Chemicals and reagents
1,3-Dimethylamylamine (1) (CAS No. 105-41-9, >99%) and 1,4-dimethylamylamine (2) (CAS No. 28292-43-5, >99%) were purchased from Sigma- Aldrich (St. Louis, MO, USA). Formic acid (88%), methanol (HPLC grade), hexane (pesticide residue grade) and hydrochloric acid (37% HCl) were all obtained from Fisher Scientific (Waltham, MA, USA). All other chemicals are analytical grades. Water (18.2 MΩ-cm) was prepared with a Barnstead NanoPure Diamond System (Lake Balboa, CA, USA).
Apparatus and instruments
Agilent 1100 HPLC system with quaternary pump (Santa Clara, CA, USA) was coupled to a Micromass Quattro Ultima mass spectrometer with electrospray ionization (ESI) source (Manchester, England). Masslynx (version 4.1) were used to control the system of LC-triple quadrupole mass spectrometer and for data acquisition and processing. The analytical column is a Phenomenex Kinetex C18 column (4.6 × 150 mm, 2.6 μm) (Torrance, CA, USA). High Speed Grinder DFY-200 was from Gaoyi In. ( Wenzhou, China). Commercial Blender 200G was from Waring Co. (Torrington, CT, USA).
Geranium plants and geranium oils
The geranium plants were procured by and obtained from Dr. Yi Jin of Yunnan University (Kunming, Yunnan Province, China) and were authenticated by Professor Xu Youkai of the Xishuangbanna Tropical Botanical Garden-Chinese Academy of Sciences (Mengla, Yunnan Province, China). The plant samples were collected from different areas of China (see
), shipped to this lab fresh and stored at −20 °C immediately upon receipt in the lab. All geranium oil samples were obtained from Jiangxi Ji’an Hengcheng Flavor Oil Factory (Ji’an, Jiangxi Province, China). The geranium oil was stored at room temperature.
Levels of 1,3-DMAA (1) and 1,4-DMAA (2) in geranium (Pelargonium graveolens) plant and geranium oil from different sources.
The stock solutions of 1,3-DMAA (1) and 1,4-DMAA (2) were prepared separately in methanol at a concentration of 1.00 mg/mL and 1.068 mg/mL, respectively. Working solutions composed of the two DMAAs were prepared by serial dilution of the stock solution with 0.5 M HCl to obtain a set of standard concentrations of 0.10–10.00 ng/mL of 1,3- DMAA (1), and 0.11– 10.68 ng/mL of 1,4-DMAA (2). All working standard solutions were stored at 4 °C and used within one week after preparation, although no significant degradation was observed in one month of storage.
Sample preparation and extraction
Geranium plant (wet leaves and stems) was thawed at room temperature and cut into pieces at about 1–2 cm and mixed well. After about five hundred grams of the plant was ground into fine pieces with a high speed grinder, 10 g of sample was weighed into a stainless steel blender cup. To this cup, 80 mL of 0.5 M HCl was added and mixed. The mixture was homogenized at high speed for 2 min. The homogenate was then transferred into a 100-mL volumetric flask. The blade and cup were washed with additional 15 mL of 0.5 M HCl. The solution was collected into the flask and extracted by sonication at 50 °C for 1 hour. After being cooled to room temperature, the volume was adjusted to the mark with 0.5 M HCl. This solution was centrifuged at 4000 × g for 10 min, and the supernatant was further purified as below.
For geranium oil, 1 mL of sample was mixed with 1 mL of hexane in a 10-mL glass tube with screw cap. Five mL of 0.5 M HCl was added and shaken with a vortex shaker for 5 min at high speed. The aqueous layer (lower) was diluted with 0.5 M HCl as necessary, filtered with a 0.45-μm nylon filter and applied to LC/MS/MS without further purification.
Four mL of supernatant and 2 mL of hexane were added to a 10-mL glass tube with screw cap. The mixture was shaken by a vortex shaker for 30 sec. The mixture was centrifuged at 2000 × g for 5 min. The aqueous layer was diluted as necessary and filtered for LC/MS/MS analysis.
The mobile phase of HPLC is composed of Water:acetonitrile (85:15) containing 0.1% formic acid. Flow rate was 0.5 mL/min; column temperature was 35 °C; the flow was diverted 0.2 mL/min to MS. Injection volume was 50 μL.
The mass spectrometer was operated in positive ESI and multiple reaction monitoring (MRM) mode. Nitrogen was used as the nebulizer, heater, and cone gas. Argon was used as the collision induced dissociation (CID) gas. The precursor-to-product ion transitions were monitored at m/z 116 [M + H] → 57 (quantification) and m/z 116 → 99 (qualification) for both 1,3- DMAA (1) and 1,4-DMAA (2). ESI parameters were optimized for maximizing the generation and stability of the precursor and fragment ions by infusion as follows: Capillary 2.5 kV, Cone 20V, Source temperature 120 °C, Desolvation temperature 360 °C, Cone gas 120 L/hour, Desolvation gas 850 L/hour, CID 11 eV, collision cell pressure 2 × e-3 mbar.
The analytical method was validated according to guidelines for United States Pharmacopeia (USP). The parameters validated include linearity, specificity, limit of detection, limit of quantification, accuracy, precision and reproducibility.
To evaluate the linearity, calibration curves of 1,3- DMAA (1) and 1,4-DMAA (2) were established using concentrations in the range from 0.1 to 10 ng/mL. The responses of each compound against its respective concentration were plotted. Linear regression analysis was performed to obtain calibration equation and correlation coefficients (R2).
Two ion transitions coupled with a high resolution column were used to enhance the method selectivity. Specificity was assessed by comparing the chromatograms of blanks (glassware and reagent blanks), standard, spiked samples and their peak purity (peak shape and relative intensity of transitions). The peak was identified by retention time and relative intensity of transitions against the reference standard.
The matrix effects (ion suppression or enhancement) were evaluated by comparing peak area of the standard, sample extract and the extract directly spiked at corresponding concentrations of DMAAs, which were set at the medium spiked concentration (20 ng/g).
Accuracy of the method was determined by assaying spiked samples of geranium plants at four different levels: about 5, 10, 20 and 40 ng/g for both 1,3-DMAA (1) and 1,4-DMAA (2) (ie, adding 50, 100, 200 or 400 ng DMAA in solution to about 10 g of sample). Each concentration level had three replicates. All samples were extracted, purified and determined as described above. For the blank, 10 mL of water instead of sample was included and carried through the same procedures of sample preparation.
Precision and reproducibility
Precision was performed by assaying a geranium sample in six subsamples. The concentration of each subsample, and average and RSD of the analyses were calculated to access the precision of the method.
Reproducibility of this method was evaluated by a second chemist beside the primary chemist in this laboratory. The geranium sample was assayed in six subsamples. The concentrations, average and RSD of the six analyses were compared with the results obtained by the primary chemist to assess the reproducibility of the method by a different chemist.
The concentrations of 1,3-DMAA (1) and 1,4- DMAA (2) in the sample preparations were obtained from their corresponding standard curves. The mean, standard deviation (SD) and relative standard deviation (RSD) of spike recoveries were calculated for assessing the accuracy of the method. Mean and RSD of repeated analyses of the geranium samples were used for precision evaluation. The mean concentration of DMAAs from the precision experiments was used as the original value to calculate recoveries of DMAAs. The recoveries from spiked samples were calculated by the following formula:
where, Fc is the concentration (ng/g) found in the spiked sample; Bc is the original value of the sample (ng/g) prior to spiking; Sc (ng/g) is the concentration spiked to the sample.