Simultaneous determination of six toxic alkaloids in human plasma and urine using capillary zone electrophoresis coupled to time‐of‐flight mass spectrometry

A novel capillary zone electrophoresis separation coupled to electro spray ionization time‐of‐flight mass spectrometry method was developed for the simultaneous analysis of six toxic alkaloids: brucine, strychnine, atropine sulfate, anisodamine hydrobromide, scopolamine hydrobromide and anisodine hydrobromide in human plasma and urine. To obtain optimal sensitivity, a solid‐phase extraction method using Oasis MCX cartridges (1 mL, 30 mg; Waters, USA) for the pretreatment of samples was used. All compounds were separated by capillary zone electrophoresis at 25 kV within 12 min in an uncoated fused‐silica capillary of 75 μm id × 100 cm and were detected by time‐of‐flight mass spectrometry. This method was validated with regard to precision, accuracy, sensitivity, linear range, limit of detection (LOD), and limit of quantification (LOQ). In the plasma and urine samples, the linear calibration curves were obtained over the range of 0.50–100 ng/mL. The LOD and LOQ were 0.2–0.5 ng/mL and 0.5–1.0 ng/mL, respectively. The intra‐ and interday precision was better than 12% and 13%, respectively. Electrophoretic peaks could be identified by mass analysis.     

Identification and estimation of the levo isomer in raw materials and finished products containing atropine and/or hyoscyamine

The belladonna alkaloids atropine sulfate and hyoscyamine sulfate, occasionally used as anticholinergic and antimuscarinic agents, have identical molecular formulas but different stereo configurations. Hyoscyamine sulfate contains almost 100% of the levo isomer, whereas atropine sulfate is composed of equal parts of dextro and levo isomers. It is believed that the therapeutic properties of these alkaloids are due exclusively or primarily to the levo isomer. Currently available methods determine only the total amount of atropine (hyoscyamine) sulfate. A method has been developed and is reported for the identification and estimation of the levo and dextro isomers of atropine and hyoscyamine. Reference solutions are prepared in methanol at the following weights per 100 mL: 8.0 mg atropine sulfate; 4.0 mg hyoscyamine sulfate; 7.0 mg scopolamine hydrobromide; and 10.0 mg homatropine methylbromide. Samples of raw materials are similarly prepared in methanol, commercial products are also extracted or diluted with methanol, and solutions are filtered. Liquid chromatography is used for separations on a 25 cm Chirobiotic T2 column. The mobile phase is prepared by mixing 3.0 mL acetic acid and 2.0 mL triethylamine with 1000 mL methanol. The injection volume is 100 or 200 microL; the flow rate is about 0.35 mL/min. Fluorescence detection is at 255 nm excitation and 285 nm emission. Scopolamine hydrobromide and hyoscyamine eluted after 20 and 60 min, respectively. Atropine sulfate generated 2 peaks after 60 and 65 min. Homatropine methylbromide also produced 2 peaks after 70 and 85 min. Samples tested in this study included raw materials and commercial tablets or injections containing belladonna alkaloids. In all cases, the percentage calculated was that of the levo isomer relative to the total amount of atropine (hyoscyamine) present.     

Determination of atropine (hyoscyamine) sulfate in commercial products by liquid chromatography with UV absorbance and fluorescence detection: multilaboratory study

A liquid chromatographic (LC) method with 2 detection systems for determining atropine (hyoscyamine) sulfate in commercial products was tested in a multilaboratory study. Depending on the type of product, sample solutions are prepared in methanol or methanol-water (1 + 1). The standard solution contains about 1.0 mg atropine sulfate/100 mL and is prepared in the same solvent used in sample preparation. LC separations are performed on a 7.5 cm Novapak silica column. The mobile phase is prepared by mixing 970 mL methanol with 30 mL of a 1% aqueous solution of 1-pentanesulfonic acid, sodium salt. Detection is by 2 systems, UV absorbance detection at 220 nm and fluorescence detection with excitation at 255 nm and emission at 285 nm. The injection volume is 100 or 200 microL. The following materials were used for the study: 2 separate samples of tablets labeled to contain 0.4 mg atropine sulfate, 2 separate samples of extended-release tablets labeled to contain 0.375 mg hyoscyamine sulfate, one sample of atropine sulfate injection labeled to contain 2 mg/mL, and one sample of 1% (v/v) atropine sulfate ophthalmic. Eight participants analyzed 2 separate portions of the 6 samples by both detection systems. A ninth participant analyzed the samples in duplicate but only by UV absorbance detection because of the unavailability of a fluorescence detector. The relative standard deviation (RSD) between laboratories ranged from 1.4 to 3.3% for samples of tablets and injections but higher for ophthalmic solutions (5.1-5.2%). A linearity study was conducted in the originating laboratory before the multilaboratory study with 5 solutions ranging in concentration from 0.80 to 1.20 mg atropine sulfate in 100 mL. Average recoveries were 100.0% by UV absorbance detection and 99.9% by fluorescence detection; the RSDs were 1.1 and 1.2%, respectively.     

Simultaneous determination of atropine,scopolamine, and anisodamine from Hyoscyamus niger L. in rat plasma by high‐performance liquid chromatography with tandem mass spectrometry and its application to a pharmacokinetics study

In order to investigate the pharmacokinetics of tropane alkaloids in Hyoscyamus niger L., a sensitive and specific high‐performance liquid chromatography with tandem mass spectrometry method for the simultaneous determination of atropine, scopolamine, and anisodamine in rat plasma is developed and fully validated, using homatropine as an internal standard. The separation of the four compounds was carried out on a BDS Hypersil? C₁₈ column using a mobile phase consisting of acetonitrile and water (containing 10 mmol ammonium acetate). Calibration curves were linear from 0.2 to 40 ng/mL for atropine, scopolamine, and from 0.08 to 20 ng/mL for anisodamine. The precision of three analytes was <5.89% and the accuracy was between ?1.04 to 2.94%. This method is successfully applied to rat pharmacokinetics analysis of the three tropane alkaloids after oral administration of H. niger extract. The maximum concentration of these three tropane alkaloids was reached within 15 min, and the maximum concentrations were 31.36 ± 7.35 ng/mL for atropine, 49.94 ± 2.67 ng/mL for scopolamine, and 2.83 ± 1.49 ng/mL for anisodamine. The pharmacokinetic parameters revealed areas under the curve of 22.76 ± 5.80, 16.80 ± 3.08, and 4.31 ± 1.21 ng/h mL and mean residence times of 2.08 ± 0.55, 1.19 ± 0.45, and 3.28 ± 0.78 h for atropine, scopolamine, and anisodamine, respectively.     

Determination of nikethamide by micellar electrokinetic chromatography

A simple, fast, sensitive and reproducible micellar electrokinetic chromatography (MEKC)–UV method for the determination of nikethamide (NKD) in human urine and pharmaceutical formulation has been developed and validated. The method exhibits high trueness, good precision, short analysis time and low reagent consumption. NKD is an organic compound belonging to the psychoactive stimulants used as an analeptic drugs. The proposed analytical procedure consists of few steps: dilution of urine or drug in distilled water, centrifugation for 2 min (12,000 g ), separation by MEKC and ultraviolet‐absorbance detection of NKD at 260 nm. The background electrolyte used was 0.035 mol/L pH 9 borate buffer with the addition of 0.05 mol/L sodium dodecyl sulfate and 6.5% ACN. Effective separation was achieved within 5.5 min under a voltage of 21 kV (~90 μA) using a standard fused‐silica capillary (effective length 51 cm, 75 μm i.d.). The determined limit of detection for NKD in urine was 1 μmol/L (0.18 μg/mL). The calibration curve obtained for NKD in urine showed linearity in the range 4–280 μmol/L (0.71–49.90 μg/mL), with R² 0.9998. The RSD of the points of the calibration curve varied from 5.4 to 9.5%. The analytical procedure was successfully applied to analysis of pharmaceutical formulation and spiked urine samples from healthy volunteers.     

Development and Validation of a Sensitive LC-MS/MS Method for the Simultaneous Analysis of Three-Tropane Alkaloids in Blood and Urine

A simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous analysis of three tropane alkaloids in blood and urine. After 1 mL of a blood or urine sample was extracted using a liquid–liquid extraction method with ethyl acetate at pH 8 and homatropine as the internal standard, the tropane alkaloids were separated. An Allure PFP propyl column (50 mm × 2.1 mm, 5 µm) separated the tropane alkaloids using an acetonitrile-buffer solution (20 mmol/L ammonium acetate and 0.1% formic acid, pH 4) (70:30) as the mobile phase at a flow-rate of 0.2 mL/min in isocratic mode, with the LC-MS/MS in the positive ionization mode. For each compound, detection was related to two daughter ions (scopolamine: m/z 304.4 → 138.1 and 155.9; atropine: m/z 290.3 → 124.0 and 93.1; anisodamine: m/z 306.3 → 140.1 and 91.1; and homatropine: m/z 276.3 → 124.3 and 142.1). The tropane alkaloids exhibited excellent linearity in the range of 0.05–100 ng/mL in blood and 0.2–100 ng/mL in urine, with a limit of detection range from 0.02 to 0.05 ng/mL for biological materials. The extraction recoveries of atropine, scopolamine, and anisodamine were more than 53% in the blood and urine; the interday and intraday RSDs were less than 10%; the within-day and between-day accuracy were between ?9.8% and +8.8%. The present method is simple and rapid, as shown by its application to a clinical case. This method is useful for routine analysis of tropane alkaloids in cases of suspected tropane alkaloid poisoning.     

Determination of scopolamine, atropine and anisodamine in Flos daturae by capillary electrophoresis.

A capillary electrophoresis method was developed for the separation and determination of tropane alkaloids in Flos daturae plants. Separation was performed on a fused silica capillary(42.1 cm x 50 microm i.d.) at an applied voltage of 20 kV. Scopolamine, atropine and anisodamine were well separated in the buffer of 50 mmol/L phosphate buffer (pH 5.0) containing 20% (v/v) tetrahydrofuran (THF). Beer's law was obeyed in the range of concentration of 2.4-21.8 microg/mL for scopolamine, 4.0-36.0 microg/mL for atropine and 2.6-23.7 microg/mL for anisodamine, respectively, and the correlation coefficients were over 0.999 (n = 6). The developed method was applied for the analysis of herb samples.     

Capillary electrophoretic enantioselective determination of zopiclone and its impurities

A capillary electrophoretic enantioselective method with UV detection was developed and validated for the simultaneous quantification of zopiclone enantiomers and its impurities, zopiclone-N-oxide enantiomers, and 2-amino-5-chloropyridine, in tablets. The analytes were extracted from the tablets using ACN and were separated in an uncoated fused-silica capillary (50 μm, 42 cm effective length, 50 cm total length) using 80 mM sodium phosphate buffer pH 2.5 and 5 mM carboxymethyl-β-cyclodextrin as running buffer. The analytes and the internal standard (trimethoprim) were detected at 305 and 200 nm, respectively. A voltage of 27 kV was applied and the capillary temperature was maintained at 25°C. All enantiomers were analyzed within 8 min and linear calibration curves over the concentration range of 0.4-0.8 mg mL?1 for each zopiclone enantiomer, 0.8-1.6 μg mL?1 for 2-amino-5-chloropyridine and 0.4-0.8 μg mL?1 for each zopiclone-N-oxide enantiomer were obtained. The coefficients of correlation obtained for the linear curves were greater than 0.99. The intra-day and inter-day accuracy and precision were lower than 2% for all analytes. This validated method was employed to study the degradation and racemization of zopiclone under stress conditions. This application demonstrated the importance of a stability-indicating assay method for this drug.     

Determination of ephedrine alkaloids in human urine and plasma by liquid chromatography/tandem mass spectrometry: collaborative study

A collaborative study was conducted to evaluate the accuracy and precision of a method for ephedrine-type alkaloids (i.e., norephedrine, norpseudoephedrine, ephedrine, pseudoephedrine, methylephedrine, and methylpseudoephedrine) in human urine and plasma. The amount of ephedrine-type alkaloids present was determined using liquid chromatography (LC) with tandem mass selective detection. The test samples were diluted to reflect a concentration of 5.00-100 ng/mL for each alkaloid. An internal standard was added and the alkaloids were separated using a 5 microm phenyl LC column with an ammonium acetate, glacial acetic acid, acetonitrile, and water mobile phase. Eight blind duplicates of human urine and eight blind duplicates of human plasma were analyzed by 10 collaborators. In addition to negative controls, test portions of urine and plasma were fortified at 3 different levels with each of the 6 ephedrine-type alkaloids at approximately 1, 2, and 5 microg/mL for urine and 100, 200, and 500 ng/mL for plasma. On the basis of the accuracy and precision results for this collaborative study, it is recommended that this method be adopted Official First Action for the determination of 6 different ephedrine-type alkaloids in human urine and plasma.     

电堆积在线扫集胶束电动色谱法测定苦参中的生物碱

建立了电堆积在线扫集胶束电动色谱法测定苦参碱和槐定碱的新方法.考察了pH值、磷酸二氢钠浓度、CTAB浓度、电压、有机溶剂和进样时间对分离效果的影响.采用未涂层熔融石英毛细管,以20mmol/L磷酸二氢钠-0.8 mmol/L CTAB-100mmol/L Tris(含10%异丙醇,pH 8.9)为缓冲液,在进样电压-1...     

Analysis of alendronate in human urine and plasma by magnetic solid-phase extraction and capillary electrophoresis with fluorescence detection

A simple, rapid and sensitive CE-fluorescence (FL) detection method for the analysis of alendronate (ALEN), a bisphosphonate drug, has been developed. Using a buffer solution of 20 mM sodium phosphate (pH 10.0) and a voltage of 24 kV, separation of ALEN in a 55-cm length (35-cm effective length) capillary was achieved in 5 min. FL detection of ALEN was performed via pre-column derivatization with 2,3-naphthalene dicarbox-yaldehyde (NDA). Linear correlation (r=0.9981, n=6) between FL intensity and analyte concentration was obtained in the range of 7-200 ng/mL ALEN. The developed CE-FL method was applied to the analysis of ALEN in human urine and plasma samples. In order to eliminate the interfering matrix components, SPE using magnetic Fe(3) O(4) @Al(2) O(3) nanoparticles as solid sorbents was employed to clean the biological fluids before CE-FL analysis. The linear ranges of ALEN in urine and plasma were 5-100 ng/mL (r = 0.9982, n = 7) and 5-70 ng/mL (r = 0.9954, n = 7), respectively. The LOD and LOQ in both urine and plasma samples were 1.5 and 5 ng/mL ALEN, respectively. Total analysis time including sample pre-treatment and CE separation was less than 1.5 h.     

Determination of quinolizidine alkaloids in Sophora flavescens and its preparation using capillary electrophoresis

A simple and accurate capillary electrophoresis method was developed for the determination of four quinolizidine alkaloids in Sophora flavescens and Kuhuang injection. Optimum separation of the analytes was obtained on a 65 cm x 75 microm i.d. uncoated fused-silica capillary using a aqueous buffer system of 60 mmol L(-1) sodium borate at pH 8.5, with applied voltage and capillary temperature of 12 kV and 25 degrees C, respectively. Detection wavelength was set at 204 nm and jatrorrhizine was used as the internal standard. Good linear relationships between peak-area ratios and concentrations of the analytes were observed over the concentration range 0.044-0.792 mg mL(-1) for matrine, 0.142-1.926 mg mL(-1) for oxymatrine, 0.0377-0.3393 mg mL(-1) for sophocarpine and 0.0664-1.062 mg mL(-1) for sophoridine. The recoveries of four alkaloids ranged between 93.08 and 101.4% with relative standard deviations from 0.7 to 9.2% (n = 6) as determined by standard addition. The limits of detection for four alkaloids were determined to be over the range 8.8-48.0 microg mL(-1). Contents of four alkaloids in Sophora flavescens and three alkaloids in Kuhuang injection were successfully determined under the optimum conditions.     

Sample stacking microemulsion electrokinetic capillary chromatography induced by reverse migrating pseudostationary phase for the quantification of phenobarbital and its p-hydroxyphenobarbital metabolite in rat urine

For the first time, a capillary electrophoretic (CE) method with sample stacking induced by a reverse migrating pseudostationary phase (SRMP) technique has been developed and validated for sensitive determination of phenobarbital (PB) and its p-hydroxyphenobarbital (PHPB) metabolite in rat urine samples. Separation and determination were optimized on a fused-silica capillary with a total length of 50 cm (effective length 40 cm) and 75 μm ID. The microemulsion background electrolyte consisted of 0.8% (v/v) ethyl acetate, 6.6% (v/v) butan-2-ol, 1.0% (v/v) acetonitrile, 2.0% (w/v) sodium n-dodecyl sulfate (SDS), and 89.6% (v/v) of 7.5 mM ammonium formate at pH 8. When this preconcentration technique was used, the sample stacking and the separation processes took place successively with changing the voltage with an intermediate polarity switching step. For practical application, a solid-phase extraction (SPE), C(18) sorbent with n-hexane/ethyl acetate (1?:?1%, v/v) as the elution solvent was used for sample purification and concentration. The SPE method gave good extraction yields for all the analytes, with absolute recovery values of 96.9% and 99.1% for PB and PHPB, respectively. The regression equations for PB and PHPB showed excellent linearity over a concentration range of 55-1386 ng mL(-1) for PB and PHPB (r = 0.998). The developed microemulsion electrokinetic capillary chromatography (MEEKC) method for separation of the studied compounds with SRMP as the electrophoretic preconcentration technique allowed detection limits in urine samples at 16.8 ng mL(-1) for PB and PHPB which are 15-fold lower than the reported CE method in the literature. The precision results, expressed by the intra-day and inter-day relative standard deviation (RSD) values range from 3.6 to 7.1% (repeatability) and from 3.2 to 7.2% (intermediate precision) for PB and PHPB, respectively, which were in line with Food and Drug Administration (FDA) criteria.     

High-performance liquid chromatographic method for the determination of mangiferin in rat plasma and urine

A reversed-phase high-performance liquid chromatography assay for mangiferin in rat plasma and urine was developed. Rutin was employed as an internal standard. The mobile phase consisted of acetonitrile-water (16:84, v/v) containing 3% acetic acid at a flow rate of 1 mL/min. Detection was at 257 and 365 nm for mangiferin in plasma and urine, respectively. The limit of quantitation (LOQ) of mangiferin was 0.6 microg/mL in plasma, and 0.48 microg/mL in urine. The standard curve was linear from 0.6 to 24 microg/mL in plasma, and 0.48 to 24 microg/mL in urine, both intra- and inter-day precision of the mangiferin were determined and their RSD did not exceed 10%. The method provides a technique for rapid analysis of mangiferin in rat plasma and urine, which can be used in pharmacokinetic studies.     

An HPLC method for the determination of ethacridine lactate in human urine

An HPLC method was developed and validated for the determination of ethacridine lactate in human urine. Solid-phase extraction cartridges were used to extract urine samples. Separation was carried out on a C(18) column maintained at 30 degrees C with methanol-0.05% sodium dodecylsulfonate (70:30, v/v, pH 3) as mobile phase at a flow rate of 1.0 mL/min. Detection was at UV 272 nm. The calibration curve was linear in the concentration range of 4-4000 ng/mL, with linear correlation coefficient r equal to 0.9998. The limit of detection for the assay was 1.1 ng/mL. The within-day accuracy ranged from 94.8 to 101.6% and precision from 2.3 to 5.4%. The between-day accuracy ranged from 96.8 to 102.6% and precision from 4.0 to 5.3%. The absolute recovery was 95.4-101.2%. Urine samples were stable for at least 15 days if stored in the dark at -20 degrees C. This simple and accurate method allows the sensitive determination of ethacridine lactate in human urine. It was successfully applied to assess the urine level of ethacridine lactate in women received intra-amniotic injection.     

Determination of ephedra alkaloids in urine and plasma by HPLC-UV: collaborative study

Ten collaborating laboratories determined the ephedra alkaloid content (ephedrine, pseudoephedrine, norephedrine, norpseudoephedrine, methylephedrine, and methylpseudoephedrine) in 8 blind duplicates of human plasma and urine using high-performance liquid chromatography (HPLC) with UV detection. In addition to negative urine and plasma controls, urine samples were spiked with individual ephedra alkaloids ranging in concentration from about 1 to 5 microg/mL. Plasma samples were spiked with individual ephedra alkaloids ranging in concentration from about 100 to 400 ng/mL. Sample solutions were treated to solid-phase extraction using a strong-cation exchange column to help remove interferences. The HPLC analyses were performed on a polar-embedded phenyl column using UV detection at 210 nm. The ephedra alkaloids were not consistently detected in any of the spiked plasma samples. When ephedra alkaloids were detected in the plasma samples, reproducibility between blind replicate samples was very poor. Repeatability, reproducibility, and accuracy were also very poor for the spiked urine samples. On the basis of these results, the HPLC-UV method for the determination of ephedra alkaloids in human urine and plasma is not recommended for adoption as Official First Action.     

Analysis of lamotrigine and its metabolites in human plasma and urine by micellar electrokinetic capillary chromatography

A reliable micellar electrokinetic capillary chromatographic method was developed and validated for the determination of lamotrigine and its metabolites in human plasma and urine. The variation of different parameters, such as pH of the background electrolyte (BGE) and Sodium dodecyl sulfate (SDS) concentration, were evaluated in order to find optimal conditions. Best separation of the analytes was achieved using a BGE composed of 10 mM borate and 50 mM SDS, pH 9.5; melatonin was selected as the internal standard. Isolation of lamotrigine and its metabolites from plasma and urine was accomplished with an original solid-phase extraction procedure using hydrophilic-lypophilic balance cartridges. Good absolute recovery data and satisfactory precision values were obtained. The calibration plots for lamotrigine and its metabolites were linear over the 1-20 microg/mL concentration range. Sensitivity was satisfactory; the limits of detection and quantitation of lamotrigine were 500 ng/mL and 1 microg/mL, respectively. The application of the method to real plasma samples from epileptic patients under therapy with lamotrigine gave good results in terms of accuracy and selectivity, and in agreement with those obtained with an high-performance liquid chromatography (HPLC) method.     

On the perspectives of capillary electrophoresis modes for the determination of morphine in human plasma without sample pretreatment

Screening and confirmation of drugs of abuse in body fluids are important for the medicinal treatment and form the legal basis of court judgments. A fast and precise identification of toxic substances is necessary. Morphine was determined in human plasma by capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC) using sample stacking mode. The electrophoretic separation was performed in an uncoated fused-silica capillary, 70 cm long to the detector, with an additional 10 cm to the cathode (75 microm i.d. and 360 microm o.d.). The UV absorbance detection was set at 190 nm. The electrophoretic buffers were prepared from 60 to 300 mm disodium tetraborate decahydrate, pH 10.5. Sodium dodecyl sulfate was added to the final solution in a concentration of 60 mm for MECC. All electrophoretic separations were carried out at 10 kV and the capillary temperature was ambient (25 degrees C). A linear calibration graph was obtained in the concentration range studied (50-5000 ng/mL). Several samples of drug-free plasma were checked for potential endogenous interference and the results showed no interference from the endogenous components, which co-migrated with morphine. As little as 50 ng/mL of morphine could be successfully analyzed by MECC in the concentration mode with acceptable precision. It is possible to determine morphine directly in plasma at therapeutic concentrations.     

Determination of calycosin-7-O-beta-D-glucopyranoside in rat plasma and urine by HPLC

A reversed-phase high-performance liquid chromatographic (HPLC) assay for calycosin-7-O-beta-D-glucopyranoside in rat plasma and urine with solid-phase extraction (SPE) was developed. Rutin was employed as an internal standard. The mobile phase consisted of acetonitrile-water (16:84, v/v) at a flow rate of 1.0 mL/min. Detection was set at 280 nm. The limit of quantitation of calycosin-7-O-beta-D-glucopyranoside was 0.2 microg/mL in both plasma and urine. The standard curve was linear from 0.2 to 10.0 microg/mL in plasma, and 0.2 to 5.0 microg/mL in urine. Both intra- and inter-day precision of the calycosin-7-O-beta-d-glucopyranoside were determined and their RSD did not exceed 10%. The method was successfully applied to the analysis of samples obtained from a basic pharmacokinetic study, in which calycosin-7-O-beta-d-glucopyranoside was administered orally to rats.     

Simultaneous determination of atropine,scopolamine, and anisodamine in Flos daturae by capillary electrophoresis using a capillary coated by graphene oxide

A novel CE method was developed for the separation and determination of three main tropane alkaloids in Flos daturae with a capillary coated by graphene oxide (GO). The GO‐coated capillary was characterized by SEM, energy dispersive X‐ray spectroscopy, and Raman spectroscopy, and the results indicated that the inner surface of the capillary was partially coated by GO. A phosphate solution (40 mM, pH7.0) containing 20% v/v methanol and 30% v/v acetonitrile was used as the running buffer for the analysis of the atropine, scopolamine, and anisodamine. The linear ranges of atropine, scopolamine, and anisodamine was 0.5–200 μg/mL with satisfactory correlation coefficients (R²) > 0.9987, and this novel method provided an efficient separation for three tropane alkaloids as well as a good reproducibility and stability. Finally, the method was successfully applied for the determination of these three tropane alkaloids in plant extracts.