1. Hailey Benson, Nathaniel Holte, and Catherine Haustein* Department of Chemistry, Central College, Pella, IA Mucuna pruriens, velvet beans, have been used as a nerve tonic for nervous system disorders because of the high concentration of L-dopa found in the seed. This seed has been used in many studies to help fight the symptoms of Parkinson’s disease (1,4). Velvet beans have also been used as a testosterone booster and muscle builder because of their androgenic properties (2). Velvet beans have been tested and proven to help with fertility properties of diabetic male rats (3). In other countries, Mucuna pruriens have been used to treat snake bites (4). The purpose of this study was to develop a method for the determination of L-dopa in a natural sample. The goal was to have minimal sample preparation and to minimize using hazardous solvents. This provides a “green” HPLC laboratory for an Analytical Chemistry class. Stock solution should be made in advance 3-4 g of velvet beans from Georgia Vines or Hawaiian Baby Woodrose Vines Beans soaked in a 600 mL beaker with 300 mL DI water for ca. 72 h at varied temperature conditions, with the exception of the beans treated with EtOH Bean liquid water placed in a 500 mL volumetric flask and diluted with DI water at ca. 48 h Syringe filtration was performed before injection into HPLC at ca. 48 h and ca. 72 h Remaining liquid was poured back into 600 mL beaker for beans to continue soaking HPLC method conditions: Agilent 1100 series HPLC with internal variable UV wavelength detector (set at 280 nm) and Luna C-18 non-polar, reverse-phase column. Chromatographic conditions were as follow: injection volume, 20µ; column temperature 30 °C; flow rate 1.0 mL/min. The mobile phase was pH 2.4 citrate-phosphate buffer, and run time was 12 minutes, t R =8.3. 8 trials of the standard solution generated the calibration curve 38 trials of the Mucuna pruriens were run Method and Mucuna pruriens were given to the fall 2014 analytical, spring 2015 analytical, and spring 2015 instrumental analysis classes. The students had no previous experience with HPLC. The results from each class were compared to the individual results obtained by Hailey Benson, shown in Table 1. Stock solution should be made in advance 3-4 g of velvet beans from Georgia Vines or Hawaiian Baby Woodrose Vines Beans soaked in a 600 mL beaker with 300 mL DI water for ca. 72 h at varied temperature conditions, with the exception of the beans treated with EtOH Bean liquid water placed in a 500 mL volumetric flask and diluted with DI water at ca. 48 h Syringe filtration was performed before injection into HPLC at ca. 48 h and ca. 72 h Remaining liquid was poured back into 600 mL beaker for beans to continue soaking HPLC method conditions: Agilent 1100 series HPLC with internal variable UV wavelength detector (set at 280 nm) and Luna C-18 non-polar, reverse-phase column. Chromatographic conditions were as follow: injection volume, 20µ; column temperature 30 °C; flow rate 1.0 mL/min. The mobile phase was pH 2.4 citrate-phosphate buffer, and run time was 12 minutes, t R =8.3. 8 trials of the standard solution generated the calibration curve 38 trials of the Mucuna pruriens were run Method and Mucuna pruriens were given to the fall 2014 analytical, spring 2015 analytical, and spring 2015 instrumental analysis classes. The students had no previous experience with HPLC. The results from each class were compared to the individual results obtained by Hailey Benson, shown in Table 1. The L-dopa calibration curve was linear with a slope of 26.624 (Figure 1). As shown in Table 1, the individual and both classroom results fit the known data of 3- 6% L-dopa (5). Because the beans were obtained over a period of time and were from different sources, this could account for slight variation between samples used in the classroom setting. As shown in Table 2, soaking the beans for 3 days produced the best overall results, however, whole beans soaking at room temperature yielded the best 2 day results. The L-dopa calibration curve was linear with a slope of 26.624 (Figure 1). As shown in Table 1, the individual and both classroom results fit the known data of 3- 6% L-dopa (5). Because the beans were obtained over a period of time and were from different sources, this could account for slight variation between samples used in the classroom setting. As shown in Table 2, soaking the beans for 3 days produced the best overall results, however, whole beans soaking at room temperature yielded the best 2 day results. Special thanks to Aleesha Godwin for original research and Samantha Wills for developing the method. 1.Taylor, Leslie. Technical Data Report for Velvet Bean Mucuna Pruriens. Austin: Sage, 2003. Web. 13 Sept. 2014.. 2. "Mucuna Pruriens Extract Benefit, Side Effects, Herb Supplement for Parkinson's Disease, L Dopa." Mucuna Pruriens Extract Benefit, Side Effects, Herb Supplement for Parkinson's Disease, L Dopa. N.p., n.d. Web. 09 Dec. 2014. 3. Prakash, Seppan. "Effect of Mucuna Pruriens (Linn.) on Sexual Behavior and Sperm Parameters in Streptozotocin-Induced Diabetic Male Rat." J Sex Med 9 (2012): 3066- 078. Print. 4. Tan, Nget Hong. "The Protective Effect of Mucuna Pruriens Seeds against Snake Venom Poisoning." Journal of Ethnopharmacology 123 (2009): 356-58. Print. 5. Siddhuraju, Perumal. "Effect of Various Domestic Processing Methods on Antinutrients and in Vitro Protein and Starch Digestibility of Two Indigenous Varieties of Indian Tribal Pulse, Mucuna Pruriens Var. Utilis." J. Agric. Food Chem 49.49 (2001): 3058- 067. Print. 1.Taylor, Leslie. Technical Data Report for Velvet Bean Mucuna Pruriens. Austin: Sage, 2003. Web. 13 Sept. 2014.. 2. "Mucuna Pruriens Extract Benefit, Side Effects, Herb Supplement for Parkinson's Disease, L Dopa." Mucuna Pruriens Extract Benefit, Side Effects, Herb Supplement for Parkinson's Disease, L Dopa. N.p., n.d. Web. 09 Dec. 2014. 3. Prakash, Seppan. "Effect of Mucuna Pruriens (Linn.) on Sexual Behavior and Sperm Parameters in Streptozotocin-Induced Diabetic Male Rat." J Sex Med 9 (2012): 3066- 078. Print. 4. Tan, Nget Hong. "The Protective Effect of Mucuna Pruriens Seeds against Snake Venom Poisoning." Journal of Ethnopharmacology 123 (2009): 356-58. Print. 5. Siddhuraju, Perumal. "Effect of Various Domestic Processing Methods on Antinutrients and in Vitro Protein and Starch Digestibility of Two Indigenous Varieties of Indian Tribal Pulse, Mucuna Pruriens Var. Utilis." J. Agric. Food Chem 49.49 (2001): 3058- 067. Print. All results obtained fell within the known range of L-dopa concentrations. The curves for individual and class results matched each other and the individual standard curve. This shows that this is a lab that can be easily performed by students with no prior training in HPLC. The mobile phase is aqueous and much less hazardous than laboratories using organic solvents. This procedure represents an improvement on other HPLC laboratories found in analytical chemistry text books, while providing a more sustainable analytical chemistry lab. Georgia Vines Velvet Beans Days soakedL-dopa %StDev L-dopa Individual Spring 201523.8%0.1 Analytical Fall 201423.7%2.5 Instrumental Spring 2015 23.4%0.03 Analytical Spring 201535.5%0.3 Table 1. Comparison of individual and class results for percentages of L-dopa in whole Mucuna pruriens. Table 2. Most to least effective method of extraction. MethodL-dopa % Three-day, room temperature, whole bean, water soak 4.69 Three-day, room temperature, ground bean, water soak 4.09 Two-day, room temperature, whole bean, water soak3.76 Two-day, boiled, whole bean, water soak3.48 Three-day, refrigerated, whole bean, water soak3.41 One-day, boiled, whole bean, water soak2.98 Two-day, refrigerated, whole bean, water soak2.86 Two-day, room temperate, ground bean, water soak2.11 Two-day, heated, whole bean, water soak1.31 Three-day, heated, whole bean, water soak1.16 Two-day, room temperature, whole bean, ethanol soak 0.897 Three-day, room temperature, whole bean, ethanol soak 0.627 Figure 1. Calibration curve of L- dopa. Figure 2. Chromatogram of bean water.