The development of a detection method of 15 pyrrolizidine alkaloids in weed, soil, fresh tea leaves and dry tea sample can provide technical support for exploring the source of pyrrolizidine alkaloids contamination in tea. Compared with the solid phase extraction, the adsorbent method for soil sample can save time, lower cost and better recoveries for pyrrolizidine alkaloids analysis. To start the extraction on the preheated dried tea products, fresh tea leaves or weeds, weigh one gram of a sample in a 50 milliliter centrifuge tube, and add 10 milliliters of 0.1 moles per liter sulfuric acid solution into the tube.
Next, vortex the sample for two minutes if it is to be analyzed using solid phase extraction, while for one minute if the same is to be analyzed using adsorbent cleanup. Then subject the sample to ultrasonic extraction for 15 minutes, followed by centrifugation at 9, 390 G for 10 minutes at room temperature. Then using a plastic-tipped dropper, transfer the supernatant solution into a 50 milliliter centrifuge tube.
After performing the extraction twice, combine the supernatants in the tube. To start the extraction on the prepared soil, weigh one gram of a sample in a 50 milliliter centrifuge tube. Add 0.1 milliliters of 0.1 moles per liter trisodium citrate solution to adjust the soil pH to 6.0, and allow the sample to stand for two minutes.
Then add 10 milliliters of 0.1 moles per liter sulfuric acid methanol solution into the tube, and vortex the sample for two minutes, followed by shaking for 30 minutes. Next, perform ultrasonic extraction for 30 minutes as demonstrated before. Then centrifuge the sample at 9, 390 G for 10 minutes.
And using a plastic-tipped dropper, transfer the supernatant into a 50 milliliter centrifuge tube. After performing the extraction twice, combine the supernatants. Activate the solid phase extraction or SPE cartridge with five milliliters of methanol followed by five milliliters of deionized water.
After activation, add 10 milliliters of the supernatant from the sample extraction to the pre-activated cartridge. After the sample solution level reach the upper layer of cartridges, elute the sample matrix with five milliliters of 1%formic acid solution, followed by five milliliters of methanol, and discard the eluents. Next, elute the analytes with five milliliters of methanol containing 0.5%ammonia water.
Filter the eluent through a 0.22 micron membrane filter to analyze by UPLC-MS/MS. To perform sample cleanup using adsorbent, prepare a 10 milliliter centrifuge tube with the adsorbent compromising GCB, PSA and C18 in ratio 10 to 20 to 15 milligrams. Then add two milliliters of the supernatant obtained after the sample treatment.
Next, vortex the sample and adsorbent mixture for one minute and centrifuge at 9, 390 G for eight minutes at room temperature. Filter one milliliter of the resulting supernatant through a 0.22 micron membrane filter into a sample vial before analyzing it by UPLC-MS/MS. In the study, a higher percentage recovery of 15 pyrrolizidine alkaloids from fresh tea leaves, dried tea samples and weed were obtained using the adsorbent purification technique as compared to the SPE cartridge method.
The recoveries of the alkaloids in soil using adsorbent purification were 79 to 111%In the fresh tea leaves collected from the five sampling sites, higher content of intermedine N-oxide was detected than senecionine. Senecionine was not detected in mature leaves from sampling sites one and two. Among the 11 pyrrolizidine alkaloids, only intermedine N-oxide was detected in the soil, while senecionine and intermedine N-oxide were detected in different parts of the tea plants.
Additionally, the content of the Intermedine N-oxide in one bud with two leaves was found to be the highest. While attempting this first, the most important thing is to add 0.1 milliliters of 0.1 moles per liter trisodium citrate solution to adjust your soil pH to 6.0. This method can be applied to the analysis of alkaloids in other edible plants, and it also provides natural technique for exploring the source of alkaloids in them.
