An Efficient and Rapid HPLC-QQQ-MS Method for the Quantitation of Tropane Alkaloids in Medicinal Plants

We developed an HPLC-QQQ-MS method to rapidly quantify three major tropane alkaloids in Anisodus tanguticus. Conventional HPLC lacks sensitivity for tropane alkaloids. This method uses highly sensitive mass spectrometry to solve this problem.

To begin, accurately weigh 10.0 milligrams of hyoscyamine, anisodamine hydrobromide, and scopolamine hydrobromide reference standards. Place them into a separate 10 milliliter volumetric flask. Add chromatographic grade methanol to the marked line in each volumetric flask.

Dissolve the standards thoroughly with ultrasonic treatment to prepare single standard stock solutions with a concentration of 1.0 milligram per milliliter. Then, transfer the stock solutions to brown reagent bottles and store it four degrees Celsius in the dark. To prepare a mixed standard intermediate solution of 10 micrograms per milliliter, pipette 100 microliters of each previously prepared stock solution into a 10 milliliter volumetric flask.

Then, add chromatographic grade methanol to the marked line to dilute the solution. Next, perform freeze drying of fresh Sophora flavescens material. Then, using a high-speed grinder, pulverize the dried material into a homogeneous powder.

Sieve the powder through a 0.25 millimeter standard test sieve. Accurately weigh 20 milligrams of the sieved powder. Transfer it into a 100 milliliter volumetric flask and add chromatographic grade methanol to just below the marked line.

Next, perform ultrasonic extraction at room temperature for 30 minutes. Then, remove the volumetric flask and allow it to cool to 25 degrees Celsius at room temperature. Add chromatographic grade methanol to the 100 milliliter marked line and vortex to mix for two minutes.

Then, pipette one milliliter of the prepared extract into a 100 milliliter volumetric flask. Add methanol to the marked line. Shake well to mix thoroughly and obtain the mother solution for HPLC injection.

For sample filtration, prepare a one milliliter disposable needle-free syringe and a 0.22 micrometer organic-phase microporous membrane. Aspirate 1.2 milliliters of the mother solution for injection into the syringe and filter the solution by pushing it slowly through the membrane. Collect the filtrate into a two milliliter sample vial with an inner insert and seal the vial tightly with a cap.

Prepare mobile phase A by measuring 1000 milliliters of ultrapure water. Add 1.0 milliliter of chromatographic grade formic acid and mix uniformly using a magnetic stirrer for 10 minutes. Next, prepare mobile phase B by using chromatographic grade methanol directly without additional treatment.

Degas mobile phases A and B at 40 kilohertz for 15 minutes using separate ultrasonic degassing units. Connect the degassed mobile phases in separate solvent bottles to the corresponding A and B lines of the HPLC system. Use a C18 column for chromatographic separation.

Equilibrate the column with methanol and water at a ratio of 80 to 20 for 30 minutes at a flow rate of 0.3 milliliters per minute. Set the column oven temperature to 30 degrees Celsius and preheat for 10 minutes in advance. Then, set the flow rate to 0.60 milliliters per minute, the injection volume to two microliters, and the detection wavelength to 210 nanometers.

After that, set the gradient elution program with the presented conditions. Perform mass spectrometric detection using a triple quadrupole mass spectrometer equipped with an electrospray ionization source operating in positive ionization mode. Set the nebulizer pressure to 15 pounds per square inch, capillary voltage to 4, 000 volts, gas temperature to 300 degrees Celsius, and gas flow to 11 liters per minute.

Then, set the parameters for multiple reaction monitoring ion pairs. To identify precursor ions, directly infuse individual standard solutions in MS2 scan mode. After method validation, perform data acquisition in multiple reaction monitoring mode.

Use the optimized parameters, including precursor ion, product ion transitions, fragmentor voltage, and collision energy for each analyte as presented here. Launch the qualitative analysis software. Select file and choose import data to import all collected data files.

Check the peak symmetry and retention time consistency of the target peaks in each extracted ion chromatogram. In the quantitation module, select MRM transition. Input the precursor ion and product ion pairs for each component and extract the corresponding extracted ion chromatograms.

Calculate the peak area for quantitative analysis. All three target tropane alkaloids were successfully separated within 20 minutes under the optimized HPLC conditions. The resolution between scopolamine and anisodamine was 8.67.

While that between anisodamine and hyoscyamine reached 11.58, all resolution values significantly exceeded the baseline separation criterion of 1.5. All analytes showed good linearity over a concentration range of 0.014 to 0.320 micrograms per milliliter with correlation coefficients greater than 0.99. Mean recoveries were 101.44%for scopolamine, 100.25%for anisodamine, and 99.56%for hyoscyamine, with relative standard deviations of 1.85%2.35%and 1.9%respectively.

Quantification of three tropane alkaloids in 10 Anisodus tanguticus samples showed that scopolamine content ranged from 0.3667 milligrams per gram in sample six to 1.0356 milligrams per gram in sample seven. Anisodamine content ranged from 0.0269 milligrams per gram in sample six to 0.3590 milligrams per gram in sample 10. Hyoscyamine content ranged from 0.2930 milligrams per gram in sample two to 1.4989 milligrams per gram in sample 10, with higher levels observed in samples four, seven, nine and 10.

This protocol allows rapid, accurate quantification of three major tropane alkaloids. The key challenge is ensuring instrument procedure and standardized sample preparation for accurate measurement. This method can be extended to explore and quantify other metabolites in Anisodus tanguticus.