Six aristolochic acids were identified in the Chilean species
The aristolochic acids (AAs) are derivatives of nitrophenanthrene present in plants of the genus
Methodologies in the analysis by HPLC reported in the literature used sample quantities ranging from 40 to 200 g [
In the present work, we determined better conditions to extract and detect aristolochic acids from lower biomass samples. Changes in some parameters to improve the extraction are made, for example, using an Ultra-Turrax®, and the concentration of acids are determined by using Soxhlet extraction equipment. In addition, solid phase extraction was applied as a purification technique. These changes allow proper identification and quantification of AAs using limited amounts of plant biomass and in low concentrations, obtaining good resolution and well-defined chromatography signals.
The measurements were carried in a Jasco HPLC-DAD model MD-2015 plus, controlled with the chrompass software. An InertSustain C18 column 250 × 4.6 mm, 3
The correct order of elution was confirmed by MS. First, the sample was separated using HPLC-DAD, and full fraction collected was measured by direct injection in mass spectrometer Q Exactive focus with an Orbitrap® detector (Thermo Scientific).
Each acid standard (AAI and AAII) was purchased separately from Sigma-Aldrich. HPLC-grade methanol and acetonitrile were purchased from Merck. The ultrapure water was prepared using the purification with the heal force model Smart Series. The other reagents such as chloroform, ethyl acetate, and methanol were of analytical quality and purchased from Merck.
Two standard solutions (1000
Aristolochic acid concentration of standard solutions for the construction of the calibration curve.
Standard mix | AAI ( |
AAII ( |
||
---|---|---|---|---|
|
RSD (%) |
|
RSD (%) | |
1 | 1 | 0.02 | 5 | 0.01 |
2 | 2.5 | 0.28 | 10 | 0.07 |
3 | 5 | 0.07 | 20 | 0.07 |
4 | 10 | 0.14 | 40 | 0.21 |
5 | 15 | 0.14 | 60 | 0.14 |
6 | 20 | 0.14 | 80 | 0.14 |
7 | 40 | 0.07 | 160 | 0.05 |
The calibration curves were constructed for each aristolochic acid by plotting the peak area versus concentration of each standard. The limit of quantification was calculated for each one.
The extraction of aristolochic acids was carried weighing 0.4012 g of lyophilized and pulverized leaf of Aristolochia chilensis, 20 mL of methanol was added, and it was then homogenized with Ultra-Turrax for 2 min. The extract was transferred to Soxhlet equipment where the extraction continued for 2 hours at 50°C. The methanolic extract was evaporated to dryness in vacuum in a rotary evaporator Büchi R-300. The syrupy residue was suspended in 30 mL of 5% NaHCO3 and heated for 10 min at 40°C in a thermostatic bath. This solution was filtered by simple filtration using filter paper (MN 615 ∅ 125 mm) and transferred to a decanting funnel. The solution was washed with 15 mL (×3) of CHCl3, discarding the organic phase. The aqueous phase recovered was washed with 15 mL (×3) of ethyl acetate, discarding the higher yellow phase, followed by the addition of 1 N HCl until it reaches pH 2. Then, the AAs are extracted from the aqueous phase with 15 mL (×3) of ethyl acetate.
The three fractions of the ethyl acetate were mixed and filtered by adding a spatula of anhydrous sodium sulfate. From filtered solution, an aliquot of 10 m
The separation was performed on a C18 SPE column (Waters), conditioned with 1 mL of methanol HPLC-grade. Methanolic extract (100
For HPLC-DAD detection, a mobile phase of ACN : H2O (1 : 1) acidified with 0.1% acetic acid was used and an isocratic flow to 1 mL·min−1. The oven temperature was 40°C, and 20
Aristolochic acid detection was carried out in electrospray positive-ion mode [(+) ESI]. The measurements were recorded in full scan mode (scan range: 200–400
Usually, the aristolochic acid detection is performed using two or more phases which contain acetonitrile and acidified water [
The cleanliness of the sample is necessary because interferers exist in high concentrations which mask the analyte, with intense signals of up to approximately 2500 mAU.
It can be observed (Figure
Comparison of signal between the extract of
Two columns were tested with different characteristics specified (Table
Characteristics of the columns tested.
Column C18 | Dimensions (mm) | Particle size ( |
Carbon load (%) |
---|---|---|---|
Sunniest | 4.6 × 250 | 5 | 16 |
InertSustain | 4.6 × 250 | 3 | 14 |
For comparison, 10
As can be observed in Figure
Comparison between columns for the measurement in the standard mix. Running conditions as in Section 2.5.3.
The spectra obtained were compared with the absorption spectra reported in the literature [
Chromatogram of the methanolic extract of
The commercial standards were used for AAI and AAII; therefore, for the others, only the percentage of relative abundance was calculated (Table
Relative percentage for each AA identified present in the extract of
Peak number | AAs | Area (mAU/min) | RT (min) | UV-Vis |
Relative % |
---|---|---|---|---|---|
1 | AAIVa | 6.4 | 5.23 | 221, 242, 251, 293, 329, 410 | 21.1 |
2 | AAIa | 5.7 | 5.41 | 224, 254, 320, 398 | 18.9 |
3 | AAIII | 1.9 | 6.69 | 251, 284, 314, 389 | 6.3 |
4 | AAII | 0.9 | 9.84 | 212, 251, 299 | 3.0 |
5 | AAI | 13.5 | 11.72 | 224, 251, 320, 395 | 44.7 |
6 | AAIV | 0.8 | 12.57 | 221, 242, 251, 293, 329, 410 | 2.6 |
Total area | 30.2 |
However, the absorption characteristics of some acids do not entirely coincide with the described characteristics. For example, the reported absorptions for IV acid are 244, 251, 285, 315, 364, and 393 [
(a) Mass spectrum of standard AAI, full scan in positive-mode electrospray ionization. (b) Mass spectrum of collection the peak number 5 from the sample, corresponding to AAI, full scan in positive-mode electrospray ionization.
Mass of the AAs and confirmatory fragments in
Peak number | AAs | Molecular weight |
|
---|---|---|---|
1 | AAIVa | 357 | 358.05 [M+H]+ |
312.06 [M+H-H2O-OCH3]+ | |||
314.06 [M+H-H2O-CO]+ | |||
340.04 [M+H-H2O]+ | |||
|
|||
2 | AAIa | 327 | 328.23 [M+H]+ |
284.06 [M+H-CO2]+ | |||
310.04 [M+H-H2O]+ | |||
|
|||
3 | AAIII | 341 | 342.13 [M+H]+ |
282.27 [M+H-CO2-CH3]+ | |||
|
|||
4 | AAII | 311 | 312.36 [M+H]+ |
284.33 [M+H-CO]+ | |||
294.04 [M+H-H2O]+ | |||
|
|||
5 | AAI | 341 | 342.06 [M+H]+ |
296.07 [M+H-H2O-OCH3]+ | |||
298.07 [M+H-H2O-CO]+ | |||
324.05 [M+H-H2O]+ | |||
|
|||
6 | AAIV | 371 | 372.12 [M+H]+ |
328.06 [M+H-H2O-CO]+ | |||
354.06 [M+H-H2O]+ |
As an example, Figure
Mass spectrum of collection peak number 1. For these masses, AAIVa was assigned.
The summary of masses and majority fragments for each peak and respective elution order are shown in Table
The calibration curves for AA I (Figure
Calibration curve for AAI. LD AAI = 0.066 ± 0.010
Calibration curve for AAII. LD AAII = 0.266 ± 0.042
By interpolating the areas of each peak in Figures
The amount of aristolochic acid (
The volume of ethyl acetate can be a variable according to the extraction. Considering a volume of 40 mL, the extract of
Methodology for identifying aristolochic acids present in
Since all AA standards or possible aristolactams present in the plant are not commercially available, it is only possible to identify each peak of the chromatogram by comparing it with the absorption spectra given in the literature; however, with these conditions, it was possible to confirm the presence of each acid using an MS detector and establishing its correct elution order.
The authors declare that there are no conflicts of interest regarding the publication of this paper.
This work is supported by internal project of the Center of Research and Technological Development of Algae (CIDTA) of University Católica del Norte (UCN), Chile. Also, this study was partially supported by FONDEQUIP EQM 150055 (CONICYT, Chile).
Figure 8: mass spectrum of collection peak number 2. For these masses, AAIa was assigned.
Figure 9: mass spectrum of collection peak number 3. For these masses, AAIII was assigned.
Figure 10: mass spectrum of collection peak number 4. For these masses, AAII was assigned.
Figure 11: mass spectrum of collection peak number 6. For these masses, AAIV was assigned.