in Figure 1. e detailed experimental procedure is as follows: MOR base (1 g), methyl chloroformate (5 mL), and sodium bicarbonate (4.2 g) were added into chloroform solution (50 mL). e reaction solution was refluxed under constant 68°C for 20 h and filtering and purifying the residue for several times with chloroform (5 mL). e removing water of the residue was performed by the dried sodium sulfate. At last, pure yellow foam (1.47 g) could be acquired after repeating the same operation several times. e foam was slowly dropwise added into 3 mL hydrazine hydrate (volume fraction φ is 0.97), then mixed with 4 ml of hydrazine hydrate (φ is 0.64), and refluxed for 63 h in an oil bath. Subsequently, cooling, crystallization, filtration, and washing with water, acetone, and chloroform three times, and red solid precipitate was obtained in 68.5% yield.
Step 2: synthesis of amine-functionalized normorphine, as shown as Compound No.3 in Figure 1. Compound 3 is N-4aminobutyl normorphine. e detailed experimental procedure is as follows: Normorphine (0.3 g), N-(4-bromobutyl) phthalimide (0.5 g), and anhydrous sodium carbonate (0.5 g) were mixed together, placed into the three-neck round-bottom flask containing dimethylformamide (7 mL) and refluxed for 2 h, and the reaction solution was vacuum filtered to reduce the liquid concentration. 5 ml hydrazine hydrate (φ is 0.90) was putted into the reaction solution and refluxed for 1 h under nitrogen atmosphere. After vacuum filtration, the white compound was precipitated and washed with a small amount of water. Finally, drying naturally and recrystallized in methanol, the white precipitate had a 73.2% yield.
Step 3: synthesis of MOR-BSA conjugate, as shown as Compound No. 4 in Figure 1.
e detailed experimental procedure is as follows: N-(4-aminobutyl) normorphine (20 mg) and BSA (20 mg) were dispersed in the double distilled water (20 mL), and the pH value of the mixture is adjusted to neutral with the aid of acid-base buffer, adding aqueous glutaraldehyde (2.2 mL) and stirred overnight at 4°C. e resulting MOR hapten was purified with a SephadexG-25 column, and the first peak wavelength of 280 nm was collected, concentrated, and lyophilized.

Verification of MOR Hapten.
Firstly, structural identification of MOR hapten was analyzed by infrared spectroscopy. Lyophilized MOR hapten was mixed with dry potassium bromide, and the ground mixture was pressed into the pellet, and then the pellet was analyzed using the infrared spectrometer. Lyophilized BSA standards and MOR were also tested as references.
Secondly, structural identification of MOR hapten was analyzed by the MALDI-TOF MS (Bruker Daltonics). e MALDI-TOF MS can detect positively charged peptide ions. Automatic detection of the protein was implemented using the FlexAnalysis software (Bruker Daltonics). A mixture solution of 0.5 μL BSA and 0.5 μL sinapinic acid (10 mg/mL in 50 : 50 ACN/H 2 O 0.1% TFA) was spotted on a MALDI-TOF MTP 384 polished steel. e protein-matrix samples were allowed to crystallize at room temperature. Prior to measurements, the instrument was calibrated against a BSA MALDI-TOF calibration standard; therefore, the ionization of the MorHap-BSA (MOR hapten-BSA) conjugates was expected to be similar, and any mass shift was attributed to hapten incorporation into the protein.

MOR Antibody Preparation.
e lymphatic subcutaneous tissue of 8-week-old female rat (BALB/c) was immunized. After centrifugation and precipitation, the celiac cell suspension was obtained and the good growing myeloma cells (SP2/0) status was obtained by subculture. Taking off the lymph node that has been immunologically treated, the cell culture medium IMDM containing lymphatic particles was obtained. en the lymphocyte, myeloma cell, and celiac cell suspension were mixed together in proportion and fused by the polyethylene glycol (PEG) pure fusion method to obtain hybridoma cells. e hybridoma cell line 3E7 with the highest inhibition rate (100%) was prepared by competitive inhibition ELISA. e monoclonal antibody with positive rate up to 100% was purified by HiTrap PROTEING HP affinity column.

Conjugation of Fluorescein Isothiocyanate and Anti-MOR
Monoclonal Antibody. According to the method in the literature [32], anti-MOR monoclonal antibodies were conjugated with FITC. Anti-MOR monoclonal antibody was dispersed in PBS buffer, FITC was dissolved in the CBS buffer (pH 9.6), and the two are mixed together, stirring at constant 20°C for 4 hours. e resulting conjugates were dialyzed against PBS (pH 7.4) for 4 h and treated by Sephadex G-50 for filtration of the remaining free dye [33]. en, the absorption of FTIC-labeled antibody was measured by using the UV spectrophotometer, shown in Figure  Journal of Analytical Methods in Chemistry 3 the pure FITC standard, the concentration of FITC was estimated to be 1.105 µg/mL and anti-MOR monoclonal antibody was 0.0886 mg/mL. e approximate molar ratio of FITC and anti-MOR monoclonal antibody (n FITC /n MOR-Ab ) was estimated to be 8.1.

Fluorescence Immunoassay Process.
e fluorescence immunoassay process was operated on the 96-well microtiter immunoassay plate. roughout the experiment, PBST was used as the rinsing solution. By slow dropping, MOR hapten conjugation solution was coated to the surface of the orifice and incubated at 4°C for 12 h, nonspecific binding sites were blocked with OVA in PBS (350 µL, 1%) at constant 37°C for 0.5 h, and then FITC (100 µL) labeled anti-MOR monoclonal antibody (diluted in PBS) was mixed with sample solutions or various MOR standard solutions (known concentration, diluted in 0.01 mol/L PBS, pH 7.4). en, the reaction was incubated at 37°C for 2.0 h, which are the optimum temperature and time. e plate was read by the automatic detection microplate reader being excited at 488 nm (λ ex ) and recorded at 525 nm (λ em ) (λ ex � 488 nm, λ em � 525 nm). F max was defined as the fluorescence intensity measured in a MOR-free sample; F was defined as the fluorescence intensity measured in the presence of MOR in the sample; F 0 was defined as the fluorescence intensity measured in a condition that no FITC-labeled antibody is available to bind to the surface-immobilized hapten because of the excessive MOR in the sample. e fluorescence intensity was plotted against the concentration of MOR, yielding to a competitive curve. All values are averaged from five parallel measurements. e half maximal inhibitory concentration (IC 50 value) was detected by the analyte concentration at 50% of the maximal quenching. e limit of detection (LOD) is defined as three times over the background, the signal-to-noise ratio.

GC-MS/MS Analysis.
e GC-MS method is a standard method in FDA China. Our lab is a certified testing lab. All the GC-MS testing followed the exact procedure as the standard method. e urine sample of MOR abusers was collected from Fujian HuaMin forensic center, a large forensic center in the province. Samples were sealed in Teflon centrifuge tubes, and sample amounts were greater than 5 mL. Sample pretreatment was conducted by first placing urine sample (1 mL) into the centrifuge tube (10 mL). 0.2 mL concentrated hydrochloric acid was dropped into urine sample and then heated by water-bath at 70°C for 30 min. Reaction solution was cooled, and 8 mol/L natrium hydroxydatum was added to adjust the pH to 9.0. CBS buffer (pH 8.9) solution (1 mL) was added and leached out by the mixture of chloroform and isopropanol (3 mL, volume ratio 9 : 1). e mixture was sonicated for 10 min, and then centrifuged at 10000 rpm for 3 min.
is process was repeated twice, and the supernatant was collected and combined in a separate test tube. 5 mg anhydrous sodium sulfate was added. e supernatant fluid was transferred into another centrifuge tube and dried in nitrogen atmosphere at 40°C. e pellet in centrifuge tube was dissolved with the mixture of ethyl acetate (200 μL) and derivatization reagents (150 μL).
e test tube was sealed with parafilm and underwent derivatization reaction for 30 min at 75°C. By centrifugation, filtration, and nitrogen drying at 40°C, final product was dissolved in ethyl acetate (200 μL) and injected (1 µL) into the GC-MS/MS for testing.
Chromatographic analysis was performed on an apparatus model Agilent 7890 A/7000QQQB equipped with HP-5 capillary column (30 m × 0.25 mm × 0.25 μm). e initial oven temperature was 150°C and was maintained for 1 min; then the temperature was raised to 280°C at a rate of 60°C/ min and maintained for 6 min, resulting in a total run time of 9.17 min. e carrier gas (He) was used, and the GC was operated in splitless mode at a constant flow rate of 1 mL/ min. e temperature of the gasification chamber was 250°C. e mass spectrometer was equipped with EI source operated at 70 eV with source temperature of 230°C. e temperature of the tandem quadrupole was 150°C, and nitrogen was utilized as the collision gas. Mass spectra were monitored by the multiple reactions monitoring (MRM) acquisition mode. e MS/MS parameters of MOR were optimized by selecting unique precursor ions, product ions, and the collision energies (CE) that achieved the highest selectivity and maximum area counts. ree optimal MOR precursor-product transitions were 429-429.  e infrared spectrum suggested that MOR-BSA was successfully synthesized.

Results and Discussion
Synthesis of MOR-BSA was further verified by MALDI-TOF-MS. Compared to the mass spectrum peak of BSA (solid line) in Figure 4, that MOR-BSA (dotted line) was shifted to right.
is indicated the molecular weight of MOR-BSA was larger than BSA, which further confirmed that MOR-BSA was synthesized successfully. Furthermore, Figure 4 shows that the molecular weight of MOR-BSA is 68467.3 and that BSA is 66304.5. erefore, the numbers of BSA combining with MOR were average of six.

Fluorescence Immunoassay Optimization.
With the improvement of analysis technology, the demand for largeamount analysis of small molecular substances such as drugs and hormones in clinical analysis experiments has never been larger than now. Different from biochemical test or enzyme analysis, immune analysis has gained credibility in its correctness and accuracy, due to its targeting and precision. As MOR structure is a small molecule structure, immune analysis must be performed under competitive mechanisms in order to be accurately tested. By coupling MOR with antibody-FITC, a new competitive fluorescence analysis method was used for recording the fluorescence intensity changes of MOR. FITC-labeled antibody mixed with sample solution or various MOR standard solutions was added into the MOR hapten-coated plates. In this immunoassay, the more MOR was mixed into the MOR antibody-FITC solution, the less MOR antibody-FITC can combine with MOR hapten coated in plates, and the lower the fluorescence intensity displayed. In order to monitor the low dosage of MOR, highly sensitive detection conditions were required, such as coating MOR hapten concentration, time and temperature of the hapten coated, pH value and ionic strength of buffer solution, incubation time, temperature of antigen-antibody reaction, etc.

Concentration of Coating MOR Hapten.
In order to select the optimal coated concentration of MOR hapten, different concentrations of MOR hapten diluted by CBS buffer (pH 9.6) solution were coated on microtiter plates. e equal quantity of anti-MOR monoclonal antibodies labeled with FITC was added into every well, and the fluorescence intensities were measured. Titration curves of different concentrations of coated MOR hapten (0.3, 3, 6.25, 12.5, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, and 400 μg/mL, respectively), and corresponding acquired fluorescence intensity, which is the average value of five times' measurements, were made. As shown in Figure 5, the corresponding fluorescence intensity immediately increased progressively as soon as the concentration of coated MOR hapten was increased and reached a plateau at higher concentration (more than 100 µg/ml). e optimal working concentration of coating MOR hapten was the inflection Journal of Analytical Methods in Chemistry point of titration curve. Ultimately, 50 µg/ml was selected as the optimal MOR hapten concentration. Incubation time and temperature are also important in the reaction of antigens and antibodies. In this study, two parts are involved. One is the binding reaction of MOR hapten coated on microporous plate with antibodies labeled FITC, and the other is the binding reaction of MOR as antigen in real samples with antibodies labeled FITC. Low temperatures could raise their binding fraction, and high temperatures could accelerate the reaction. Incubation time for antigen-antibody reactions was studied (shown in Figure 6), specifically from 0.5 h to 4 h at 37°C. e fluorescence intensity of the antigen-antibody reaction has been increasing rapidly until 2 h at 37°C, then strength slowly weakened.

Incubation Time and Temperature of MOR Hapten
is result suggested that the optimal incubation time is 2 hours.

Influence of pH.
PBS buffer has widely used in immunoassays. Different pH values were tested from 4.0 to 10.0 and found the assay performed better in neutral media, as shown in Figure 7. e sensitivity was lower in both acidic and basic conditions. erefore, the pH condition for the study was 7.4.

Ionic
Strength. Different salt concentrations of PBS, ranging between 0.5X and 4X of the original PBS buffer salt concentration, were examined (Figure 8).
e results indicated that the fluorescence intensity increased sharply with the increase of buffer salt concentration before 0.01 mol/L. When the salt concentration was equal to 0.01 mol/L, it reached the highest and then decreased gradually. In the experiment, the concentration of 0.01 mol/L phosphate was the best buffer.    Journal of Analytical Methods in Chemistry

Fluorescence Competitive Curve and Calibration.
e fluorescence competitive curve for MOR with different concentrations was obtained under optimized conditions. FITC-labeled antibody mixed with various MOR standard solutions (0, 0.001, 0.005, 0.01, 0.05, 0.2, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, and 10 μg/mL, respectively) were inoculated on the MOR hapten-coated plates (50 µg/ml). e fluorescence values and fluorescence competitive curve for MOR standard solutions with concentrations ranging from 0 to 10 μg/mL are shown in Figure 9. In this curve, the x-axis is the concentration of MOR. e y-axis is the average value of five time's measurements. From the titration curve, the IC 50 value (the concentration of the chemical used to reduce fluorescence by 50%) of 0.497 μg/mL was measured. e calibration curve was analyzed for inflexion of fluorescence competitive curve, and MOR determination was done by plotting the relative fluorescence intensity curve based on the concentration of MOR. e curve has a linear response ranging from 0.2-2.5 μg/mL. e linear regression curve was y � −0.3326x + 0.9329, where y is the relative fluorescence intensity (F-F 0 ) and x is the concentration of MOR. e coefficient of correlation degree (R 2 ) is 0.9949. e limit of detection was calculated to be 1 ng/mL (Figure 9 inset).
Most detection techniques serviced for determination of MOR in urine had been based on gas chromatography coupled to simple quadrupole mass spectrometry (GC-MS) with electron ionization [12][13][14][15]. However, conventional analytical instruments usually have weak recognition capabilities and limited accuracy when dealing with samples containing trace levels of MOR. In recent years, gas chromatography-tandem mass spectrometry (GC-MS/MS) technology has developed rapidly, providing higher potential for targeted analysis in terms of recognition and accuracy. As seen on Table 1, data acquired by GC-MS/MS method were compared to results obtained by the new competitive fluorescence immunoassay method. Although the linear range of MOR detection using the new method is narrower than measuring range of the GC-MS/MS, the detection limit of the new method is two times lower than that of the GC-MS/ MS. Standard addition results of MOR in three different concentrations (low, middle, and high) of quantitative curves in urine samples can be seen from Table 2. e recovery and reproducibility of the new method is better than that of the GC-MS/MS method, and the RSD value was lower than 10%. In addition, sample pretreatment for the GC-MS/MS method is complex and time-consuming. In comparison, the new method dispenses with sample pretreatment, thus the new method is more ideal.

Immunoassay Specificity.
In order to study the selectivity of new immunoassay, drug analogues with similar molecular structure including 6-acetylmorphine, 3-acetylmorphine, heroine, codeine, cocaine, ketamine, and ephedrine were evaluated for their potential cross-reactivity (CR). CR is used as a parameter to evaluate the selectivity of immunoassay and is usually calculated based on the IC 50 value in the competition curve of each drug analogues. ese seven drugs were also commonly abused like MOR and are thus likely to also be present in urine from drug abusers. In the experiment, these drugs were dissolved in ethanol and prepared as mentioned above. e CR rate was the ratio of IC 50 value of MOR to that of coexisting drugs.
Results obtained under optimum conditions are summarized in Table 3. 6-Acetylmorphine had the highest CR rate with MOR, followed by 3-acetylmorphine. However, heroine, codeine, cocaine, ketamine, and ephedrine exhibited negligible CR rate. e data indicate that the selectivity of antibodies is based on the degree of molecular structure similarity. All the CR values of the other seven drugs were below 10%, and the antibody bound to MOR with the greatest affinity. e results suggested the MOR antibody prepared in our experiment has the highest specificity compared to others found in the literature. Susan et al. reported a series of monoclonal antibodies against MOR, in which CR values for codeine, apomorphine, heroin, papaverine, naloxone, and natrexone were 100%, 16.5%, lower than 0.05%, lower than 0.05%, lower than 0.05%, and lower than 0.05%, respectively [34]. Compared to the specificity of MOR antibody in our experiment, the CR rate with codeine was 1.6% and heroine was lower than 0.05%. e results demonstrate the MOR antibody acquired in our experiment had better specificity. Matyas et al. made MOR antibody bound with 6-acetylmorphine, that had a CR rate of 20.1% and 0.8% with heroin, the lowest in six experiments of different animals immunized [35]. Compared with the specificity of MOR antibody in our experiment, the CR rate with 6-acetylmorphine was 8.8% and heroine was lower than 0.05%. e results demonstrate the MOR antibody acquired in our experiment had better specificity.
us, the new competitive fluorescence immunoassay method developed using this antibody can ensure that MOR was detected exclusively without interference from other coexisting compounds.

Forensic Sample Analysis.
e urine samples were collected from the Fujian HuaMin forensic center. Urine samples from people of different age and sex were used to perform this study and analyzed with the optimized immunoassay. e concentration of MOR in human urine decreased over time. e concentration of MOR in urine obtained from MOR abusers is typically 1-100 µg/mL under normal conditions [36,37]. Using this new method, the samples were diluted 0-10 times as needed before being analyzed. Furthermore, the urine samples were also analyzed using the GC-MS/MS method. As seen in Table 4, the detection results of urine samples using the two different methods were almost the same. e relative deviations of results from these two different methods were lower than 10%. In conclusion, MOR in urine was detected with satisfactory results by the competitive fluorescence immunoassay method.

Conclusion
In this work, a competitive fluorescence immunoassay method for the determination of MOR in human urine has Journal of Analytical Methods in Chemistry

<0.05
Journal of Analytical Methods in Chemistry been described. In order to get the most accurate analysis results, various factors affecting the specific immunochemical interactions, such as reaction time, reaction temperature, environmental pH, and ionic strength, were investigated. Under the optimized conditions, the immunoassay yielded a detection limit of 1 ng/mL for MOR, with a working range of 0.2-2.5 μg/mL. e detection limit for MOR in our new method is two times lower than conventional GC-MS/MS method. e immunoassay showed high selectivity to MOR and discriminated it against seven coexisting drugs that have similar molecular structures. eir CR rates were lower than 10%. e quantification results from our immunoassay had been verified by the GC-MS/MS method. Specifically, in the examination of MOR in urine samples, the relative deviation of six actual samples by these two methods is lower than 10%. Beneficial from simple, fast, sample-free pretreatment process, our method can be used to accurately detect MOR in human urine. Due to the existence of multifarious drug metabolites in human urine, simultaneous and multitarget testing is highly desirable. Since this assay is based on fluorescence detection, it can be easily integrated into microarray platform.

Data Availability
e data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.