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.     

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.     

Simultaneous Electrochemiluminescence Detection of Anisodamine,Atropine, and Scopolamine in Flos daturae by Capillary Electrophoresis Using β‐Cyclodextrin as Additive

This work developed a simple and sensitive method for simultaneous determination of three effective ingredients, atropine, scopolamine and anisodamine, in Flos daturae based on capillary electrophoresis coupled with electrochemiluminescence detection. β‐Cyclodextrin was used as an additive to the running buffer for obtaining the absolute separation. The proposed method displayed the linear ranges from 0.2 to 100, 0.2 to 100 and 20 to 200 μM for anisodamine, atropine and scopolamine with correlation coefficients more than 0.99, respectively. This method showed the relative standard deviations less than 4% and 6% for detection of migration time and peak height, respectively, and was suitable for the determination of these tropane alkaloids in plants and valuable in clinical and biochemical laboratories for quality control.     

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.     

Simultaneous quantification of three tropane alkaloids in goji berries by cleanup of the graphene/hexagonal boron nitride hybrids and ultra‐high‐performance liquid chromatography tandem mass spectrometry

A modified quick, easy, cheap, effective, rugged and safe method was established for simultaneous determination of atropine, anisodamine, and scopolamine in goji berries by using ultra‐high‐performance liquid chromatography with tandem mass spectrometry. The graphene/hexagonal boron nitride hybrids were prepared and first applied as a cleanup adsorbent. Compared to classical cleanup adsorbent (C₁₈), the graphene/hexagonal boron nitride hybrids as adsorbent had better extraction efficiency for the detection of analytes. Under the optimal conditions, the proposed analytical method achieved satisfactory linearity (R² > 0.995), and obtained desirable recoveries ranged from 77.4 to 94.0% with the relative standard deviation of 1.2–6.1% at the concentration levels of 3.2–13.4 µg/kg. The limits of quantitation of atropine, anisodamine, and scopolamine were, respectively, 3.2, 4.6, and 4.5 µg/kg with linearity ranged from 3.2 to 25.4 µg/kg. The modified quick, easy, cheap, effective, rugged, and safe sample preparation with ultra‐high‐performance liquid chromatography and tandem mass spectrometry method was successfully applied to evaluate the safety of goji berries collected from 30 plant areas in China, suggesting its applicability and suitability for the routine analysis of three tropane alkaloids in goji berries.     

毛细管电泳-电致化学发光检测法分离测定中药马尿泡中的托烷类生物碱成分

以铕离子掺杂类普鲁士蓝(Eu-PB)化学修饰铂电极为工作电极,采用毛细管电泳-电致化学发光检测法对4种托烷类生物碱成分（如山莨菪碱、东莨菪碱、阿托品和樟柳碱）进行了分离检测。考察了氧化电位值、运行缓冲液酸度、盐浓度和甲醇含量等实验条件对电泳分离效果及检测灵敏度的影响。在优化的实验条件下,以20 mmol/L的磷酸盐(pH 8.0)-7%(体积分数）甲醇为运行液,各组分在6 min内可达到基线分离,其峰面积的相对标准偏差小于5.0%,迁移时间的相对标准偏差小于1.1% (n=12)。并将该法成功地应用于测定中药马尿泡根茎中的山莨菪碱和东莨菪碱的含量,其含量平均值分别为27.8 g/kg和4.43 g/kg。样品的加标回收率为97.8%～102%。     

Chromatographic determination of some tropane alkaloids in Datura metel

An analytical scenario has been developed for the determination of some tropane alkaloids in Datura metel, including sample preparation by solid-phase extraction and chromatographic (GC and HPLC) determination of the analytes. The proposed scenario is rapid and provides the complete recovery of alkaloids; the analytical range for atropine and scopolamine is 0.01–30 mg/mL. The procedure ensures the simultaneous determination of compounds from other classes of substances.     

Simultaneous determination of atropine,anisodamine, and scopolamine in plant extract by nonaqueous capillary electrophoresis coupled with electrochemiluminescence and electrochemistry dual detection

A rapid and simple method was demonstrated for the analysis of atropine, anisodamine, and scopolamine by nonaqueous capillary electrophoresis (NACE) coupled with electrochemiluminescence (ECL) and electrochemistry (EC) dual detection. The mixture of acetonitrile (ACN) and 2-propanol containing 1 M acetic acid (HAc), 20 mM sodium acetate (NaAc), and 2.5 mM tetrabutylammonium perchlorate (TBAP) was used as the electrophoretic buffer. Although a short capillary of 18 cm was used, the decoupler was not needed and the separation efficiency was good. The linear ranges of atropine, anisodamine, and scopolamine were 0.5–50, 5–2000, and 50–2000 μM, respectively. For six replicate measurements of 100 μM scopolamine, 15 μM atropine, and 200 μM anisodamine, the RSDs of ECL intensity, EC current, and migration time were less than 3.6%, 4.5%, and 0.3%, respectively. In addition, because the organic buffer was used, the working electrode (Pt) was not easily fouled and did not need reactivation. The method was also applied for the determination of these three alkaloids in Flos daturae extract.     

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.     

Development and validation of a rapid and sensitive assay for the determination of anisodamine in 50 μL of beagle dog plasma by LC–MS/MS

A simple, rapid, high‐throughput, and highly sensitive LC–MS/MS was developed to determine anisodamine in a small volume (50 μL) of beagle dog plasma using atropine sulfate as the internal standard. The analyte and internal standard were isolated from 50 μL plasma samples after a one‐step protein precipitation using Sirocco 96‐well protein precipitation filtration plates. The separation was accomplished on a Hanbon Hedera CN column (100 × 4.6 mm, 5 μm) and the run time was 4 min. A Micromass Quatro Ultima mass spectrometer equipped with an ESI source was operated in the multiple reaction monitoring mode with the precursor‐to‐product ion transitions m/z 306.0→140.0 (anisodamine) and 290.0→123.9 (atropine) used for quantitation. The method was sensitive with a low LOQ of 0.05 ng/mL, and good linearity in the range 0.05–50 ng/mL for anisodamine (r² ≥ 0.995). All the validation data, such as accuracy, intra‐ and interrun precision, were within the required limits. The method was successfully applied to the pharmacokinetic study of anisodamine hydrochloride injection in beagle dogs.     

Development of CE-C4D Method for Determination Tropane Alkaloids

A fast method for the determination of tropane alkaloids, using a portable CE instrument with a capacitively coupled contactless conductivity detector (CE-C⁴D) was developed and validated for determination of atropine and scopolamine in seeds from Solanaceae family plants. Separation was obtained within 5 min, using an optimized background electrolyte consisting of 0.5 M acetic acid with 0.25% (w/v) β-CD. The limit of detection and quantification was 0.5 µg/mL and 1.5 µg/mL, respectively, for both atropine and scopolamine. The developed method was validated with the following parameters—precision (CV): 1.07–2.08%, accuracy of the assay (recovery, RE): 101.0–102.7% and matrix effect (ME): 92.99–94.23%. Moreover, the optimized CE-C⁴D method was applied to the analysis of plant extracts and pharmaceuticals, proving its applicability and accuracy.     

A sensitive and efficient procedure for the high throughput determination of banned aromatic amines in textiles and leather products aided by advanced sample composition

We have developed and validated a quantitative liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI MS/MS) procedure for the simultaneous determination of seven natural and semisynthetic tropane alkaloids in plasma: atropine (d-hyoscyamine/l-hyoscyamine), cocaine, homatropine, ipratropium, littorine, N-butylscopolamine, and scopolamine. Plasma and serum samples were precipitated for deproteinization (recovery 88–94%), followed by reversed-phase-based liquid chromatography prior to positive electrospray ionization for detection by multiple reaction monitoring using a linear ion trap quadrupole mass spectrometer. All analytes were quantified using cocaine-d3 as an internal standard suitable and reliable for robust, precise (coefficient of variation 2–13%), and accurate (87–122%) measurement within a linear range of 3 orders of magnitude (0.05–50 ng/ml plasma). The method was exemplarily applied to stability studies in phosphate-buffered saline, human serum, and rabbit serum. Each alkaloid was incubated separately and samples were taken at distinct incubation time points. Supernatants of diverse alkaloids at corresponding time points were pooled and subjected to simultaneous LC-ESI MS/MS quantification. This combinatorial analysis design allowed us to analyze the stability of samples with a drastically reduced number of chromatographic runs. In the presence of rabbit serum, all tropane alkaloids tested were degraded significantly within minutes to hours, with the exception of the stable semisynthetic compounds ipratropium and N-butylscopolamine. In contrast, in the presence of equal concentrations of human serum, no degradation was observed for any of the compounds, with the exception of cocaine. Relevant enzymes involved in enzymatic degradation are discussed.     

Determination of sertraline in rat plasma by HPLC and fluorescence detection and its application to in vivo pharmacokinetic studies

A simple, rapid, and sensitive HPLC method based on 9H‐fluoren‐9‐ylmethyl chloroformate derivatization for the quantification of sertraline in rat plasma has been developed, requiring a plasma sample of only 0.1 mL, which was deproteinized and derivatized for 5 min in two single steps. The obtained derivative was stable at room temperature and was determined by HPLC using a fluorescence detector. The analytical column was a C(18) column and the mobile phase was acetonitrile and water (80:20, v/v). Calibration curves were linear in the range of 10–500 ng/mL. The limit of detection was approximately 3 ng/mL, and the lower limit of quantification was established at 10 ng/mL. The bias of the method was lower than 10%, and the within day as well as between day, relative standard deviations were lower than 12%. This analytical method was successfully applied to characterize sertraline pharmacokinetics in rats following intravenous (t_1/2 = 213 ± 48 min, Cl = 43.1 ± 8.7 mL/min, V_d = 11560 ± 1861 mL) and oral (C_max = 156 ± 76 ng/mL, t_max = 63.8 ± 16.3 min) administration of 2 and 5 mg, respectively.     

Application of high performance capillary electrophoresis on toxic alkaloids analysis

We employed CE to identify mixtures of the toxic alkaloids lappaconitine, bullatine A, atropine sulfate, atropine methobromide, scopolamine hydrobromide, anisodamine hydrobromide, brucine, strychnine, quinine sulfate, and chloroquine in human blood and urine, using procaine hydrochloride as an internal standard. The separation employed a fused-silica capillary of 75 microm id x 60 cm length (effective length: 50.2 cm) and a buffer containing 100 mM phosphate and 5% ACN (pH 4.0). The sample was injected in a pressure mode and the separation was performed at a voltage of 16 kV and a temperature of 25 degrees C. The compounds were detected by UV absorbance at wavelengths of 195 and 235 nm. All the ten alkaloids were separated within 16 min. The method was validated with regard to precision (RSD), accuracy, sensitivity, linear range, LOD, and LOQ. In blood and urine samples, the detection limits were 5-40 ng/mL and linear calibration curves were obtained over the range of 0.02-10 microg/mL. The precision of intra- and interday measurements was less than 15%. Electrophoretic peaks could be identified either by the relative migration time or by their UV spectrum.     

Ligand fishing and identification of acetylcholinesterase inhibitors in Mahonia bealei (Fort.) Carr. using high performance liquid chromatography-mass spectrometry

Abstract

A fishing platform was developed using immobilized capillary enzyme reactors in combination with liquid chromatography-mass spectrometry (LC-MS) to fish acetylcholinesterase inhibitor (AChEI) from Mahonia bealei (Fort.) Carr. (M. bealei) extract. Four potential AChEIs (columbamine, jatrorrhizine, berberine, and palmatine) were successfully screened out and identified. Their AChE inhibitory activities were further verified by an in vitro assay with IC₅₀ values of 0.93?±?0.12?μg/mL (columbamine), 3.50?±?0.15?μg/mL (jatrorrhizine), 2.51?±?0.12?μg/mL (berberine), and 1.52?±?0.13?μg/mL (palmatine). A synergy study of these AChEIs was subsequently investigated. In comparison with the IC₅₀ value of M. bealei extract (IC₅₀=0.83?±?0.21?μg/mL), it can be stated that M. bealei extract is much more active than any single AChEI. Thereafter, the determination of these four alkaloids were investigated and AChE inhibitory effect were compared in terms of the extract and corresponding contents of these four alkaloids. The inhibitory effects of extract at each concentration were stronger than four alkaloids mixture. The results demonstrated that not the four AChEI mixture cause the synergistic effect but rather than the concentrations or ratios of these AChEIs play a vital role in their synergy study.     

Determination of bakkenolide A in rat plasma using liquid chromatography/tandem mass spectrometry and its application to a pharmacokinetic study

A sensitive rapid analytical method was established and validated to determine the bakkenolide A (BA) in rat plasma. This method was further applied to assess the pharmacokinetics of BA in rats receiving a single dose of BA. Liquid chromatography tandem mass spectrometry in multiple reaction monitoring mode was used in the method, and costundide was used as internal standard. A simple protein precipitation based on methanol was employed. The combination of a simple sample cleanup and short chromatographic running time (2.4 min) increased the throughput of the method substantially. The method was validated over the range of 1–1000 ng/mL with a correlation coefficient > 0.99. The lower limit of quantification was 1 ng/mL for BA in plasma. Intra‐ and inter‐day accuracies for BA were 93–112% and 103–104%, respectively, and the inter‐day precision was less than 15%. After a single oral dose of 20 mg/kg of BA, the mean peak plasma concentration (C_max) of BA was 234.7 ± 161 ng/mL at 0.25 h. The area under the plasma concentration–time curve (AUC_{0–24 h}) was 535.8 ± 223.7 h·ng/mL, and the elimination half‐life (T_1/2) was 5.0 ± 0.36 h. In case of intravenous administration of BA at a dosage of 2 mg/kg, the area under the plasma concentration–time curve (AUC_{0–24 h}) was 342 ± 98 h?ng/mL, and the elimination half‐life (T_1/2) was 5.8 ± 0.7 h. Based on the results, the oral bioavailability of BA in rats at 20 mg/kg is 15.7%. Copyright © 2012 John Wiley & Sons, Ltd.     

LC‐MS/MS determination of bakkenolide D in rats plasma and its application in pharmacokinetic studies

A sensitive and rapid liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed and validated for determination of bakkenolide D (BD), which was further applied to assess the pharmacokinetics of BD. In the LC‐MS/MS method, the multiple reaction monitoring mode was used and columbianadin was chosen as internal standard. The method was validated over the range of 1–800 ng/mL with a determination coefficient >0.999. The lower limit of quantification was 1 ng/mL in plasma. The intra‐ and inter‐day accuracies for BD were 91–113 and 100–104%, respectively, and the inter‐day precision was <15%. After a single oral dose of 10 mg/kg of BD, the mean peak plasma concentration of BD was 10.1 ± 9.8 ng/mL at 2 h. The area under the plasma concentration–time curve (AUC_{0–24 h}) was 72.1 ± 8.59 h ng/mL, and the elimination half‐life (T_1/2) was 11.8 ± 1.9 h. In case of intravenous administration of BD at a dosage of 1 mg/kg, the AUC_{0–24 h} was 281 ± 98.4 h?ng/mL, and the T_1/2 was 8.79 ± 0.63 h. Based on these results, the oral bioavailability of BD in rats at 10 mg/kg is 2.57%. Copyright © 2013 John Wiley & Sons, Ltd.     

Therapeutic monitoring of amphotericin B in Saudi ICU patients using UPLC MS/MS assay

Amphotericin B (AmB) is the first‐line agent for the treatment of life‐threatening invasive fungal infections. The aim of this study was to monitor AmB in critically ill Saudi patients in ICU after i.v. administration of 0.68 ± 0.1 mg/kg/day Fungizone®. A selective, sensitive and precise UPLC MS/MS method was developed to measure AmB concentrations in these patients. Seven ICU patients with creatinine clearance (ClCr) >40 mL/min were included. AmB levels were analyzed using a Waters Aquity UPLC MS/MS system, a BEH Shield RP₁₈ column and detection via electrospray ionization source with positive ionization mode. The precision and accuracy of the developed UPLC method in the concentration range of 200–4000 ng/mL show no significant difference among inter‐ and‐intra‐day analysis (p > 0.05). Linearity was observed over the investigated range with correlation coefficient, r > 0.995 (n = 6/day). The pharmacokinetics of AmB in these patients, at steady state, showed a high terminal half‐life of 124.6 ± 73.4 h, with a highest concentration of 513.9 ± 281.1 ng/mL, a lowest concentration 316.4 ± 129.0 ng/mL and a mean clearance 91.1 ± 39.2 mL/h/kg. The pharmacokinetics of AmB in critically ill Saudi patients in ICU was studied using a fully validated assay. A weak correlation (r = ?0.22) of AmB Cl with ClCr was obtained, which suggests the need for further investigation in a larger population. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of a hydrophilic paclitaxel derivative, 7‐xylosyl‐10‐deacetylpaclitaxel in rat plasma by LC–MS/MS

A LC‐MS/MS method for the determination of a hydrophilic paclitaxel derivative 7‐xylosyl‐10‐deacetylpaclitaxel in rat plasma was developed to evaluate the pharmacokinetics of 7‐xylosyl‐10‐deacetylpaclitaxel in the rats. 7‐Xylosyl‐10‐deacetylpaclitaxel and docetaxel (IS for 7‐xylosyl‐10‐deacetylpaclitaxel) were extracted from rat plasma with acetic ether and analyzed on a Hypersil C₁₈ column (4.6 × 150 mm i.d., particle size 5 µm) with the mobile phase of ACN/0.05% formic acid (50:50, v/v). The analytes were detected using an ESI MS/MS in the multiple reaction monitoring mode. The standard curves for 7‐xylosyl‐10‐deacetylpaclitaxel in plasma were linear (r >0.999) over the concentration range of 2.0–1000 ng/mL with a weighting of 1/concentration². The method showed a satisfactory sensitivity (2.0 ng/mL using 50 µL plasma), precision (CV ≤ 10.1%), accuracy (relative error ?12.4 to 12.0%), and selectivity. This method was successfully applied to the pharmacokinetic study of 7‐xylosyl‐10‐deacetylpaclitaxel in rat plasma after intravenous administration of 7‐xylosyl‐10‐deacetylpaclitaxel to female Wistar rats. Copyright © 2008 John Wiley & Sons, Ltd.     

Quantitative determination of a novel enantiomeric tropane analog, (−)‐satropane,in biological fluids using liquid chromatography/tandem mass spectrometry

A sensitive, accurate and precise liquid chromatography–tandem mass spectrometry method was developed for the determination of (?)‐satropane (3α‐paramethyl‐benzenesulfonyloxy‐6β‐acetoxy‐tropane) in rabbit aqueous humor. Since (?)‐satropane may be absorbed from the aqueous humour with resultant systemic side effects, the LC‐MS/MS method was also evaluated for its applicability in analyzing plasma samples containing this compound. (?)‐Satropane and phentolamine (the internal standard, represented as IS) were detected by multiple reaction monitoring using the transitions m/z 354–182 and 282–212, respectively. The calibration curve was linear over the ranges 2–500 and 5–1000 ng/mL, and the values of the lower limit of quantification were 2 and 5 ng/mL for the microdialysis dialysate and rat plasma samples, respectively. The intra‐day and inter‐day precision and accuracy were better than 8.6 and 6.00%, respectively, in both matrices investigated. The absolute recovery of the plasma samples was more than 76.30%. The average matrix effects of (?)‐satropane were 91.72 and 83.05% in the microdialysis dialysate and plasma samples, respectively. The validated method was successfully applied to analyze (?)‐satropane in microdialysis dialysate and rat plasma samples, and this assay has been used to quantify (‐)‐satropane in the pharmacokinetic and toxicokinetic studies in our laboratory. Copyright © 2009 John Wiley & Sons, Ltd.