Sulfonamide drugs: Low-cost spectrofluorometric determination using a computer monitor calibrator for detection.

A possibility of the use of a common monitor calibrator as a portable and inexpensive tool for the fluorometric determination of sulfonamide drugs after their reaction with fluorescamine was examined. The luminescence measurements with a calibrator are based on irradiation of a test sample by the device lamp with a broadband spectrum in the visible and near UV regions and simultaneous registration of the secondary radiation by the device detector. Two types of cuvettes with black light absorbing sides eliminating the reflected self-radiation were tested. The commercially available Eppendorf-type black plastic microtubes ("LightSafe") were suggested as a good option for such measurements. It was shown that a monitor calibrator can be applied for optimization of the determination conditions. By the example of sulfanilamide and sulfamethazine, it was shown that the procedure should be carried out at pH 4-6 and fluorescamine concentration of 200 µmol L-1, and 40 min of the interaction. The limit of detection of sulfanilamide and sulfamethazine using a monitor calibrator is 0.9 µmol L-1 and 0.8 µmol L-1, respectively, which is comparable with their spectrophotometric determination.

A feasible fluorimetric approach anchored in diaryl pyrrolone derivative for the facile analysis of milnacipran in tablets; evaluation of the method greenness.

In the present work a new, feasible, and green approach was employed for the analysis of milnacipran. A drug is used in the management of depression in addition to fibromyalgia. It inhibits of the reuptake of two essential neurotransmitters serotonin and nor-adrenaline. In slightly alkaline buffer (pH 8.5) the primary amino group of milnacipran reacted with fluorescamine to give a substituted pyrrolone derivative which exhibited high fluorescence activity. The fluorescence of produced derivative was measured at (λex 385 nm, λem 477 nm), and the experimental factors were cautiously optimized. The measured intensity of fluorescence was plotted versus the respective concentration of milnacipran to setup the calibration plot which has a linear concentration range of 50-300 ng/mL. The ICH guidelines were utilized to totally validate the presented approach. In addition the method could be efficiently incorporated in the analysis of commercial milnacipram tablets with no considerable effect on the results of the assay for milnacipran. The developed approach is characterized by its high simplicity and greenness of the procedure which make it suitable for routine analysis.

Protective actions of bioactive flavonoids chrysin and luteolin on the glyoxal induced formation of advanced glycation end products and aggregation of human serum albumin: In vitro and molecular docking analysis.

The post-translational modification of proteins by nonenzymatic glycation (NEG) and the accumulation of AGEs are the two underlying factors associated with the long-term pathogenesis in diabetes. Glyoxal (GO) is a reactive intermediate which has the ability to modify proteins and generate AGEs at a faster rate. Human serum albumin (HSA) being the most abundant serum protein has a higher chance to be modified by NEG. The key objective of the present study is to investigate the potency of chrysin and luteolin as antiglycating and antifibrillating agents in the GO-mediated glycation and fibril formation of HSA. AGEs formation were confirmed from the absorption and fluorescence spectral measurements. Both the flavonoids were able to quench the AGEs fluorescence intensity in vitro indicating the antiglycating nature of the molecules. The formation of fibrils in the GO-modified HSA was confirmed by the Thioflavin T (ThT) fluorescence assay and the flavonoids were found to exihibit the antifibrillation properties in vitro. Docking results suggested that both the flavonoids interact with various amino acid residues of subdomain IIA including glycation prone lysines and arginines via non-covalent forces and further stabilized the structure of HSA, which further explains their mechanisms of action as antiglycating and antifibrillating agents.

The convenient use of fluorescamine for spectrofluorimetric quantitation of pramipexole in pure form and pharmaceutical formulation; application to content uniformity testing.

Pramipexole is a selective dopamine receptor agonist which is used in the treatment of Parkinson's disease and restless legs syndrome. The present work illustrates the development and validation of a sensitive and selective spectrofluorometric method for quantitation of pramipexole (PMP) through its interaction with fluorescamine at pH 7.5 using aqueous borate buffer to produce a highly fluorescent product. The fluorescent intensity of the formed product was measured at 480 nm after excitation at 391 nm. Experimental factors that could influence the formation, stability and the fluorescence intensity of the formed product were investigated and optimized. The linearity of the proposed method was achieved in the concentration range of 0.05-2.0 µg/mL. The quantitation and detection limits were 47 and 15 ng/mL, respectively. The proposed method has been validated in respect to guidelines of ICH and pharmaceutical tablets of PMP were successfully analyzed. Moreover, the method was applied for studying the content uniformity test according to the guidelines of United States Pharmacopeia.

Fluorescamine-based fluorophore for spectrofluorimetric determination of heptaminol in human plasma; application to spiked human plasma.

An innovative approach to determine heptaminol spectrofluorimetrically was developed, determining the optimum conditions needed, then validated for determination of heptaminol in its pure form, its tablets and in spiked human plasma. The presented method is based on the reaction between fluorescamine reagent with the primary amine group found in heptaminol, using a borate buffer at pH 9.0 that yields a highly fluorescent product, fluorescence was measured at 471 nm after excitation at 393 nm. The linearity of the constructed calibration curve was (75-850 ng/ml) with LOD and LOQ values 23.85 and 72.29 ng/ml respectively. The method was validated following the International Council for Harmonisation (ICH) guidelines indicating good accuracy and precision. Finally, the presented approach was adapted for in vitro study of heptaminol in spiked human plasma with a mean percentage recovery 100.52 ±â€¯1.19% as well as in its tablets with a mean percentage recovery 99.47 ±â€¯1.25%.

Determination of Sulfonamides in Feeds by High-Performance Liquid Chromatography after Fluorescamine Precolumn Derivatization.

A new multi-residue method for the analysis of sulfonamides (sulfadiazine, sulfamerazine, sulfamethazine, sulfaguanidine and sulfamethoxazole) in non-target feeds using high-performance liquid chromatography-fluorescence detection (HPLC-FLD) and precolumnderivatization was developed and validated. Sulfonamides (SAs) were extracted from feed with an ethyl acetate/methanol/acetonitrile mixture. Clean-up was performed on a Strata-SCX cartridge. The HPLC separation was performed on a Zorbax Eclipse XDB C18 column with a gradient mobile phase system of acetic acid, methanol, and acetonitrile. The method was validated according to EU requirements (Commission Decision 2002/657/EC). Linearity, decision limit, detection capability, detection and quantification limits, recovery, precision, and selectivity were determined, and adequate results were obtained. Using the HPLC-FLD method, recoveries were satisfactory (79.3⁻114.0%), with repeatability and reproducibility in the range of 2.7⁻9.1% to 5.9⁻14.9%, respectively. Decision limit (CCα) and detection capability (CCß) were 197.7⁻274.6 and 263.2⁻337.9 µg/kg, respectively, and limit of detection (LOD) and limit of quantification (LOQ) were 34.5⁻79.5 and 41.3⁻89.9 µg/kg, respectively, depending on the analyte. Results showed that this analytical procedure is simple, rapid, sensitive, and suitable for the routine control of feeds.

Optimisation of a high-throughput fluorescamine assay for detection of N-acyl-l-homoserine lactone acylase activity.

N-acyl-l-homoserine lactone (AHL) acylases are a well-known group of enzymes that disrupt quorum sensing in Gram-negative bacteria by degrading AHL signalling molecules. This degradation of signalling molecules (termed 'quorum quenching') has potential uses in the prevention or reduction of biofilm formation and/or bacterial infections. Therefore, there is a great deal of interest in the identification and characterisation of quorum quenching enzymes. Here, we present an optimised fluorescamine-based assay for the detection of AHL acylase activity and demonstrate it can be used in a high-throughput screening format.

The convenient use of fluorescamine for spectrofluorimetric determination of midodrine hydrochloride in pure form and its tablets formulation: Application to content uniformity testing.

A novel sensitive and simple spectrofluorimetric method was developed then validated for determination of midodrine in both its authentic pure form and its tablets. This method is based on the reaction between midodrine's aliphatic primary amine moiety with fluorescamine reagent, using borate buffer at pH 7.8 and yielding a highly fluorescent product whose fluorescence intensity was measured at 462 nm after excitation at 388 nm. This method represents the first attempt for determination of midodrine spectrofluorimetrically. A calibration curve was constructed showing that the linear range was 0.2-3.0 µg/ml. The limit of detection and limit of quantitation values were 0.06 and 0.19 µg/ml respectively. The correlation coefficient (r) and the determination coefficient (r2 ) values were 0.9992 and 0.9984 respectively. The proposed method was validated according to ICH guidelines and successfully applied for determination of midodrine in its tablets with an overall % recovery of 99.56 ± 0.95. Finally, the presented method was adapted to study the content uniformity test according to United States Pharmacopeia guidelines.

In-capillary derivatization with fluorescamine for the rapid determination of adamantane drugs by capillary electrophoresis with UV detection.

In-capillary derivatization using fluorescamine as the labeling reagent was proposed to enhance the detectability of adamantine drugs (memantine, amantadine and rimantadine) by spectrophotometric detection. Fluorescamine and the drugs were delivered to the capillary electrophoresis instrument at a ratio of 10:1 by zone injection. The derivatization reaction occurred following the application of voltage (20 kV). The derivatized products, hydrolyzed- fluorescamine and excess fluorescamine were separated in 7 min using 100 mM borate buffer (pH 10.0) containing 0.1% w/v of Brij®-35 and 20% v/v of acetonitrile. Validation data showed good linearity (r2  > 0.98), precision (%RSDs < 3.4), and accuracy (recoveries ranging from 98.0 to 102.0%). The detection and quantitation limits are in the range of 6.0-8.5 and 18-25 µM, respectively. The validation data is comparable to reported methods, however, the current method offers better precision with enhanced sensitivity (up to six times). Applications of the method show percent labeled amounts found in the studied samples within 100.6-109.3%, which complied with the United States Pharmacopeia limit (90.0-110.0%). The method was simple, rapid and, automated, which required no extra instrumentation or skillful operators.

Development of a dual luciferase activity and fluorescamine protein assay adapted to a 384 micro-well plate format: Reducing variability in human luciferase transactivation cell lines aimed at endocrine active substances.

There is a need to adapt cell bioassays to 384-well and 1536-well formats instead of the traditional 96-well format as high-throughput screening (HTS) demands increase. However, the sensitivity and performance of the bioassay must be re-verified in these higher micro-well plates, and verification of cell health must also be HT (high-throughput). We have adapted two commonly used human breast luciferase transactivation cell bioassays, the recently re-named estrogen agonist/antagonist screening VM7Luc4E2 cell bioassay (previously designated BG1Luc4E2) and the androgen/glucocorticoid screening MDA-kb2 cell bioassay, to 384-well formats for HTS of endocrine-active substances (EASs). This cost-saving adaptation includes a fast, accurate, and easy measurement of protein amount in each well via the fluorescamine assay with which to normalize luciferase activity of cell lysates without requiring any transfer of the cell lysates. Here we demonstrate that by accounting for protein amount in the cell lysates, antagonistic agents can easily be distinguished from cytotoxic agents in the MDA-kb2 and VM7Luc4E2 cell bioassays. Additionally, we demonstrate via the fluorescamine assay improved interpretation of luciferase activity in wells along the edge of the plate (the so-called "edge effect"), thereby increasing usable wells to the entire plate, not just interior wells.

Fluorescamine Labeling for Assessment of Protein Conformational Change and Binding Affinity in Protein-Nanoparticle Interaction.

Protein adsorption alters the "biological identity" of nanoparticles (NPs) and could affect how biosystems respond to invading NPs. Study of protein-NP interaction can help understand how the physicochemical properties of NPs impact the interaction and thus potentially guide the design of safer and more effective NPs for biomedical or other applications. Binding affinity between proteins and NPs and the occurrence of protein conformational change upon binding to NPs are two important aspects to be learned, but few methods are currently available to assess both simultaneously in a simple way. Herein, we demonstrated that the fluorescamine labeling method developed by our group not only could reveal protein conformational change upon adsorption to NPs, owing to its capability to label the primary amines exposed on protein surface, but also could be applied to measure the binding affinity. By screening the interaction between a large number of proteins and four types of NPs, the present study also revealed that protein adsorption onto NPs could be strongly affected by structure flexibility. The proteins with high structure flexibility experienced high degrees of conformation change when binding to the polystyrene NPs, which could potentially influence protein function. Overall, we demonstrate that our assay is a quick, simple, and high-throughput tool to reveal potential impacts on protein activity and evaluate the strength of protein-NP binding.

Multi-modal Binding of a 'Self' Peptide by HLA-B*27:04 and B*27:05 Allelic Variants, but not B*27:09 or B*27:06 Variants: Fresh Support for Some Theories Explaining Differential Disease Association.

A self-derived-peptide with the same amino acid sequence (N-RRYLENGKETLQR-C) as residues 169-181 of the human leukocyte antigen (HLA) B27 heavy chain is known to bind to MHC Class I complexes containing the HLA-B27 heavy chain. This observation has been invoked previously in at least two different (but related) molecular explanations for the disease-association of the HLA-B27 allele. Here, we use a combination of fluorescence polarization, competitive inhibition and gel filtration chromatographic studies to show that a fluorescently-labeled peptide of the above sequence binds to two disease-associated subtypes of HLA-B27 (namely HLA-B*27:04 and HLA-B*27:05) but not to non-disease-associated subtypes (HLA-B*27:06 or HLA-B*27:09). This differential binding behavior is seen both in (a) peptide binding to complexes of heavy chain (HLA-B27) and light chain (ß2 microglobulin), and in (b) peptide binding to ß2 microglobulin-free heavy chains in the aggregated state. Such subtype-specific differences are not seen with two other control peptides known to bind to HLA-B27. Our results support the likelihood of differential peptide binding holding at least one of the keys to HLA-B27's disease association.

Fluram-Kemptide-Lys8 Non-radioactive Assay for Protein Kinase A.

The cAMP-dependent protein kinase (PKA) is the best understood member of the superfamily of serine-threonine protein kinases and is involved in controlling a variety of cellular processes. Measurements of PKA activity traditionally relied on the use of [(32)P]-labeled ATP as the phosphate donor and a protein or peptide substrate as the phosphoaceptor. Recently non-isotopic assays for the PKA have been developed and this paper presents an improvement of a fluorometric assay for measuring the activity of PKA. Three peptides were synthesized with the following sequences: LRRASLG (Kemptide), LRRASLGK (Kemptide-Lys8) and LRRASLGGGLRRASLG (Bis-Kemptide), these have in common the substrate sequence recognized by the PKA (RRXS/TΨ), where X is any amino acid and Ψ is a hydrophobic amino acid. Optimal conditions were established for the non-radioactive assay to detect the PKA activity by phosphorylation of these three peptides that are covalently linked to fluorescamine at their N-terminus. The phosphorylated and non-phosphorylated peptides were easily separated by electrophoresis, identified and quantified with optical densitometry and ultraviolet light. The fluorescamine-labeled Kemptide-Lys8 substrate (Fluram-Kemptide-Lys8) was used to calculate the Km and Vmax of the catalytic subunit of PKA from pig heart and showed a detection limit of 260 pmol, a linear range between 700 and 1150 pmol with a linear regression R (2) = 0.956.

Phase Transfer and Surface Functionalization of Hydrophobic Nanoparticle using Amphiphilic Poly(amino acid).

Functionalization of nanoparticles with chemical and biochemical is essential for their biomedical and other application. However, most of the high quality nanoparticles are hydrophobic in nature due to surfactant capping and their conversion into water-soluble functional nanoparticle via appropriate coating and conjugation chemistry is extremely critical issue. Here we report amphiphilic poly(amino acid)-based one-pot coating and conjugation approach that can transform hydrophobic nanoparticle into water-soluble nanoparticle functionalized with primary amine, thiol, and biomolecule. We have designed amphiphilic polyaspartimide that can anchor hydrophobic nanoparticle through octadecyl groups, leaving the polar polyethylene glycol and aspartimide groups exposed outwards. The aspartimide group is then reacted with primary amine containing chemical/biomolecule with the formation of water-soluble functional nanoparticle. This approach has been extended to different hydrophobic nanoparticles and biomolecules. The present approach has advantages over existing approaches as coating and functionalization can be performed in one pot and functional nanoparticles have <12 nm hydrodynamic size, high colloidal stability, and biocompartibility. This developed approach can be used to derive biocompatible nanobioconjugates for various biomedical applications.

KDAC8 with High Basal Velocity Is Not Activated by N-Acetylthioureas.

Lysine deacetylases (KDACs) are enzymes that reverse the post-translational modification of lysine acetylation. Recently, a series of N-acetylthioureas were synthesized and reported to enhance the activity of KDAC8 with a fluorogenic substrate. To determine if the activation was general, we synthesized three of the most potent N-acetylthioureas and measured their effect with peptide substrates and the fluorogenic substrate under multiple reaction conditions and utilizing two enzyme purification approaches. No activation was observed for any of the three N-acetylthioureas under any assayed conditions. Further characterization of KDAC8 kinetics with the fluorogenic substrate yielded a kcat/KM of 164 ± 17 in the absence of any N-acetylthioureas. This catalytic efficiency is comparable to or higher than that previously reported when KDAC8 was activated by the N-acetylthioureas, suggesting that the previously reported activation effect may be due to use of an enzyme preparation that contains a large fraction of inactive enzyme. Further characterization with a less active preparation and additional substrates leads us to conclude that N-acetylthioureas are not true activators of KDAC8 and only increase activity if the enzyme preparation is below the maximal basal activity.

Highly sensitive spectrofluorimetric method for determination of doxazosin through derivatization with fluorescamine; Application to content uniformity testing.

A highly sensitive, simple and selective spectrofluorimetric method has been developed and validated for determination of doxazosin mesylate in pure form, pharmaceutical formulations and human plasma. The method is based on the reaction between doxazosin mesylate and fluorescamine in Teorell buffer solution (pH 3) to give highly fluorescent derivative that can be measured at 489 nm using excitation wavelength of 385 nm. Different experimental parameters affecting the reaction were carefully studied and optimized. The calibration plot was constructed over the concentration range of 16-400 ng mL(-1) with quantitation limit of 14.3 ng mL(-1). The developed procedure was validated according to ICH guidelines and the results were satisfactory. The proposed method has been successfully applied to the analysis of the cited drug in its pharmaceutical preparations as well as for content uniformity testing. The results showed excellent agreement with the reported method with respect to precision and accuracy. In addition, the drug concentration was determined in the spiked human plasma by the suggested method with % recovery in the range of 96.2-98.3% (SD; 0.76-0.93, n=5).

KDAC8 substrate specificity quantified by a biologically relevant, label-free deacetylation assay.

Analysis of the human proteome has identified thousands of unique protein sequences that contain acetylated lysine residues in vivo. These modifications regulate a variety of biological processes and are reversed by the lysine deacetylase (KDAC) family of enzymes. Despite the known prevalence and importance of acetylation, the details of KDAC substrate recognition are not well understood. While several methods have been developed to monitor protein deacetylation, none are particularly suited for identifying enzyme-substrate pairs of label-free substrates across the entire family of lysine deacetylases. Here, we present a fluorescamine-based assay which is more biologically relevant than existing methods and amenable to probing substrate specificity. Using this assay, we evaluated the activity of KDAC8 and other lysine deacetylases, including a sirtuin, for several peptides derived from known acetylated proteins. KDAC8 showed clear preferences for some peptides over others, indicating that the residues immediately surrounding the acetylated lysine play an important role in substrate specificity. Steady-state kinetics suggest that the sequence surrounding the acetylated lysine affects binding affinity and catalytic rate independently. Our results provide direct evidence that potential KDAC8 substrates previously identified through cell based experiments can be directly deacetylated by KDAC8. Conversely, the data from this assay did not correlate well with predictions from previous screens for KDAC8 substrates using less biologically relevant substrates and assay conditions. Combining results from our assay with mass spectrometry-based experiments and cell-based experiments will allow the identification of specific KDAC-substrate pairs and lead to a better understanding of the biological consequences of these interactions.

High-throughput profiling of nanoparticle-protein interactions by fluorescamine labeling.

A rapid, high throughput fluorescence assay was designed to screen interactions between proteins and nanoparticles. The assay employs fluorescamine, a primary-amine specific fluorogenic dye, to label proteins. Because fluorescamine could specifically target the surface amines on proteins, a conformational change of the protein upon interaction with nanoparticles will result in a change in fluorescence. In the present study, the assay was applied to test the interactions between a selection of proteins and nanoparticles made of polystyrene, silica, or iron oxide. The particles were also different in their hydrodynamic diameter, synthesis procedure, or surface modification. Significant labeling differences were detected when the same protein incubated with different particles. Principal component analysis (PCA) on the collected fluorescence profiles revealed clear grouping effects of the particles based on their properties. The results prove that fluorescamine labeling is capable of detecting protein-nanoparticle interactions, and the resulting fluorescence profile is sensitive to differences in nanoparticle's physical properties. The assay can be carried out in a high-throughput manner, and is rapid with low operation cost. Thus, it is well suited for evaluating interactions between a larger number of proteins and nanoparticles. Such assessment can help to improve our understanding on the molecular basis that governs the biological behaviors of nanomaterials. It will also be useful for initial examination of the bioactivity and reproducibility of nanomaterials employed in biomedical fields.

Chitosan coated polylactic acid nanoparticle-mediated combinatorial delivery of cisplatin and siRNA/Plasmid DNA chemosensitizes cisplatin-resistant human ovarian cancer cells.

Development of resistance toward anticancer drugs results in ineffective therapy leading to increased mortality. Therefore, overriding resistance and restoring sensitivity to anticancer drugs will improve treatment efficacy and reduce mortality. While numerous mechanisms for drug resistance in cancer have previously been demonstrated, recent studies implicate a role for proteasome and the autophagy regulatory protein P62/SQSTM1 (P62) in contributing to drug resistance. Specifically, reduction in the expression of the ß5 subunit of the proteasome and/or enhanced P62 protein expression is known to contribute to cancer drug resistance such as cisplatin (CDDP) in ovarian cancer cells. Therefore, we hypothesized that restoration of ß5 expression and/or suppression of P62 protein expression in CDDP-resistant ovarian cancer cells will lead to restoration of sensitivity to CDDP and enhanced cell killing. To test our hypothesis we developed a biodegradable multifunctional nanoparticle (MNP) system that codelivered P62siRNA, ß5 plasmid DNA, and CDDP and tested its efficacy in CDDP resistant 2008/C13 ovarian cancer cells. MNP consisted of CDDP loaded polylactic acid nanoparticle as inner core and cationic chitosan (CS) consisting of ionically linked P62siRNA (siP62) and/or ß5 expressing plasmid DNA (pß5) as the outer layer. The MNPs were spherical in shape with a hydrodynamic diameter in the range of 280-350 nm, and demonstrated encapsulation efficiencies of 82% and 78.5% for CDDP and siRNA respectively. MNPs efficiently protected the siRNA and showed superior serum stability compared to naked siRNA as measured by gel retardation and spectrophotometry assays. The MNPs successfully delivered siP62 and pß5 to cause P62 knockdown and restoration of ß5 expression in 2008/C13 cells. Combined delivery of siP62, pß5, and CDDP using the MNPs resulted in a marked reduction in the IC50 value of CDDP in 2008/C13 cells from 125 ± 1.3 µM to 98 ± 0.6 µM (P < 0.05; 21.6% reduction) when compared to the reduction in the IC50 of CDDP observed in cells that had only siP62 delivered (IC50 = 106 ± 1.1 µM; P < 0.05; 15.2% reduction) or pß5 delivered (IC50 = 115 ± 2.8 µM; 8% reduction) via MNPs. Finally, our studies showed that the CDDP resistance index in 2008/C13 cells was reduced from 4.62 for free CDDP to 3.62 for MNP treatment. In conclusion our study results demonstrated the efficacy of our MNP in overcoming CDDP resistance in ovarian cancer cells.

A lab-on-a-chip device for analysis of amlodipine in biological fluids using peroxyoxalate chemiluminescence system.

A highly sensitive, rapid and economical method for the determination of amlodipine (AM) in biological fluids was developed using a peroxyoxalate chemiluminescence (CL) system in a lab-on-a-chip device. Peroxyoxalate-CL is an indirect type of CL that allows the detection of native fluorophores or compounds derivatized with fluorescent labels. Here, fluorescamine was reacted with AM, and the derivatization product was used in a bis-(2,4,6-trichlorophenyl)oxalate-CL system. Fluorescamine reacts selectively with aliphatic primary amine at neutral or basic pH. As most of the calcium channel blocker and many cardiovascular drugs do not contain primary amine, the developed method is highly selective. The parameters that influenced the CL signal intensity were studied carefully. These included the chip geometry, pH, concentration of reagents used and flow rates. Moreover, we confirmed our previous observation about the effects of imidazole, which is commonly used in the bis-(2,4,6-trichlorophenyl)oxalate-CL system as a catalyst, and found that the signal was significantly improved when imidazole was absent. Under optimized conditions, a calibration curve was obtained with a linear range (10-100 µg/L). The limit of detection was 3 µg/L, while the limit of quantification was 10 µg/L. Finally the method was applied for the determination of AM in biological fluids successfully.