Core Capabilities

Many projects involve the quantitation of predetermined analytes in a complex biological matrix. This type of targeted analysis requires that analytical methods not only be developed, but also validated for specificity, linearity, range, precision, detection limit, quantitation limit, accuracy, robustness, and solution stability. An internal standard is used during quantitation. This is done to correct for the loss of analyte during sample preparation or introduction into the instrument. Stable isotopes are often utilized as internal standards. The calibration curve below is a plot of the ratio of the analyte signal to the internal standard signal (y-axis) as a function of the analyte concentration of the standards.

Indole Acetic Acid (IAA)

Indole-3-acetic acid, also known as IAA, is a member of the group of phytohormones called auxins. IAA is generally considered to be the most important native auxin, inducing cell elongation and cell division. Amber Jannasch in the MPF has developed protocols to successfully extract IAA from plant tissue (Arabidopsis and poplar), utilizing solid phase extraction (SPE) for sample clean-up and methylation prior to GC/MS analysis. Quantitation is performed using a 13C6-IAA analog. The detection limit is low picograms injected on-column.

Amino Acids

Amino Acids Amino Acids have been successfully analyzed using GC/MS. The samples are derivatized to produce a TBDMS (tert-butyldimethylsilyl) derivative. This label will derivatize both the carboxyl and amino group of the amino acid, as well as any hydroxyl, carboxyl, thiol or primary and secondary amine that may appear in the side chain. These derivatives are 10,000 times more stable than traditional TMS ethers. The separation can be conducted in 30 minutes. The detection limit is low picograms injected on-column. Useful internal standards are 2-aminobutyric acid and beta-alanine.

Carbohydrate Analysis

Carbohydrate Analysis Sugars (monosaccharides) represent a challenging class of compounds to analyze. We have been successful in analyzing this class using GCxGC/MS. The samples are derivatized, by performing a methoxymation to prevent ring formation followed by a trimethylsilylation with MSTFA.

Plant Hormones

Different strategies have been evaluated for the analysis of common plant hormones, such as abscisic acid, jasmonic acid, indoleacetic acid, salicylic acid, and gibberellins. For GC/MS analysis, two different derivatization agents were compared. TMSI generates the common TMS (trimethylsilyl) derivative, whereas MTBSTFA offers a more stable but bulkier TBDMS (tert-butyl-dimethylsilyl) derivative. The TMSI agent produced overall better results, as the GAs did not derivatize using MTBSTFA. LC/MS could be used on these compounds without derivatization, but sensitivity is improved by derivatization. A CMP (3-acyl-oxymethyl-1-methylpyridinium iodide) reagent was chosen, generating a derivatized product with a permanent positive charge. The results are shown below: