DEVELOPMENT AND CHARACTERIZATION OF GENETIC RESOURCES FOR AGRONOMIC AND QUALITY TRAITS USING GENOMIC TOOLS
Location: Dale Bumpers National Rice Research Center
Title: Single kernel method for detection of 2-acetyl-1-pyrroline in aromatic rice germplasm using SPME-GC/MS
Submitted to: American Society for Mass Spectrometry
Publication Type: Abstract Only
Publication Acceptance Date: May 2, 2010
Publication Date: May 23, 2011
Citation: Bryant, R.J., Mcclung, A.M., Grimm, C.C. 2011. Single kernel method for detection of 2-acetyl-1-pyrroline in aromatic rice germplasm using SPME-GC/MS. American Society for Mass Spectrometry. Poster 481.
Aromatic rice or fragrant rice, (Oryza sativa L.), has a strong popcorn-like aroma due to the presence of a five-membered N-heterocyclic ring compound known as 2-acetyl-1-pyrroline (2-AP). To date, existing methods for detecting this compound in rice require the use of several kernels. The lack of an efficient method to reliably detect the compound in individual kernels prevents the detection of expensive aromatic rice that has been blended with inexpensive non-aromatic rice. Such a method will also aid rice breeders in developing new cultivars by screening progenies segregating for aroma and prevent contamination of seedlots. SPME-GC/MS was used to develop a method that could distinguish aromatic from non-aromatic rice kernels using brown or milled rice.
Rice kernels were dehulled and milled prior to analysis. One kernel was placed in a 2 ml vial and 10 µl of water containing 1 ng of TMP was placed on the kernel. After preheating the samples for 5 min at 80oC, volatile compounds, including 2-AP, were adsorbed onto a SPME fiber. The fiber was desorbed for 25 sec on a Varian Chrompack CP-3800 GC with a Saturn 2000 MS attached. The MS was operated in the scan mode from m/z 40-200. The target ion used for 2-AP was 83 m/z and 121 m/z for TMP with qualifying ions being 111 m/z at 43% relative abundance and 79 m/z at 26% relative abundance for 2-AP and TMP, respectively.
Retention times for 2-AP and TMP were 5.5 min and 6.6 min, respectively. The response for 2-AP was linear up to 46 ng with a R2=0.988 and 0.986 in the presence of and without a rice kernel, respectively. Reproducibility (N=3) over a range of 1–46 ng shows that this method is suitable for relative comparison of 2-AP concentration in rice kernels. Relative recovery factor of 2-AP in the presence of a rice kernel was found to be 0.823 ± 0.006. Ten kernels of Jasmine 85 (aromatic rice) were mixed with 10 kernels of non-aromatic rice in a zip-locked bag and kept at room temperature (21oC) overnight. Kernels were randomly removed and each kernel was analyzed using the method. The method correctly detected the aromatic kernels in three different runs. The concentration of 2-AP was determined for 10 milled kernels of Jasmine 85. The concentration of 2-AP in the headspace above each kernel ranged from 137-276 ng g-1. The concentration using the area of the 83 m/z ion of 2-AP to the concentration obtained using the above method were compared and, although the value using the graph was higher (521-1305 ng g-1), they were found to have a correlation of 0.954. The method was tested on paddy rice and brown rice. Paddy rice gave the lowest recovery and highest variability. Most of the paddy rice samples showed area counts 50,000 for the 83 m/z ion. The low recovery from paddy rice may be due to the hull insulating the kernels thus keeping it from gelatinizating and releasing 2-AP. Recovery from brown rice was similar to that of milled rice, this would increase the efficiency of the evaluation by elimination the milling step.
This method can distinguish aromatic from non-aromatic rice kernels and it can be performed accurately on milled or brown rice.