Determination of amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxyethylamphetamine, and 3,4-methylenedioxymethamphetamine in urine by online solid-phase extraction and ion-pairing liquid chromatography with detection by electrospray tandem mass spectrometry

A method using an online solid-phase extraction (SPE) and ion-pairing liquid chromatography with electrospray tandem mass spectrometry (LC/ES-MS/MS) was developed for determination of amphetamine (Amp), methamphetamine (mAmp), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxyethylamphetamine (MDEA), and 3,4-methylenedioxymethamphetamine (MDMA) in urine samples. A SPE cartridge column with both hydrophilic and lipophilic functions was utilized for online extraction. A reversed-phase C18 LC column was employed for LC separation and MS/MS was used for detection. Trifluoroacetic acid was added to the mobile phase as an ion-pairing reagent. This method was fully automated and the extraction and analysis procedures were controlled by a six-port switch valve. Recoveries ranging from 85-101% were measured. Good linear ranges (10-500 ng/mL) for Amp and mAmp were determined. For MDA, MDMA and MDEA, dual linear ranges were obtained from 5-100 and 100-500 ng/mL, respectively. The detection limit of each analytical compound, based on a signal-to-noise ratio of 3, ranged from 1-3 ng/mL. The applicability of this newly developed method was examined by analyzing several urine samples from drug users. Good agreement was obtained between the results from this method and a literature GC/MS method.     

Hydrophilic interaction chromatography combined with tandem-mass spectrometry to determine six aminoglycosides in serum

An ion-pair liquid chromatography-electrospray mass spectrometry (LC-ESI-MS) method with in vivo microdialysis for the determination of free-form amphetamine in rat brain has been developed. A microdialysis probe was surgically implanted into the striatum of the rat and artificial cerebrospinal fluid (aCSF) was used as the perfusion medium. Samples were collected and then analyzed off-line by LC-ESI-MS. A reversed phase C18 column was employed for LC separation. Trifluoroacetic acid (TFA) was added in the mobile phase (acetonitrile-water, 10:90, v/v) as an ion-pair reagent. The ion-pair process disguises the protonated amphetamine cations from the ESI-MS electric field as neutral molecules. Post-column addition of volatile organic acid was utilized to minimize TFA signal suppression effect on ESI-MS detection. More than six-fold enhancement of ESI-MS response was achieved by the post-column addition of propionic acid. Good linearity (0.01-1.00 microg/ml, r2 = 0.99) and detection limit (0.002 microg/ml) were determined. Good precision and accuracy were obtained. The applicability of this newly developed method was demonstrated by continuous monitoring of amphetamine concentrations in rat brain after a single 3.0 mg/kg i.p. administration.     

Quantitative determination of acetylcholine in microdialysis samples using liquid chromatography/atmospheric pressure spray ionization mass spectrometry

A fast, simple and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the determination of acetylcholine in rat brain microdialysis samples. The chromatographic separation was achieved in 3 min on a reversed-phase column with isocratic conditions using a mobile phase containing 2% (v/v) of acetonitrile and 0.05% (v/v) of trifluoroacetic acid (TFA). A stable isotope-labeled internal standard was included in the analysis and detection was carried out with a linear ion trap mass spectrometer using selected reaction monitoring (SRM). Analyte ionization was performed with an atmospheric pressure chemical ionization (APCI) source without applying discharge current (atmospheric pressure spray ionization). This special ionization technique offered significant advantages over electrospray ionization for the analysis of acetylcholine with reversed-phase ion-pairing chromatography. The lower limit of quantification was 0.15 nM (1.5 fmol on-column) and linearity was maintained over the range of 0.15-73 nM, providing a concentration range that is significantly wider than that of the existing LC/MS methods. Good accuracy and precision were obtained for concentrations within the standard curve range. The method was validated and has been used extensively for the determination of acetylcholine in rat brain microdialysis samples.     

Quantification of rat brain neurotransmitters and metabolites using liquid chromatography/electrospray tandem mass spectrometry and comparison with liquid chromatography/electrochemical detection

Analytical methodology based on solid-phase extraction, polar reversed-phase liquid chromatography, and electrospray tandem mass spectrometry (LC/MS/MS) with isotope dilution was developed and validated for quantifying the neurotransmitters, dopamine and serotonin, and their major metabolites in brain tissue. Limits of detection (0.1-20 pg/mg tissue) were sufficient for analysis of multiple neurotransmitters in rat brain regions, including parietal cortex, hypothalamus, pituitary, substantia nigra, and striatum. Method performance was compared with contemporaneous measurements using a well-established procedure based on ion-pairing reversed-phase liquid chromatography and amperometric detection. The principal advantages of the LC/MS/MS method include a more robust sample purification procedure, an optimized chromatographic separation, and the qualitative and quantitative assurance that comes from coeluting isotopically labeled internal standards; however, sensitivity did not consistently improve upon that provided by amperometric detection. This methodology may be particularly useful for applications in which simultaneous determinations are required for drugs and their affected neurotransmitters in specific brain regions.     

A sensitive, rapid and specific determination of midazolam in human plasma and saliva by liquid chromatography/electrospray mass spectrometry

A rapid, sensitive and selective liquid chromatography/electrospray mass spectrometry (LC/ES-MS) method was developed for the quantitative determination of the anaesthetic benzodiazepine midazolam (MID) in human saliva and plasma from patients undergoing anesthesia procedures. Biological samples spiked with diazepam-d5, the internal standard, were extracted into diethyl ether. Compounds were separated on a Xterra RP18 column using a mobile phase of acetonitrile/formic acid 0.1% at a flow rate of 0.25 mL/min under a linear gradient. Column effluents were analyzed using MS with an ES source in the positive ionization mode. Calibration curves were linear in the concentration ranges of 1-250 and 0.2-25 ng/mL in plasma and saliva, respectively. The limits of detection were 0.5 ng/mL in plasma and 0.1 ng/mL in saliva, using a 0.5-mL sample volume. The recoveries of the spiked samples were above 65%. The method was applied to ten real samples from patients undergoing midazolam treatment.     

High-throughput determination of carbocysteine in human plasma by liquid chromatography/tandem mass spectrometry: application to a bioequivalence study of two formulations in healthy volunteers

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method to determine carbocysteine in human plasma was developed and fully validated. After methanol-induced protein precipitation of the plasma samples, carbocysteine was subjected to LC/MS/MS analysis using electrospray ionization (ESI). The MS system was operated in the selected ion monitoring (SRM) mode. Chromatographic separation was performed on a Hypurity C18 column (i.d. 2.1 mm x 50 mm, particle size 5 microm). The method had a chromatographic running time of 2.0 min and linear calibration curves over the concentration ranges of 0.1-20 microg/mL for carbocysteine. The lower limit of quantification (LLOQ) of the method was 0.1 microg/mL for carbocysteine. The intra- and inter-day precision was less than 7% for all quality control samples at concentrations of 0.5, 2.0, and 10.0 microg/mL. These results indicate that the method was efficient with a simple preparation procedure and a very short running time (2.0 min) for carbocysteine compared with methods reported in the literature and had high selectivity, acceptable accuracy, precision and sensitivity. The validated LC/MS/MS method has been successfully used to a bioequivalence study of two tablet formulations of carbocysteine in healthy volunteers.     

Determination of chloramphenicol residues in bee pollen by liquid chromatography/tandem mass spectrometry

A novel liquid chromatographic/tandem mass spectrometric (LC/MS/MS) method was developed for the trace residue determination of chloramphenicol (CAP) in bee pollen. CAP was extracted from bee pollen with a mixture of methanol and 1% metaphosphoric acid solution, followed by a 2-stage solid-phase extraction enrichment and cleanup. The first stage involved a polymeric cartridge, and the second stage involved an alumina neutral cartridge. The LC separation was performed on a C18 column with 10 mM ammonium formate-acetonitrile (7 + 3) as the mobile phase and MS detection with negative-ion electrospray ionization. CAP-d5 was used as the internal standard. The method was validated according to Commission Decision 2002/657/EC. The calibration curves were linear between 0.1 and 5.0 ng/mL, and overall recoveries ranged from 98 to 113%. Decision limits (CCalpha) ranged from 0.05 to 0.07 microg/kg, and detection capabilities (CCbeta) ranged from 0.08 to 0.12 microg/kg. The developed method was applied to 11 samples.     

Determination of glufosfamide in rat plasma by liquid chromatography/tandem mass spectrometry

A sensitive and selective high-performance analytical method based on liquid chromatography with tandem mass spectrometric detection (LC/MS/MS) was developed for the quantification of glufosfamide in rat plasma. Zidovudine was employed as internal standard. Glufosfamide was determined after methanol-mediated plasma protein precipitation using LC/MS/MS with an electrospray ionization interface in negative ion mode. Two sets of standard curves were developed, from 0.005 to 1.0 microg/mL and from 1.0 to 50.0 microg/mL. The assay was accurate (% deviations from nominal concentrations < 5%), precise and reproducible (intra- and inter-day coefficients of variation < 10%). Glufosfamide in rat plasma was stable over three freeze/thaw cycles, and at ambient temperatures, for at least 2 h. The validated method was successfully applied to the determination of glufosfamide plasma concentrations in rats for 24 h following an intravenous administration of 25 mg/kg.     

Quantification of cyclophosphamide and its metabolites in urine using liquid chromatography/tandem mass spectrometry

A reliable and easy to use liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the simultaneous quantification of urinary concentrations of cyclophosphamide (CP) and its main metabolites excreted in urine, i.e. N-dechloroethylcyclophosphamide (DCL-CP), 4-ketocyclophosphamide (4KetoCP), and carboxyphosphamide (CarboxyCP). Sample preparation consisted of dilution of urine with an aqueous solution of the internal standard D(4)-CP and methanol, and centrifugation. LC/MS/MS detection was performed using a triple-quadrupole mass spectrometer working in selected reaction monitoring mode. All analytes were quantified in a single run within 11.5 min. The limits of detection were 5 ng/mL for CP and 4KetoCP, 1 ng/mL for DCL-CP, and 30 ng/mL for CarboxyCP. Quantification ranges were adjusted to the expected concentrations in 24-h urine collections of patients treated with a polychemotherapy regimen (3-175 microg/mL for CP, 0.5-27 microg/mL for 4KetoCP and 0.17-9 microg/mL for CarboxyCP and DCL-CP, respectively). The method was validated according to international guidelines of the ICH and the FDA.     

Sensitive and simultaneous determination of HIV protease inhibitors in rat biological samples by liquid chromatography-mass spectrometry

A sensitive and simultaneous liquid chromatographic-mass spectrometric (LC/MS) method for the determination of current four HIV protease inhibitors (PIs), indinavir (IDV), saquinavir (SQV), nelfinavir (NFV) and amprenavir (APV) in rat plasma and liver dialysate by a microdialysis method was described. An isocratic LC/MS method in combination with atmospheric pressure chemical ionization was developed for the determination of these four PIs in biological samples in the same run. The analytes including an internal standard were extracted from 100 microL of plasma or 150 microL of liver dialysate samples by salting-out with 100 microL of ice-cold 2 M K(3)PO(4) followed by ether extraction. The separation of analytes was carried out on a reversed-phase semi-micro column using 50% of acetonitrile containing 1% acetic acid as mobile phase at a flow rate of 0.2mL/min(-1). The separation was completed within 5 min. Precision, recovery and limits of detection indicated that the method was suitable for the quantitative determination of these PIs in rat plasma or liver dialysate. This simple, sensitive and highly specific LC/MS method is suitable for pharmacokinetic studies and therapeutic drug monitoring in AIDS patients who receive double protease therapy.     

Validation of liquid chromatography and liquid chromatography/tandem mass spectrometry methods for the determination of etoricoxib in pharmaceutical formulations

Reversed-phase liquid chromatography (LC) and LC/tandem mass spectrometry (LC/MS/MS) methods were developed and validated for the determination of etoricoxib in pharmaceutical dosage forms. The LC method was performed by reversed-phase chromatography on a Synergi fusion C18 column (150 x 4.6 mm id) maintained at ambient temperature. The mobile phase consisted of 0.01 M phosphoric acid, pH 3.0-acetonitrile (62 + 38, v/v) at a flow rate of 1.0 mL/min, and photodiode array detection at 234 nm was used. The chromatographic separation was obtained within 7.0 min, and calibration curves were linear in the concentration range of 0.02-150 microg/mL. The LC/MS/MS method was performed on a Luna C18 column (50 x 3.0 mm id). The mobile phase consisted of acetonitrile-water (95 + 5)-0.1% acetic acid (90 + 10, v/v). Detection was performed by positive electrospray ionization in the multiple reaction monitoring mode, monitoring the transitions 359.3 > 280.0 and 332.0 > 95.0 for etoricoxib and piroxicam (internal standard), respectively. The chromatographic separation was obtained within 2.0 min, and calibration curves were linear in the concentration range of 1-5000 ng/mL. Validation parameters, such as specificity, linearity, precision, accuracy, and robustness, were evaluated, which gave results within the acceptable range for both methods. Moreover, the proposed methods were successfully applied for routine quality control analysis of pharmaceutical products and showed significant correlation (r = 0.9999) of the results.     

In vivo microdialysis and reverse phase ion pair liquid chromatography/tandem mass spectrometry for the determination and identification of acetylcholine and related compounds in rat brain

A method using liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been developed for the determination of basal acetylcholine (ACh) in microdialysate from the striatum of freely moving rats. A microdialysis probe was surgically implanted into the striatum of the rats and Ringer's solution was used as the perfusion medium at a flow rate of 2 microL per minute. The samples were then analyzed off-line by LC/MS/MS experiments. The separation of ACh and choline (Ch) was carried out using reverse phase ion pair liquid chromatography with heptafluorobutyric acid as a volatile ion pairing reagent. Analytes were detected by electrospray ionization tandem mass spectrometry in the positive ion mode. The detection limit for ACh was 1.4 fmol on column, which is at least three times lower than previously reported. Three quaternary ammonium compounds in the rat brain microdialysate were also identified by tandem mass spectrometry experiments in which the unknown mass spectra were compared with standard reference compounds. These compounds were identified as carnitine, acetylcarnitine and (3-carboxypropyl)trimethylammonium. This is the first known report of the compound (3-carboxypropyl)trimethylammonium being found in rat brain.     

Determination of iodoacetic acid using liquid chromatography/electrospray tandem mass spectrometry

A rapid analytical method based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) using electrospray ionization in negative ion detection mode was developed for the analysis of underivatized iodoacetic acid in water. The method was applied to model reaction mixtures in the study of the formation of iodoacetic acid after chlorinated tap water was boiled in the presence of potassium iodide or iodized table salt. Samples can be directly analyzed by the LC/MS/MS system without extraction or chemical derivatization. Limit of detection was determined to be 0.3 microg/L (or 0.3 ng/mL) and limit of quantitation was about 1 microg/L (1 ng/mL).     

Liquid chromatography with electrospray ion-trap mass spectrometry for the determination of anatoxins in cyanobacteria and drinking water

Anatoxin-a (AN) and homoanatoxin-a (HMAN) are potent neurotoxins produced by a number of cyanobacterial species. A new, sensitive liquid chromatography/multiple tandem mass spectrometry (LC/MS(n)) method has been developed for the determination of these neurotoxins. The LC system was coupled, via an electrospray ionisation (ESI) source, to an ion-trap mass spectrometer in positive ion mode. The [M+H](+) ions at m/z 166 (anatoxin-a) and m/z 180 (homoanatoxin-a) were used as the precursor ions for multiple MS experiments. MS(2)bond;MS(4) spectra displayed major fragment ions at m/z 149 (AN), 163 (HMAN), assigned to [Mbond;NH(3)+H](+); m/z 131 (AN), 145 (HMAN), assigned to [Mbond;NH(3)bond;H(2)O+H](+), and m/z 91 [C(7)H(7)](+). Although the chromatographic separation of these neurotoxins is problematic, reversed-phase LC, using a C(18) Luna column, proved successful. Calibration data for anatoxin-a using spiked water samples (10 mL) in LC/MS(n) modes were: LC/MS (25-1000 microg/L), r(2) = 0.998; LC/MS(2) (5-1000(microg/L), r(2) = 0.9993; LC/MS(3) (2.5-1000 microg/L), r(2) = 0.9997. Reproducibility data (% RSD, N = 3) for each LC/MS(n) mode ranged between 2.0 at 500 microg/L and 7.0 at 10 microg/L. The detection limit (S/N = 3) for AN was better than 0.03 ng (on-column) for LC/MS(3) which corresponded to 0.6 microg/L.     

Determination of amphetamine and methamphetamine in urine by solid phase extraction and ion-pair liquid chromatography-electrospray-tandem mass spectrometry

A method using a solid phase extraction (SPE) and ion-pair liquid chromatography-electrospray-tandem mass spectrometry (LC-ES-MS/MS) was developed for determination of amphetamine (Amp) and methamphetamine (mAmp) in urine samples. A reversed phase C₁₈ column was utilized for LC separation and MS/MS was used for detection. Trifluoroacetic acid was added to the mobile phase as an ion-pairing reagent. MS² was employed for quantitative determination. In addition, d₈-amphetamine and d₈-methamphetamine were used as internal standards. An Oasis HLB SPE cartridge, which has hydrophilic and lipophilic functions, was utilized for sample pre-treatment. Recoveries ranging from 97.3 to 102.1% were measured. Good linear ranges, 5-500 ng/ml, for Amp and mAmp were determined. The detection limit of each analytical compound, based on a signal-to-noise ratio of 3, was approximately 1 ng/ml. The applicability of this newly developed method was examined by analyzing several urine samples from drug users.     

Analysis of acetylcholine and choline in microdialysis samples by liquid chromatography/tandem mass spectrometry

A sensitive liquid chromatography/electrospray ionisation tandem mass spectrometric (LC/ESI-MS/MS) method was developed for the analysis of acetylcholine and choline in microdialysis samples. A Ringer's solution that contains high (150 mM) concentrations of inorganic salts was used to extract acetylcholine and choline from a rat or mouse brain. The separation of acetylcholine, choline, an internal standard acetyl-beta-methylcholine, endogenous compounds and inorganic cations was achieved with hydrophilic interaction chromatography using a diol column. The eluent consisted of 20 mM ammonium formate (pH 3.3) and acetonitrile (20:80) which is favourable for the ESI process. Limits of detection (signal-to-noise (S/N) ratio = 3) of 0.02 nM (0.2 fmol) for acetylcholine and 1 nM (10 fmol) for choline were observed using standards diluted in Ringer's solution. A good linearity was obtained from the limit of quantitation: 0.1 nM (S/N ratio = 10) to 50 nM (r = 0.999) for acetylcholine and within the concentration range of 100-3500 nM (r = 0.998) for choline. The between-day repeatability of the method was good; RSD was 3.1% at 1 nM level of acetylcholine and 3.5% at 1000 nM level of choline. The recoveries for addition of 1 or 2.5 nM acetylcholine and 0.2 or 1 microM choline in microdialysis balancing samples were between 93 and 101% indicating that no suppressing endogenous compounds were co-eluting with acetylcholine or choline. The developed method was applied to the analysis of microdialysis balancing samples collected from rat and mouse brains.     

Determination of phenothiazines in human body fluids by solid-phase microextraction and liquid chromatography/tandem mass spectrometry

Eleven phenothiazine derivatives with heavy side-chains were found to be extractable from human whole blood and urine samples by solid-phase microextraction (SPME) with a polyacrylate-coated fiber. The fiber was then injected into the desorption chamber of an SPME-liquid chromatography (LC) interface for LC/tandem mass spectrometry (MS/MS) with positive ion electrospray (ES) ionization. All compounds formed base peaks due to [M + 1](+) ions by LC/ES-MS/MS. By use of LC/ES-MS/MS, the product ions produced from each [M + 1](+) ion showed base peaks due to side-chain liberation. Selected reaction monitoring (SRM) and selected ion monitoring (SIM) were compared for the detection of the 11 phenothiazine derivatives from human whole blood and urine. SRM showed much higher sensitivity than SIM for both types of sample. Therefore, a detailed procedure for the detection of drugs by SRM with SPME-LC/MS/MS was established and carefully validated. The extraction efficiencies of the 11 phenothiazine derivatives spiked into whole blood and urine were 0. 0002-0.12 and 2.6-39.8%, respectively. The regression equations for the 11 phenothiazine derivatives showed excellent linearity with detection limits of 0.2-200 ng ml(-1) for whole blood and 4-22 pg ml(-1) for urine. The intra- and inter-day precisions for whole blood and urine samples were not greater than 15.1%. The data obtained after oral administration of perazine or flupentixol to a male subject are presented.     

Development of a novel HPLC‐MS/MS method for the determination of aconitine and its application to in vitro and rat microdialysis samples

A sensitive and selective LC‐MS/MS method was developed and validated for the determination of aconitine in microdialysate and rat plasma. Extraction of plasma sample was conducted by use of 1% trichloracetic acid and acetonitrile solution with 10 ng/mL internal standard (propafenone) spiked. Microdialysates were analyzed without sample purification. After sample preparation, 2 µL were injected and separated with an isocratic mobile phase consisting of acetonitrile:0.1% formic acid (60:40, v/v) at a flow rate of 0.3 mL/min. The Agilent G6410A triple quadrupole LC/MS system was operated under the multiple‐reaction monitoring mode (MRM) using the electrospray ionization technique in positive mode. Overall, the assay exhibited good precision and accuracy. The diffusion properties of aconitine investigated in in vitro microdialysis experiments revealed unfavourable concentration dependence avertable by keeping a constant pH 5.77 using isotonic phosphate buffer solution as perfusate. The mean relative recoveries were 48.23% [coefficient of variation (CV 4.47%)] and 55.38% (CV 2.89%) for retrodialysis and recovery experiments, respectively. The in vivo recovery of aconitine was 34.48% (CV 3.05%) and was stable over the 6 h study period. Following characterization of aconitine both in vitro and in vivo microdialysis, the developed setting is suitable for application in pharmacokinetics and pharmacodynamics studies. Copyright © 2009 John Wiley & Sons, Ltd.     

Quantification of lurasidone, an atypical antipsychotic drug, in rat plasma with high-performance liquid chromatography with tandem mass spectrometry

A liquid chromatography–tandem mass spectrometric (LC/MS/MS) method was developed for the determination of an atypical antipsychotic drug, lurasidone, in rat plasma. The method involves the addition of acetonitrile and ziprasidone (internal standard) solution to plasma samples, followed by centrifugation. An aliquot of the supernatant was diluted with water and directly injected into the LC/MS/MS system. The separations were performed on a column packed with octadecylsilica (5 μm, 2.0 × 50 mm) with 0.1% formic acid and 0.1% formic acid in acetonitrile as mobile phase and the detection was performed using tandem mass spectrometry by multiple‐reaction monitoring via an electrospray ionization source. The standard curve was linear (r = 0.9982) over the concentration range 0.002–1 μg/mL. The intra‐ and inter‐assay precisions were 1.7 and 8.6%, respectively. The accuracy range was from 90.3 to 101.8%. The lower limit of quantification was 2.0 ng/mL using 50 μL of rat plasma sample. The developed analytical method was successfully applied to the pharmacokinetic study of lurasidone in rats. Copyright © 2011 John Wiley & Sons, Ltd.     

Enhancing capillary liquid chromatography/tandem mass spectrometry of biogenic amines by pre-column derivatization with 7-fluoro-4-nitrobenzoxadiazole

This paper describes a capillary liquid chromatography/tandem mass spectrometry (LC/MS/MS) determination of biogenic amines enhanced by pre-column derivatization with 7-fluoro-4-nitrobenzoxadiazole (NBD-F). Biogenic amines including tryptamine, N-methylsalsolinol, histamine, and agmatine were studied. The biogenic NBD-amine derivatives could be quantitatively enriched in-line on 20 x 0.25 mm capillary columns packed in-house with 5 microm C(8) silica particles. In an electrospray ionization (ESI) source these derivatives were ionized effectively, and collision-induced dissociation (CID) produced predominant characteristic ions allowing sensitive MS/MS detection. Agmatine, a potential neurotransmitter/modulator, was taken as a reference compound to study the analytical figures of merit of the procedure. The detection limit of agmatine was estimated to be 0.6 ng/mL (signal-to-noise (S/N) = 3). A linear calibration curve in the range 15-1000 ng/mL agmatine with an r value of 0.9997 was obtained. Tissue samples of rat brain, stomach, and intestine were analyzed. Minimum sample pre-treatment was needed. Each analysis was accomplished within ca. 12 min. The concentration of agmatine was found to be 0.246, 3.31, and 0.058 microg/g wet tissue in the brain, stomach, and intestine, respectively.