There has been significant discussion of 1,3-dimethylamylamine (1,3-DMAA) in the literature concerning the presence of 1,3-DMAA in geranium plants (Pelargonium graveolens
1,3-DMAA, also known as 4-methyl-2-hexaneamine (MHA), 1,3-dimethylpentylamine, or 2-amino-4-methylhexane can be labeled as geranium extract in dietary supplements. Confirming the presence or absence of 1,3-DMAA as a natural product in geranium plants has important regulatory and commercial consequences for many dietary supplement companies.7
The chemical properties and concentrations of 1,3-DMAA and the associated matrix do not allow for simpler LC detection methods (UV-visible absorption or refractive index). Typically, GC-MS analysis requires derivatization to a higher molecular weight to increase boiling point and retention time. The geranium oil and plant matrix are sufficiently complex that most universal detectors, such as refractive index and flame ionization detectors, are likely to encounter significant matrix interferences. Thus, research and analytical effort for 1,3-DMAA analysis has focused on GC-MS1
and LC-MS/MS 1
analysis protocols for matrices, such as urine, geranium oil extracts and geranium plants.
The World Anti-Doping Agency requires that compounds with chemical structure and biological activity similar to banned substances must be analyzed by anti-doping laboratories. 1,3-DMAA and 2-aminoheptane (a banned stimulant) have similar chemical structures and physiological stimulant effects (). The laboratory of Saudan1
developed a high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for detection of 1,3-DMAA in urine samples. The method was calibrated over the range of 50 to 700 ng/mL with excellent intraday precision and accuracy of less than 6%. The results from the Saudan laboratory found that 1,3-DMAA could be detected in urine samples up to 105 hours after administration of a 40 mg dose.
Chemical structures of the stereoisomers of 1,3-DMAA, 1,4-DMAA, and 2-aminoheptane with stereogenic carbons labeled (*) and their respective (R,S) configurations.
Subsequent research by Vorce et al2
used LC-MS/MS to confirm 1,3-DMAA as the cause of false positives in amphetamine screening kits used by the United States Department of Defense drug screening laboratories. 1,3-DMAA was suspected due to its inclusion in bodybuilding energy supplements available over the counter. Vorce et al reported that 1,3-DMAA would cause false positives at urine concentrations above 6.0 mg/L and confirmed the presence of 1,3-DMAA concentrations over the 6.0 mg/L limit in 92.3% of the false positive results for amphetamines.
The laboratory of Lisi3
conducted an analysis of five geranium oils which had origins in France, Egypt, and New Zealand. The geranium oils were analyzed using a derivatization and extraction procedure for 1,3-DMAA. None of these samples were reported to have 1,3-DMAA, but no limit of detection (LOD) was reported for the method. Supplements containing 1,3-DMAA were then administered and tested in a urine excretion study using a GC with a nitrogen-phosphorous detector. The results showed that 1,3-DMAA is excreted for at least 29 hours in agreement with a previous report.1
The research team of ElSohly et al4
used GC-MS, LC-MS/MS, and high resolution ultra-performance LC with quadrupole-time of flight-MS (UPLC-QTOF-MS) to analyze geranium oils and leaves from India as well as geranium leaves, stems, and freshly extracted oil from plants grown in Oxford, Mississippi. The GC-MS and LC-MS/MS-based methods used similar extraction procedures with a reported extraction efficiency of 35% (which is relatively low). However, the extraction was shown to have excellent accuracy (75%) and precision (less than 5%) on the control sample using GC-MS analysis. The limits of detection for the GC-MS, LC-MS/MS, and UPLC-QTOF-MS were 0.1 ppm, 2.5 ppb, and 10 ppb, respectively. The GC-MS analysis of the 0.1 ppm spikes of 1,3-DMAA in the geranium oil clearly showed the characteristic double peaks of the 1,3-DMAA diastereomer pairs. The authenticated geranium plant material showed a similar pattern to the spiked geranium oil, whereas the negative geranium oil and authenticated geranium oil did not. The GC-MS chromatograms of the authenticated geranium plant material suggested the presence of the 1,3-DMAA. However, the two more sensitive LC-MS/MS methods did not detect 1,3-DMAA in any of the samples analyzed. The LC-based methods do not exhibit the characteristic diastereomer double peak—possibly due to the chromatographic separation conditions.2
Zhang et al5
recently reported the analysis of eight different geranium oils, four from China and four from Egypt, and analysis of thirteen dietary supplements containing 1,3-DMAA. The goal of their paper was to determine whether the 1,3-DMAA in dietary supplements had synthetic or natural origins. The supplements were analyzed using GC-FID analysis with a chiral column. The 1,3-DMAA in the standards and supplements were derivatized by pentafluoropropionic anhydride (PFPA). The derivatized stereoisomer separation of 1,3-DMAA by GC-FID was excellent, showing all four stereoisomers present. The GC-FID analysis protocol did not have an LOD reported; however, the calibration curve range was 0.2 to 0.8 mg/mL of 1,3-DMAA. The dietary supplements were reported to contain the same stereoisomer ratios as the synthetic standards.
Zhang et al then used two LC-MS-based methods to analyze the geranium oils for 1,3-DMAA.5
The LOD of the linear ion trap method (HPLC-ESI-LIT) was 50 ppb and the LOD of the triple quadrupole instrument (HPLC-ESI-QQQ) was 10 ppb for derivatized 1,3-DMAA. The HPLC-ESI-LIT used a chiral-phase HPLC separation column. The HPLC-ESI-QQQ used a standard C18 separation phase. In both methods, 1,3-DMAA was not detected above the LOD, and both lacked the characteristic diastereomer double peak as expected (both possibly due to chromatographic separation choices).
Finally, the research team of Li et al6
developed an extraction and LC-MS/MS-based method for the analysis of 1,3-DMAA and 1,4-DMAA in geranium plants and oils (three distinct samples of each). The method validation was detailed and conducted according to United States Pharmacopeia guidelines. The traditional instrument LOD8
reported was 1 to 2 pg/g with a reported method quantification limit (LOQ) of 1 to 2 ng/g in the geranium sample. Li reported concentrations of 1,3-DMAA and 1,4-DMAA as present in three samples of geranium plants ranging from 13 to 365 ng/g and 3 to 35.3 ng/g, respectively. In the geranium oil, Li et al reported all three samples contained 1,3-DMAA ranging from 167 to 13,271 ng/g. In the sample containing 13,271 ng/g of 1,3-DMAA, 1,4-DMAA was detected at 220 ng/g. The other two geranium oil samples did not contain 1,4-DMAA above the LOD.
The research and sample analysis presented here used an adapted extraction and LC-MS-MS analysis6
to analyze both 1,3-DMAA and 1,4-DMAA in geranium plants. Linearity, method detection limit (MDL), accuracy, and precision studies were carried out followed by analysis of geranium plants from 3 distinct regions in China (Changzhou, Guiyang, and Kunming) during winter and summer months. An improved analysis protocol was developed that used standard addition analysis to re-analyze samples and confirm the reported concentrations of 1,3-DMAA and 1,4-DMAA. One of the Changzhou, China, samples was analyzed by another laboratory,6
and to the best of the authors’ knowledge, this represents the first inter-laboratory analysis and confirmation of 1,3-DMAA in an identical geranium sample. Additionally, the diastereomer ratio of 1,3-DMAA in geranium plants was measured and compared with synthetic standards and previously reported research.5