PDF
(Download)
|
- Monk Fruit (Siraitia grosvenorii, Cucurbitaceae)
- Sweeteners
- HPLC-MS
|
Date:
12-15-2017 | HC# 021724-582
|
Re: HPLC-ESI-MS/MS Method Simultaneously Quantifies Eight Mogrosides of Monk Fruit
Luo
Z, Shi H, Zhang K, Qin X, Guo Y, Ma X. Liquid chromatography with tandem mass
spectrometry method for the simultaneous determination of multiple sweet
mogrosides in the fruits of Siraitia
grosvenorii and its marketed sweeteners. J Sep Sci. November 2016;39(21):4124-4135.
Monk
fruit (MF) (luo han guo; Siraitia
grosvenorii, Cucurbitaceae) has been used for thousands of years as both a
natural sweetener and traditional medicine. Recent interest in MF as a natural
sweetener and its generally recognized as safe (GRAS) status has resulted in
new MF sweeteners in the US market. The active ingredients in MF that act as
natural sweeteners are cucurbitane-type triterpenoids known as
"mogrosides." The eight major mogrosides found in MF are mogroside
III (MIII), mogroside IVa (MIVA), mogroside IV, mogroside V (MV), mogroside VI
(MVI), iso-mogroside V, 11-oxomogroside-V, and siamenoside I (SI). Mogroside
type and quantity in MF will differ depending on ripeness. Ripe MF contains
mostly MV, the most plentiful sweet-tasting mogroside, whereas unripe MF
contains mostly mogroside IIE and MIII, which are, respectively, bitter and
tasteless.1-3 Ripe MF is difficult to distinguish by appearance only,
and quality can differ considerably among cultivation sites.1-3 This
study discusses the development and validation of a high-performance liquid
chromatography with electrospray ionization tandem mass spectrometry
(HPLC-ESI-MS/MS) method for simultaneous quantification of the eight primary mogrosides
of MF from samples of harvested fruits and marketed sweeteners.
A
mixed standard solution of all eight mogrosides was analyzed via HPLC at
varying concentrations to generate calibration curves (peak area versus
concentration) for each mogroside. Mogroside standards were purchased from
Chengdu Must Bio-Technology (Sichuan, China). A total of 10 batches of MF (MF1
through MF10) were obtained. Five batches
were supplied by the Guangxi Branch Institute of Medicinal Plant
Development (IMPLAD), Chinese Academy of Medicinal Science, Peking Union
Medical College, and five were purchased from Guangxi Province, China. Three
sweeteners were purchased from Xi'an Jinheng Chemical (Shanxi, China). Ultrasound-assisted
solid-liquid extraction of mogrosides in methanol/water (80/20, per volume) was
found to give optimal results while being convenient, cost effective, and
highly reproducible. Samples of dried MF were prepared by homogenization,
mixing with methanol/water (80/20, per volume), sonication, and filtering. Sweetener
samples were also mixed with methanol/water (80/20, per volume), sonicated, and
filtered. The HPLC system used was an Agilent Technologies 1260 Series LC
system (Agilent; Santa Clara, California). After preliminary experiments, Agilent
Poroshell 120 SB C18 columns were chosen for chromatographic
separation of the eight mogrosides for producing good results with a short analytical
time. Acetonitrile and methanol mobile phases were tested at different flow
rates (0.2-0.5 mL min−1), and acetonitrile/water eluent (both
containing 0.1% formic acid) with a 0.25 mL min−1 flow rate showed
the best separation and symmetric peak shapes. Gradient elution was used for
HPLC analysis to avoid long retention times and bad peak shapes noted on some mogrosides
with isocratic runs. Satisfactory separation of the eight mogrosides was achieved
in 10 minutes. "Good linearity (r2
≥ 0.9984) was achieved within the investigated ranges for all of the analytes,"
the authors observe. Retention times, precursor and product ions, and ion
ratios were compared to identify the mogrosides. Optimum mass spectrometric
behaviors were analyzed by introducing each standard compound individually into
the mass spectrometer to optimize the ESI source parameters. The negative
ionization mode showed higher sensitivity for determining quantity; therefore,
the [M-H]- ion was measured for
each compound. Multiple reaction monitoring (MRM) scanning was employed for
quantification and optimized for maximum [M-H]- and fragment/product
ions generated for each mogroside.
Intra-
and inter-day variability was calculated as a precision check, based on analysis
of three mogroside standards, at low, intermediate, and high concentrations
compared to sample MF3. Intra- and inter-day relative standard deviations (RSDs)
for all were less than 3.73% and 3.91%, respectively, indicating precision of
quantification. The authors summarize recovery results—"The average
recoveries of all of the compounds ranged from 91.22 to 106.58%, and the RSDs
were less than 3.79%, which demonstrated that the method was accurate for
simultaneous quantitative evaluation of the eight mogrosides in [MF]."
Stability was evaluated by analyzing the prepared solution of MF3, kept at room
temperature, every six hours for 24 hours, and it was found to be stable (RSD
values at peak area < 3.01%). Good repeatability was determined after six
independently prepared solutions of MF3 were analyzed, with RSD less than
3.42%. Matrix effects were calculated for each compound, as endogenous
substances present in a complex sample such as MF can lead to ion suppression
or signal enhancement. It was determined that any matrix effects were minor and
would not interfere with accurate analysis.
The
quantity of each mogroside and total mogrosides varied significantly in the
analyzed MF batches (8.83 to 18.46 mg/g total mogrosides). MV, present at
5.77-11.75 mg/g, made up 49.29-66.96% of total mogrosides in the MF samples. SI,
which like MV tastes sweet, was the second most abundant mogroside. MIII and
MIVA were detected only in MF3 and MF8, indicating these batches may not have
been fully ripe. MVI was not detected in any sample. MV was also the main
component in the tested sweeteners, accounting for 75% of total mogrosides. A
small amount of MIVA was present in one sweetener. No MIII or MVI was present. The
total contents of the sweeteners varied greatly, with different amounts of MF
extract added and blended with other less intensely sweet substances, so the
mogroside contents were, as expected, lower than those of MF samples.
The
developed HPLC-ESI-MS/MS method was checked for precision, repeatability,
accuracy, stability, and sensitivity, culminating in mogroside quantifications
of MF and sweetener samples analyzed. In line with current industry trends to
better establish the identity of botanical ingredients, a test to identify and quantify the mogrosides of MF before
commercial use would be a valuable tool for market standardization.
The
study was funded by the National Natural Science Foundation of China and by the
Program for Innovative Research Team at IMPLAD.
—Alexis Collins, MA,
MS
References
1Lu F, Li D, Fu C, et
al. Studies on chemical fingerprints of Siraitia
grosvenorii fruits (Luo Han Guo) by HPLC. J Nat Med. January 2012;66(1):70-76.
2Zhang H, Yang H, Zhang
M, et al. Identification of flavonol and triterpene glycosides in Luo-Han-Guo
extract using ultra-high performance liquid chromatography/quadrupole
time-of-flight mass spectrometry. J Food
Compost Anal. March 2012;25(2):142-148.
3Li D, Ikeda T, Huang
Y, et al. Seasonal variation of mogrosides in Lo Han Kuo (Siraitia grosvenori) [sic]
fruits. J Nat Med. July 2007;61(3):307-312.
|