[Determination of amantadine, rimantadine and dimethylamantadine residues in poultry by ultra-performance liquid chromatography-tandem mass spectrometry].

OBJECTIVE: To establish a method for determination of amantadine, rimantadine and dimethylamantadine residues in poultry matrix by ultra-performance liquid chromatography-tandem mass spectrometry. METHODS: Poultry samples were extracted with acid acetonitrile, salting out, and then the organic phase was cleaned up by C_(18) and PSA. A Waters ACQUITYTM UPLC HSS T3 column(100 mm×2.1 mm, 1.7 mm)was used for liquid chromatography separation, ESI positive ion scan was used with multiple reaction monitoring(MRM) mode and quantified by matrix-matched external standard method. RESULTS: At the spiked level of 0.5, 1.0 and 5.0 µg/kg, the recoveries of each compound were in the range of 81.3%-91.1% with the relative standard deviations of 6.5%-11.3%. The qualitative limits of detections were 0.06-0.2 µg/kg and the quantitative limits were 0.2-0.5 µg/kg for the 3 target compounds. The established method was applied to the detection of the 3 target compounds in 30 poultry samples, and none of the target compounds exceeded the residue limits. CONCLUSION: The method is simple, rapid, high sensitivity and good stability, with a wide variety and a certain development. It can be used for the daily monitoring of the veterinary drug residues in poultry.

Development of a molecularly imprinted microspheres-based microplate fluorescence method for detection of amantadine and rimantadine in chicken.

In this study, molecularly imprinted microspheres of a type capable of recognising amantadine and rimantadine were first synthesised, and three fluorescent tracers based on dansyl chloride, fluorescein isothiocyanate and 5-carboxytetramethylrhodamine were also synthesised. These reagents were used to develop and optimise a direct competitive fluorescence method on conventional 96-well microplate for detection of the two analytes. Results showed that this method achieved simple operation procedure, rapid assay process (30 min), high sensitivity (limits of detection 0.04-0.05 ng mL-1) and acceptable recycle performance (five times). After optimisation of several parameters, this method was used to detect amantadine and rimantadine in chicken muscle samples. Their recoveries from standards fortified blank samples were in the range of 62.3-93.7%. The analysis results for some real chicken samples were consistent with a confirmatory LC-MS/MS method. Therefore, this method could be used as a rapid, simple and accurate tool for routine screening the residues of amantadine and rimantadine in a large number of chicken muscle samples.

Simultaneous determination of amantadine and rimantadine in feed by liquid chromatography-Qtrap mass spectrometry with information-dependent acquisition.

A sensitive method for simultaneous determination of amantadine and rimantadine in feed was developed using an ultra-high-performance liquid chromatography-triple quadrupole linear ion trap mass spectrometry (UHPLC-Qtrap-MS) in the multiple reaction monitoring information-dependent acquisition-enhanced product ion (MRM-IDA-EPI) mode, and employing the mixed cation exchange (MCX) solid-phase extraction column as sample cleanup and amantadine-d15 and rimantadine-d4 as internal standards, respectively. Compared to traditional MRM mode, for the targeted drugs in feed simultaneously both the secondary mass spectra and MRM information can be obtained using UHPLC-Qtrap-MS with MRM-IDA-EPI mode, and thus more accurate qualitative confirmation results achieved even at lower concentration of 0.2 µg/L in acceptable purity fit values. After optimization of sample preparation, good linearities (R > 0.9994) were obtained over the concentration range from 1 to 200 µg/L for amantadine and rimantadine. The precision was validated by intra-day and inter-day, and the relative standard deviations were all within 9.61%. Mean recoveries ranged from 76.1 to 112% at spiked concentrations of 0.5-100 µg/kg in three types of feed samples, including formula feed and complex concentrated feed for pigs and premix feed for chicken. The limits of detection (LODs) and quantification (LOQs) were 0.2 and 0.5 µg/kg for both drugs, respectively. The application in real feed samples further proved the accuracy and reliability of the developed method. This method provides an important tool to detect illegal uses of amantadine and rimantadine in feed. Graphical abstract Simultaneous quantitation and qualitative confirmation of amantadine and rimantadine in feed by MRM-IDA-EPI.

Brompheniramine as a novel probe for indirect UV detection and its application for the capillary electrophoresis of adamantane drugs.

Brompheniramine, an antihistamine drug, was employed as a novel UV probe for capillary electrophoresis with indirect UV detection of adamantane drugs (memantine, amantadine, and rimantadine). The probe possesses high molar absorptivity of 24 × 103 L/mol cm at 6 mM, which enables the measurement of these nonchromophore analytes without derivatization. The simple background electrolyte (10 mM sodium dihydrogen phosphate (pH 5.0) containing 5 mM brompheniramine and 6 mM ß-cyclodextrin) provided the separation of the analytes in a short time (7.5 min). Under these conditions, brompheniramine had similar mobility to that of the analyte ions resulting in symmetric peaks with minimal electrodispersion. The analytes displace the probe at a one-to-one ratio with transfer values close to unity. ß-Cyclodextrin played a role in the resolution of the structurally similar adamantane derivatives. Method validation showed good linearity (r2  > 0.98), precision (%RSD ≤ 3.30), and accuracy (recoveries ranging from 98 to 109%). The proposed method was successfully applied to determine the adamantane content in pharmaceutical products.

Preparation of a monoclonal antibody against amantadine and rimantadine and development of an indirect competitive enzyme-linked immunosorbent assay for detecting the same in chicken muscle and liver.

A monoclonal antibody (mAb) was produced in order to monitor the illegal use of amantadine and rimantadine in animals. The produced mAb 2G3 exhibited an IC50 value of 15.8µgL-1 for amantadine and exhibited cross-reactivity to both amantadine (100%) and rimantadine (70.6%). Standard curves ranged from 5 to 80µgL-1 for 2G3. The limits of detection of the developed indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) ranged from 5.0µgkg-1 to 5.4µgkg-1 in chicken muscle and liver. The recoveries were 81.3% to 98.1% with a coefficient of variation less than 15.7%. Good correlations were observed between the results of the ic-ELISA and liquid chromatography-mass spectrometry in the incurred tissues. These results suggest that ic-ELISA is a sensitive, accurate, and low-cost method that could be a useful tool for screening the residues of amantadine and rimantadine in chicken muscle and liver.

[Determination of amantadine and rimantadine residues in egg and chicken samples by dispersive solid phase extraction purification-ultra high performance liquid chromatography-tandem mass spectrometry].

A method was developed for the determination of residual amantadine and rimantadine in eggs and chickens by dispersive solid phase extraction-ultra high performance liquid chromatography-tandem mass spectrometry. Egg and chicken samples were extracted with ammonia water-acetonitrile (2:98, v/v). The extraction solution was dried to 1 mL under nitrogen, and then purified by dispersive solid phase extraction method with C18 and NH2 sorbents. After purification, the extraction solution was filtered through a filter. The target compounds were analyzed by ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) on a ZORBAX C18 column using a mixture of 1 mmol/L ammonium acetate solution (containing 0.1% (v/v) formic acid) and methanol as mobile phases with gradient elution. The mass spectrometer was operated under multiple reaction monitoring (MRM) mode in positive mode. The good linearities were obtained for amantadine and rimantadine at a concentration range of 0.15-10.0 µg/L. The limits of detection for amantadine and rimantadine were all 0.05 µg/kg, and the limits of quantification were 0.20 µg/kg. The recoveries of amantadine and rimantadine in eggs and chickens at three spiked levels (0.2, 1.0 and 2.0 µg/kg) age of 89%-108% with the relative standard deviations of 5.0%-8.6%. The results demonstrated that the method is suitable for the determination of amantadine and rimantadine in eggs and chickens.

Determination of amantadine and rimantadine in chicken muscle by QuEChERS pretreatment method and UHPLC coupled with LTQ Orbitrap mass spectrometry.

A novel sample pretreatment method was developed for the quantitative determination of amantadine and rimantadine in chicken muscle tissues by ultra high performance liquid chromatography coupled with high resolution LTQ Orbitrap mass spectrometry (UHPLC-LTQ Orbitrap MS). The samples were pretreated by modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method, using acetonitrile (1% acetic acid, v/v) as extraction solution and C18 sorbent for clean-up. The separation was carried out on a Waters ACQUITY UPLC HSS T3 column (150mm×2.1mm, 1.8µm particle), using a mobile phase of acetonitrile and 0.1% aqueous formic acid solution. LTQ Orbitrap MS with resolving power of 60000 was applied for analysis of the samples. Amantadine and rimantadine were identified from their accurate mass (within 5ppm) and retention times from the acquired full-scan chromatogram and quantified by their peak areas. The linear range for the determination of the analytes was 1-100µg/kg. Limits of detection (LODs) for amantadine and rimantadine were 1.02µg/kg and 0.67µg/kg, respectively. The intra- and inter-day accuracy ranged from 87.5% to 102.4%, and 82.5% to 105.8% for amantadine, and 95.3% to 97.4%, and 89.4% to 93.2% for rimantadine, respectively. The precision of intra- and inter-day was between 3.9-6.3% and 5.95-13.9% for amantadine, 6.0-7.45% and 7.8-12.4% for rimantadine, respectively. Finally, the method was applied for the determination of these antiviral agents in routine samples, and amantadine residue was detected in some cases.

Determination of amantadine and rimantadine using a sensitive fluorescent probe.

Amantadine hydrochloride (AMA) and rimantadine hydrochloride (RIM) are non-fluorescent in aqueous solutions. This property makes their determination through direct fluorescent method difficult. The competing reactions and the supramolecular interaction mechanisms between the two drugs and coptisine (COP) as they fight for occupancy of the cucurbit[7]uril (CB[7]) cavity, were studied using spectrofluorimetry, (1)H NMR, and molecular modeling calculations. Based on the significant quenching of the supramolecular complex fluorescence intensity, a fluorescent probe method of high sensitivity and selectivity was developed to determine AMA or RIM in their pharmaceutical dosage forms and in urine samples with good precision and accuracy. The linear range of the method was from 0.0040 to 1.0 µg mL(-1) with a detection limit ranging from 0.0012 to 0.0013 µg mL(-1). This shows that the proposed method has promising potential for therapeutic monitoring and pharmacokinetics and for clinical application.

Determination of memantine in plasma and vitreous humour by HPLC with precolumn derivatization and fluorescence detection.

A new HPLC procedure with precolumn derivatization and rimantadine as the internal standard for determining memantine, a candidate agent for the treatment of glaucoma in plasma and vitreous humour, has been developed and validated. Precolumn derivatization was performed with 9-fluorenylmethyl-chloroformate-chloride (FMOC-Cl) as the derivatization reagent and followed by a liquid-liquid extraction with n-hexane. Optimal conditions for derivatization were an FMOC-Cl concentration of 1.5 mM, a reaction time of 20 min, the temperature at 30°C, the borate buffer pH 8.5, and a borate buffer-acetonitrile ratio of 1:1. The derivatives were analyzed by isocratic HPLC with the fluorescence detector λex 260 nm λem 315 nm on a Novapack C(18) reversed-phase column with a mobile phase of acetonitrile-water (73:27, v/v), 40°C, and a flow rate of 1.2 mL/min. The linear range was 10-1000 ng/mL with a quantification limit of ∼ 10 ng/mL for both types of samples. This analytical method may be suitable for using in ocular availability studies.

Validation of a capillary zone electrophoresis method for determination of rimantadine hydrochloride in Rimantadin100 tablets and the method application to dissolution test monitoring.

A capillary zone electrophoretic method with indirect UV-detection for determination of rimantadine, an antiviral drug against influenza A, in tablets was validated. Instrumental precision, the method precision, accuracy, calibration curve linearity, selectivity, robustness, and time stability of the sample and the standard were tested. The method was also applied to monitor dissolution tests of the tablets. The possibility of addition of an internal standard for improvement of the method precision was discussed.

Electrophoretic behavior of adamantane derivatives possessing antiviral activity and their determination by capillary zone electrophoresis with indirect detection.

Separation and determination of adamantane derivatives with antiviral activity, namely amantadine (1-adamantan amine), memantine (1-amino-3,5-dimethyl adamantane) and rimantadine (alpha-methyl-1-adamantane methylamine), were examined by capillary zone electrophoresis. After optimization, an indirect detection method using 5 mM 4-methylbenzylamine in ethanol/water solution (1:4) as simultaneously absorbing and buffering background electrolyte with detection at 210 nm was found suitable for determination of the individual compounds (limit of detection was 0.35 mg L(-1) for memantine hydrochloride, S/N = 3). Baseline separation of all the three compounds was reached by addition of alpha- or beta-cyclodextrins to the electrolyte in concentrations of 20 and 2 mM, respectively.

[The forensic chemical determination of remantadine in biological material]. / Sudebno-khimicheskoe opredelenie remantadina v biologicheskom materiale.

Suggests new microcrystalloscopic tests for the identification of remantadine and assesses the specificity and sensitivity of these tests. Describes a thin-layer chromatographic technique for remantadine identification.

Quantitation of the enantiomers of rimantadine in human plasma and urine by gas chromatography-mass spectrometry.

A gas chromatographic-mass spectrometric procedure has been developed for the quantitation in plasma and urine of the enantiomers of rimantadine, an antiviral drug effective against type A influenza. The assay utilizes derivatization with an optically active reagent, selective ion monitoring, methane negative-ion chemical ionization (NICI) mass spectrometry and stable isotope dilution. The method has been used to measure concentrations of each rimantadine enantiomer over a range of 2.5-250 and 12.5-1250 ng/ml in the plasma and urine, respectively, of four male volunteers administered rimantadine. In plasma and urine, no differences were observed in the disposition of the unconjugated enantiomers. In urine, one enantiomer, but not both, was released following enzymatic hydrolysis.

Quantitative determination of rimantadine in human plasma and urine by GC-MS.

A GC-MS procedure has been developed for the quantitation in plasma and urine of rimantadine, an antiviral drug effective against type A influenza. The assay utilizes selective ion monitoring, methane negative ion chemical ionization (NCI) and stable isotope dilution. Sensitivity to NCI is effected by derivation of rimantadine with pentafluorobenzoyl chloride. The method has been used to quantitate plasma concentrations of rimantadine over a range from 4.2 ng/ml to 416 ng/ml, and urinary concentrations of rimantadine over a range of 21 ng/ml to 2077 ng/ml.

[Capillary isotachophoresis--a new method in drug analysis. 3. Analytic capillary isotachophoresis for the determination of amantadine and rimantadine]. / Die Kapillarisotachophorese--eine neue Methode in der Arzneimittelanalytik. 3. Mitteilung: Analytische Kapillarisotachophorese zur Bestimmung von Amantadin und Rimantadin.

Investigations by means of analytical capillary isotachophoresis are possible not only for ampholytes, as peptides or amino acids, but generally for weak and strong electrolytes. Amantadine and rimantadine are determined by making use of a leading electrolyte including K+ ions at a pH range from 5.9 to 6.0 (terminating ion = creatinine+) at concentrations of the test solutions of greater than or equal to 600 nmol/ml and greater than or equal to nmol/ml, respectively, with a precision calculated as rel. S.D. (%) less than +/- 3 as a rule and a duration for one analysis less than 10 min. The tested discontinuous electrolyte system enables the determination of both the active substances simultaneously. The missing UV absorption of 1 and 2 is not disadvantageous by virtue of the application of the conductivity detector. The method was employed to carry out a content uniformity test for 2 tablets and informing experiments about the dissolution of 2 hydrochloride respectively 1 hydrochloride in water out of tablets and capsules, respectively.