Chemical derivatization strategies for enhancing the HPLC analytical performance of natural active triterpenoids

Triterpenoids widely exist in nature,displaying a variety of pharmacological activities.Determining triterpenoids in different matrices,especially in biological samples holds great significance.High-performance liquid chromatography(HPLC)has become the predominant method for triterpenoids analysis due to its exceptional analytical performance.However,due to the structural similarities among botanical samples,achieving effective separation of each triterpenoid proves challenging,necessitating significant improvements in analytical methods.Additionally,triterpenoids are characterized by a lack of ultraviolet(UV)absorption groups and chromophores,along with low ionization efficiency in mass spectrometry.Consequently,routine HPLC analysis suffers from poor sensitivity.Chemical derivatization emerges as an indispensable technique in HPLC analysis to enhance its performance.Considering the structural characteristics of triterpenoids,various derivatization reagents such as acid chlorides,rhodamines,isocyanates,sulfonic esters,and amines have been employed for the derivatization analysis of triterpenoids.This review comprehensively summarized the research progress made in derivatization strategies for HPLC detection of triterpenoids.Moreover,the limitations and challenges encountered in previous studies are discussed,and future research directions are proposed to develop more effective derivatization methods.

DMMIC derivatization-assisted liquid chromatography-mass spectrometry method for metabolite profiling of the glutathione anabolic pathway in esophageal cancer tissues and cells

In this work,a new pyrylium derivatization-assisted liquid chromatography-mass spectrometry(LC-MS)method was developed for metabolite profiling of the glutathione anabolic pathway(GAP)in cancer tissues and cells.The pyrylium salt of 6,7-dimethoxy-3-methyl isochromenylium tetrafluoroborate(DMMIC)was used to label the amino group of metabolites,and a reductant of dithiothreitol(DTT)was employed to stabilize the thiol group.By combining DMMIC derivatization with LC-MS,it was feasible to quantify the 13 main metabolites on the GAP in complex biological samples,which had good linearity(R^(2)=0.99810.9999),precision(interday precision of 1.6%e19.0%and intraday precision of 1.4%e19.8%)and accuracy(83.4%-115.7%).Moreover,the recovery assessments in tissues(82.5%e107.3%)and in cells(98.1%e118.9%)with GSH-^(13)C2,^(15)N,and Cys-^(15)N demonstrated the reliability of the method in detecting tissues and cells.Following a methodological evaluation,the method was applied successfully to investigate difference in the GAP between the carcinoma and para-carcinoma tissues of esophageal squamous cell carcinoma(ESCC)and the effect of p-hydroxycinnamaldehyde(CMSP)on the GAP in KYSE150 esophageal cancer cells.The results demonstrate that the developed method provides a promising new tool to elucidate the roles of GAP in physiological and pathological processes,which can contribute to research on drugs and diseases.

Polarity-regulated derivatization-assisted LC-MS method for amino-containing metabolites profiling in gastric cancer

Amino-containing compounds,including amino acids,aliphatic amines,aromatic amines,small peptides and catecholamines,are involved in various biological processes and play vital roles in multiple metabolic pathways.Previous studies indicated that some amino-containing metabolites are significant diagnostic and prognostic biomarkers of gastric cancer.However,the discovery of precise biomarkers for the preoperative diagnosis of gastric cancer is still in an urgent need.Herein,we established a polarityregulated derivatization method coupled with liquid chromatography-mass spectrometry(LC-MS)for amino-containing metabolites profiling in the serum samples of patients with gastric cancer and healthy controls,based on our newly designed and synthesized derivatization reagent(S)-3-(1-(diisopropoxyphosphoryl)pyrrolidine-2-carboxamido)-N-hydroxysuccinimidyl ester(3-DP-NHS).Enhanced separation efficiency and detection sensitivity for amino-containing metabolites were achieved after derivatization.This method exhibited good linearity,recovery,intra-and inter-day precision and accuracy.Only 5 mL serum is needed for untargeted analysis,enabling 202 amino-containing metabolites to be detected.Statistical analysis revealed altered amino acid metabolisms in patients with gastric cancer.Furthermore,ultra high performance liquid chromatography coupled with mass spectrometry(UHPLCMS/MS)analysis quantification revealed increased serum levels of tryptamine and decreased concentrations of arginine and tryptophan in patients with gastric cancer.Receiver operating characteristic(ROC)curves indicated that an increased tryptamine/tryptophan ratio could serve as a potential biomarker for gastric cancer diagnosis.This study demostrated the possibility of using serum amino acid biomarkers for gastric cancer diagnosis,providing new avenues for the treatment of gastric cancer.

Analysis of Monosaccharide Composition of Pu-erh Tea Polysaccaride by Pre-column Derivatization HPLC

[Objective] This study aimed to establish a pre-column derivatization HPLC method for the identification and analysis of monosaccharide composition of Pu-erh tea polysaccharide. [Method] Pu-erh tea polysaccharide was extracted using the wa- ter extraction method, further isolated and purified by DEAE cellulose-52 columns. The obtained tea polysaccharide and four components TPS1, TPS2, TPS3 and TPS, were first derived by 1-phenyl-3-methyl-5-pyrazolone （PMP）, and then the PMP derivatives of monosaccharide were analyzed by high performance liquid chromatog- raphy （HPLC）. [Result] Pu-erh tea polysaccharide contained eight kinds of monosac- chaddes （mannose, rhamnose, glucuronic acid, galacturonic acid, grucose, galactose, arabinose, fucose）, without xylose; so it was the same with TPS1; each of TPS2, TPS3 and TPS4 contained seven monosaccharides, while no fucose. [Conclusion] This method is simplified and rapid, which can be used to determine the monosac- charide composition of Pu-erh tea polysaccharide and monosaccharide content.

Determination of Sulphonylurea Glimepiride in Dog Serum by RP-HPLC with Pre-column Derivatization

Aim A simple, sensitive and rapid RP HPLC method with pre column derivatization has been developed for the determination of sulphonylurea glimepiride in dog serum. Methods The sulphonylurea glimepiride was extracted from the dog serum using dichlromethane followed by derivatization with DNBF for 20 min at 100℃. The solvent was then evaporated at 60℃ under nitrogen, and the residue was taken up in 100 μL of mobile phase consisting of acetonitrile water (75∶30, v/v). The separation was performed on a Hypersil BDS C18 column with a flow rate of 0 8 mL·min -1 , and the ultraviolet detector wavelength was set at 350 nm. Results Extraction recovery ranged from 75.9% to 83.2%, and methodological recovery was between 96.5% and 109.3%. Within day RSD ranged from 1.5% to 6.3%, and inter day RSD was between 2 9% and 14.8%. The method showed good linearity (R=0.9998). Conclusion The method was simple, convenient and sensitive. The reaction of derivatization was reproducible.

A Pre-column Derivatization HPLC Method for the De-termination of Peimine and Peiminine in Bulbus Fritillar-iae

A pre-column derivatization HPLC method for the determination of peimine(P)and peiminine(PE)in Bulbus Fritillariae has been developed. Under the derivatization conditions optimized,the calibration curve is Y1=-3.83×103+1.33 ×105X1,r=0.998 for P and Y2=-7.86 × 102+6.33 × 104X2,r=0995 for PE,where Y is the peak area and X is the weight of the alkaloid; the average recovery is 98.0%(n=5, RSD=2.1%) for P and 101.0%(n=5,RSD=4.1%)for PE,the linear range is from 0.504 μg to 3.126μg for P and from 0.520μgto 3.328μgfor PE,respectively. Results of the determination of the two alkaloids in several samples of different Fritillaria species from various parts ofthe country are presented.The results suggest that P and PE are two major chemical constituents in bulbs of different Fritillaria species,and that the method developed is generally appli-cable to the determination of the hydroxy group on aliphatic fused ring systems without steric hin-drance.

Precolumn derivatization LC-MS/MS method for the determination and pharmacokinetic study of glucosamine in human plasma and urine

A selective precolumn derivatization liquid chromatography–tandem mass spectrometric (LC–MS/MS) method for the determination of glucosamine in human plasma and urine has been developed and validated. Glucosamine was derivatized by o-phthalaldehyde/3-mercaptopropionic acid. Chromatographic separation was performed on a Phenomenex ODS column (150 mm 4.6 mm, 5 mm) using linear gradient elution by a mobile phase consisting of methanol (A), and an aqueous solution containing 0.2% ammonium acetate and 0.1% formic acid (B) at a flow rate of 1 mL/min. Tolterodine tartrate was used as the internal standard (IS). With protein precipitation by acetonitrile and then the simple one-step derivatization, a sensitive bio-assay was achieved with the lower limit of quantitation (LLOQ) as low as 12 ng/mL for plasma. The standard addition calibration curves suitable for clinical sample analysis showed good linearity over the range of 0.012–8.27 mg/mL in plasma and 1.80–84.1 mg/mL in urine. The fully validated method has been successfully applied to a pharmacokinetic study of compound glucosamine sulfate dispersible tablets in health Chinese volunteers receiving single oral doses at 500, 1000 and 1500 mg of glucosamine sulfate, as well as multiple oral doses of 500 mg t.i.d. for 7 consecutive days.

Determination of Lignin in Marine Sediment Using Alkaline Cupric Oxide Oxidation-Solid Phase Extraction-on-Column Derivatization-Gas Chromatography

Lignin serves as one of the most important molecular fossils for tracing Terrestrial Organic Matters （TOMs） in marine environment. Extraction and derivatization of lignin oxidation products （LOPs） are crucial for accurate quantification of lignin in marine sediment. Here we report a modification of the conventional alkaline cupric oxide （CuO） oxidation method, the modification consisting in a solid phase extraction （SPE） and a novel on-column derivatization being employed for better efficiency and reproducibility. In spiking blanks, recoveries with SPE for the LOPs are between 77.84% and 99.57% with relative standard deviations （RSDs） ranging from 0.57% to 8.04% （n=3）, while those with traditional liquid-liquid extraction （LLE） are from 44.52% to 86.16% With RSDs being from 0.53% to 13.14% （n=3）. Moreover, the reproducibility is greatly improved with SPE, with less solvent consumption and shorter processing time. The average efficiency of on-column derivatization for LOPs is 100.8%±0.68%, which is significantly higher than those of in-vial or in-syringe derivatization, thus resulting in still less consumption of derivatizing reagents.Lignin in the surface sediments sampled from the south of Yangtze River estuary, China, was determined with the established method. Recoveries of 72.66% to 85.99% with standard deviation less than 0.01mg/10g dry weight are obtained except for p-hydroxybenzaldehyde. The lignin content ∑8 （produced from 10g dry sediment） in the research area is between 0.231 and 0.587mg. S/V and C/V ratios （1.028 ± 0.433 and 0.192±0.066, respectively） indicate that the TOMs in this region are originated from a mixture of woody and nonwoody angiosperm plants; the high values or （Ad/Al）v suggest that the TOMs has been highly degraded.

Determination of Content of Amino Acid in Abalone by Precolumn Derivatization and its Nutritional Value Evaluation

[Objectives] This study was conducted to optimize the determination conditions of amino acids from abalone. [Methods] The sample was treated by acid hydrolysis method and subjected to 2,4-2 nitro fluorobenzene column derivatization. The amino acid content in abalone was determined by HLPC,and the nutritional value of the amino acids was evaluated with egg protein model put forward by Institute of Nutrition and Food Hygiene,Chinese Academy of Preventive Medicine. [Results] Abalone contains full amino acids. According to the FAO/WHO ideal,it is a high-quality protein source and suitable for supplement of protein source for human body. [Conclusions]The experimental method has simple operation and could achieve a good effect with wide linear range and correlation coefficient over 0. 999 8,and the obtained results are satisfactory.

Ionic Liquid as Useful Media for Dissolution, Derivatization, and Nanomaterial Processing of Chitin

This paper reviews studies on dissolution, derivatization, and nanomaterial processing of chitin using an ionic liquid as useful media. Because chitin is the second most abundant polysaccharide on the earth after cellulose, there is major interest in conversion of native chitin resources into various useful materials after proper dissolution in suitable solvents. For the derivatization and nanomaterial processing of chitin, the author has been focusing on ionic liquids because which have been found to be used as good solvents for cellulose in a past decade. The author found that an ionic liquid, 1-allyl-3-methylimidazolium bromide (AMIMBr), dissolved chitin in concentrations up to 4.8 wt% and mixtures of the higher amounts of chitin with AMIMBr gave ion gels. Acetylation, the simplest derivatization, of chitin using acetic anhydride was achieved in the AMIMBr solvent under mild conditions. Furthermore, the chitin nanofibers were fabricated by regeneration technique from the chitin ion gel with AMIMBr using methanol. Moreover, filtration of the chitin nanofiber dispersion with methanol was carried out to give a chitin nanofiber film. The chitin nanofiber-poly(vinyl alcohol) composite film was also prepared from the ion gel by co-regeneration method.

Determination of Sparfloxacin in Human Urine by Reversed-Phase High Performance Liquid Chromatography With Nitrous Acid and Hydroiodic Pre-Column Derivatization

Sparfloxacin can be oxidized by nitrous acid, then react with hydroiodic acid to form a fluorescent derivative. Based on this, a reversed-phase high performance liquid chromatographic pre-column derivatization new method is described for the determination of sparfloxacin in human urine. The linear range is 0.05 mg/L to 4.0 mg/L, the recoveries are 91.5%similar to 95.7% and the RSD is 1.2%similar to4.2%. The results showed that this method is suitable for the determination of sparfloxacin in human urine.

Isolation, identification, derivatization and phytotoxic activity of secondary metabolites produced by Cladosporium oxysporum DH14, a locust-associated fungus

Three main phytotoxic compounds including lunatoic acid A （1）, 5Z-7-oxozeaenol （2） and zeaenol （3） were isolated from the fermentation broth of Cladosporium oxysporum DH14, a fungus residing in the locust （Oxya chinensis ） gut. Two additional derivative compounds, compound la and lb, were synthesized by methylation and chlorination of compound 1, respectively. The structures of such compounds were identified on the basis of spectroscopic analysis and by comparison of the corresponding data to those previously reported in the literature. Compounds 1-3 exhibited significantly phytotoxic activities against the radicle growth of Amaranthus retroflexus L. with the 50% inhibitory concentrations （ICso values） of 4.51, 4.80 and 8.16 μg· mL^-1, respectively, which is comparable to that positive control 2,4-dichlorophenoxyacetic acid （IC50=1.95 μg· mL^-1）. Furthermore, the compound 1 showed selective phytotoxic activity with the inhibition rate of less than 22% against the crops of Brassica rapa L., Sorghum durra, Brassica campestris L,, Capsicum annucm and Raphanus safivus L. under the concentration of 100 μg· mL^-1 Both derivatives of compound 1 had moderate phytotoxic activity against the radicle growth of A. retroflexus L. The findings of our present study suggest that these compounds provide new promising candidates for the potential management strategies of weeds.

Enantioselective assay of S(+)- and R(-)-propafenone in human urine by using RP-HPLC with pre-column chiral derivatization

The enantioselective assay for S(+)- and R(-)-propafenone (PPF) in human urine that developed in this work involves extraction of propafenone from human urine and using S(+)-propafenone as internal standard, chiral derivatization with 2,3,4,6-tetra-O-b-D-glucopranosyl isothiocyanate, and quantitation by an RP-HPLC system with UV detection (l=220 nm). A baseline separation of propafenone enantiomers was achieved on a 5-mm reverse phase ODS column, with a mixture of methanol:water:glacial acetic acid (25:12:0.02,v/v) as mobile phase. There was good linear relationship from 24.9 ng/ml to 1875.0 ng/ml for both of enantiomers. The regression equations of the standard curves based on CS-PPF (or CR-PPF ) versus ratio of AS-PPF/AS (or AR-PPF/AS ) were y=0.0032x-0.081, (r=0.999) for S-PPF and y=0.0033x+0.0039, (r=0.998) for R-PPF, respectively. The method抯 limit of detection was 12.5 ng/ml for both enantiomers, and the method抯 limit of quantitation was 28.20.52 ng/ml for S-PPF, 30.40.53 ng/ml for R-PPF (RSD<8%, n=5). The analytical method yielded average recovery of 98.9% and 100.4% for S-PPF and R-PPF, respectively. The relative standard deviation was no more than 6.11% and 6.22% for S-PPF and R-PPF, respectively. The method enabled study of metabolism of S(+)- and R(-)-propafenone in human urine. The results from 7 volunteers administered 150 mg racemic propafenone indicated that propafenone enantiomers undergo stereoselective metabolism and that in the human body, S(+)-propafenone is metabolized more extensively than R(-)- propafenone.

Determination of 18 Kinds of Amino Acids in Fresh Tea Leaves by HPLC Coupled with Pre-column Derivatization

A rapid and accurate quantitative method of high performance liquid chromatography( HPLC) with fluorescence detector has been developed for the analysis of 18 kinds of amino acids in fresh tea leaves. The samples were minced and mixed,and extracted with ultra pure water at 90℃ for 20 min. The 6-aminoquinolyl N-hydroxy-succinimidyl carbamate( AQC) was used as pre-column derivatization reagent. Gradient HPLC separation was performed on a C_(18) column( Symmetry C_(18),3. 9 mm × 15 cm,4 μm). Good linearity between concentrations and peak areas was achieved in the concentration range of 5. 0-250 μmol/L for 18 kinds of amino acids. The method was validated by the analysis of five replicates. The 18 kinds of amino acid standards were spiked in fresh tea leaf samples and the average recovery rate was 86. 25%-109. 05% with relative standard deviations( n = 5) ranging from 6. 03% to 10. 56%. The limit of detection( LOD) for the analytes was0. 05-1. 27 μmol/L. The method was successfully applied to the analysis of the 18 kinds of amino acids in fresh tea leaves from east Dongting and west Dongting mountains in Suzhou. The results indicate that the method is simple,rapid,precise and reliable.

Quantitative‑Profling Method of Serum Steroid Hormones by Hydroxylamine‑Derivatization HPLC-MS

A sensitive and rapid high performance liquid chromatography-mass spectrometry(HPLC-MS)method was developed and validated for simultaneous quantifcation of ten steroid hormones,including estrogens,androgens,progesterones,and corticosteroids four classes of steroids.The following ten steroid hormones were analyzed:progesterone,21-deoxycortisol,estrone,4-androstenedione,testosterone,dihydro-testosterone,androstenone,dehydroepiandrosterone,corticosterone and cortisone.Stable deuterated isotopes were used as internal standards for quantifcation.Sample preparation with and without derivatization were performed after liquid-liquid extraction,and the corresponding results were compared according to sensitivity and selectivity.Hydroxylamine derivatization was found to improve the ionization efciency of the analytes for electrospray ionization MS analysis.The gradient of mobile phase and experimental parameters for HPLC separation were optimized.The lower limits of quantifcation were in the range of 0.05-5 ng mL^(−1) with wide linear range for the ten steroid hormones.The intra-day precision<11.1%and recovery of 84.5-120% with negligible matrix efect were achieved,where within the acceptance limits of the FDA guideline.Total HPLC-MS analysis time was 6 min.This method enables simultaneous quantifcation of steroids in human serum.It will be helpful for the serum steroid profling in order to understand various endocrinology diseases.

Development of Simultaneous HPLC-Fluorescence Assay of Phenol and Chlorophenols in Tap Water after Pre-Column Derivatization with 3-Chlorocarbonyl-6,7-dimethoxy-1- methyl-2(1H)-quinoxalinone

Chlorophenols (2-chlorophenol, 4-chlorophenol, 2,4-dichlorophenol, 2,6-dichlorophenol and 2,4, 6-trichlorophenol) may be presented in natural waters or drinking water as a result of disinfection processes involving chlorination, or as contaminants derived from domestic products, industrial operations and agricultural chemicals. A previous HPLC-UV method for determination of phenol and five chlorophenols in tap water using 4-fluoro-7-nitro-2,1,3-benzoxadiaole as a UV labeling reagent shows limited sensitivity. Here, we present an improved HPLC-fluorescence detection method for simultaneous determination of phenol and the above chlorophenols in tap water after pre-column derivatization with 3-chlorocarbonyl-6,7-dimethoxy-1-methyl-2(1H)-quino- xalinone (DMEQ-COCl), using a short, narrow column (50 × 2.1 mm i.d., packed with 5 μm particles of C18 material) to improve the sensitivity. Standard samples containing the compounds are derivatized with DMEQ-COCl in borate buffer (pH 9.0) at room temperature for 3 mins. The response is linear in the concentration range of 0.01 - 0.05 to 0.5 mg/L with r2 values ≥0.9967 for all compounds. The lower limits of detection are 0.001 to 0.008 mg/L, and the coefficients of variation are less than 8.8%. The recovery values from tap water spiked with standard samples are satisfactory. The present method is suitable for examining whether or not tap water samples are contaminated with phenol and chlorophenols in excess of regulatory values.

Determination of Sulfadiazine Based on Its Derivatization with Fluorescamine by Self-Ordered Ring Fluorescence Microscopic Imaging Technique

A self-ordered ring(SOR) fluorescence microscopic imaging technique has been developed for the determination of trace amounts of sulfadiazine based on its derivatization with fluorescamine.In the presence of HAc-NaAc buffer solution(pH 3.12) and polyvinyl alcohol-124(PVA-124),the droplet containing fluorescamine derivatized sulfadiazine can form a SOR on the solid support after solvent evaporation with the diameter of 1.86mm and its ring belt width of 54.9 μ m.The quantitative analysis of sulfadiazine is achieved with the linear range of 7.8×10-14 ～1.8×10-12 mol·ring-1(3.9×10-7 ～9.0×10-6 mol·L-1) and detection limit of 7.8×10-15 mol·ring-1(3.9×10-8 mol·L-1) when 0.2 μ L droplet was spotted.The technique has been satisfactorily applied to the determination of sulfadiazine in the tablet,synthetic sample and residues in six different milk samples with the recoveries of 91.0%～105.8%,respectively,and RSDs less than 4.4%.

Simple HPLC-Fluorescence Determination of Raspberry Ketone in Fragrance Mist after Pre-Column Derivatization with 4-Hydrazino-7-nitro-2,1,3-benzoxadiazole

Raspberry ketone {RK, 4-(4-hydroxyphenyl)butan-2-one} is a natural compound contained in raspberry, and is added to cosmetics for skin whitening. It is very important to measure the RK level in cosmetics for quality assessment, since RK structurally resembles 4-(4-hydroxyphenyl)-2-butanol, which causes leukoderma on consumers’ skin. Here, we present a simple HPLC-fluorescence method for determination of RK in a fragrance mist by pre-column derivatization with 4-hydrazino-7-nitro-2,1,3-benzoxadiazole hydrazine (NBD-H), which reacts with the carbonyl group of RK. The NBD-RK derivative was eluted from a reversed-phase ODS column, and detected with excitation at 470 nm and emission at 550 nm. The retention time of NBD-RK derivative obtained by reaction with NBD-H at 80°C for 20 min was 10.3 min. The standard curve was linear in the range of 0.2 to 10 μg/mL, with a correlation coefficient (r2) value of 0.9980. The lower limit of detection was 0.018 μg/mL (absolute amount of 1.8 pmol). The coefficients of variation were less than 8.1%. The content of RK in fragrance mist (1.00 mL) was 1.18 ± 0.07 mg (range: 1.12 to 1.28 mg, n = 5). Recovery tests were satisfactory (83.9% ± 3.9%;range: 79.6 to 88.8%, n = 5).

Direct hydride derivatization of methyl- and ethylmercury chlorides in aqueous solution with KBH_4

A convenient hydride derivatization procedure of methyl\|and ethylmercury chlorides to volatile hydrides was reported. In sealed vials methylmercury and ethylmercury compounds in acidic aqueous solutions were converted into their volatile forms by the reaction with potassium tetrahydroborate(KBH 4) and elvolved to the headspace of the vials. The gaseous analytes in the headspace were extracted and concentrated by solid phase microextraction(SPME) and injected into gas chromatography (GC) for separation and identified by mass selective detector(MS).

Rapid and Reproducible Dibutylation Derivatization Coupled with Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry for the Simultaneous Determination of Dopamine, Norepinephrine and 5-Hydroxytryptamine in Rat Brain Microdialysates

A rapid and reproducible method has been developed for the simultaneous quantification of dopamine (DA), norepinephrine (NE), and serotonin (5-HT) in rat brain microdialysates. Derivatization was processed through reductive amination with butyraldehyde and sodium cyanoborohydride at 60℃ for 80 min incubation. Dibutylated monoamine neurotransmitters (MANTs) were directly analyzed with ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The chromatographic run time was shorter (2.1 min/sample) than previous studies [15] [18] [20]. Good linearity (R2 > 0.99) was obtained for DA, NE and 5-HT in the range of 25 - 5000 pg/mL, 5 - 1000 pg/mL and 2.5 - 500 pg/mL, respectively. Acceptable precision (CV, 8.5% - 13.4%) as well as accuracy (recovery, 94.1% - 106.8%) could be acquired by analysis of six batches of quality control samples (QCs) at four different concentrations, which demonstrated the reliability and reproducibility of current method. This method was successfully applied to the simultaneous determination of DA, NE and 5-HT in rat brain microdialysates, where basal levels as well as elevated levels after dosing with amphetamine (AMPH) were quantified for all three MANTs. This study provides a simple and rapid way to analyze MANTs in the biofluid in the future.