RESULTS AND DISCUSSION

Limits of detection (LODs) and limits of quantification (LOQs) of OSCs

In determining the LOD and LOQ, four organosulphur compunds (OSCs) which are
(S-methylcystein (SMC), S-ethylcysteine (SEC), S-allylcystein (SAC), dan S-allylcysteinine
(Alliin) were prepared in 10 ppm (lg/mL) stock solution and diluted to different concentrations
ranging from 10 ng/mL to 5000 lg/mL for the construction of calibration curves.

To optimize the electrospray conditions for mass spectroscopy (MS), the Selcted reaction
monitoring (SRM) which was employed to select the precursor and product ion at the
fragmentation transition. This transitions were established according to the values of precursor
ion and product ion. The calibration curves of the four OSCs were illustrated by the signal area
of SRM transitions and had correlation coefficient (R2) ranging from 0.9939 to 0.9976.

The LOD and LOQ of this method were determined and calibrated using the blank
method (N = 10). The respective LODs and LOQs were 14.38 and 37.36 ng/mL for SMC, 8.02
and 16.80 ng/mL for SEC, 3.18 and 8.60 ng/mL for SAC, and 1.42 and 3.43 ng/mL for Alliin.

Absolute quantification of garlic extracted using different methods

Two extraction solvents and two pretreatment methods was used to determine the
contents of four OSCs in garlic. Therefore, we employed a total of four processing methods and
they are:

Raw garlic extracted by water (GEW)

Processed garlic extracted by water (PGEW)
Raw garlic extracted by methanol (GEM)
Processed garlic extracted by methanol (PGEM)

According to the calibration curves of the four OSCs, the statistics data of the quantification
of four OSC contents were shown and the spectra of MS in MRM of four OSCs with the
illustrated bar charts are shown where the results showed that:
 Types of Extraction OSCs Value
Extraction of raw garlic SMC 61.99 lg/g
using water SEC 25.84 lg/g
Extraction with methanol SMC 13.64 lg/g
SEC 14.56 lg/g
Extraction of raw garlic using water resulted in higher recoveries due to the water
solubility of these compounds.

Types of Extraction OSCs Value

Extraction of raw garlic SAC 1469.94 lg/g
using water Alliin 134.03 lg/g
Extraction with methanol SAC 2193.19 lg/g
Alliin 18164.20 lg/g
The recoveries of SAC and Alliin were lower after water extraction than after
methanol extraction

Of all the studied OSCs, alliin was present in the highest quantity.

The contents SMC (11.88 lg/g) and SEC (3.97 lg/g) were greater in cooked garlic
extracted by water than in cooked garlic extracted by methanol (1.84 and 3.19 lg/g,
respectively).

The results did not indicate any significant differences in OSC content between raw and
processed garlic; for example, the SEC contents were 3.97 lg/g in PGEW and 3.19 lg/g in
PGEM, the SAC contents were 830.50 lg/g in PGEW and 757.79 lg/g in PGEM, and the alliin
contents were 5118.87 lg/g in PGEW and 6300.99 lg/g in PGEM.

On comparing raw garlic extraction and cooked garlic extraction, we observed a general
phenomenon that raw garlic extractions had higher quantities than processed garlic extractions,
except for alliin in water extraction. It is possible that heat during cooking destroys OSCs.
Nevertheless, heat would make garlic have loose pieces of structures and water extraction in
processed garlic was more convenient than that in raw garlic.

CONCLUSION

In conclusion, its been demonstrated a novel synthetic method for standards and ISs
using reductive amination with isotopic formaldehydes. Reductive amination is described as
stable-isotope dimethyl labeling is a way to utilize aldehydes to interact with primary amines.
Using this novel method, the four OSCs were determined and monitored in raw and processed
garlic samples extracted by water and methanol. The utilization of isotopic formaldehyde in the
reductive amination reaction provided a novel method to cheaply and conveniently produce
standards and ISs.

The standards and ISs using reductive amination with isotopic formaldehydes. OSCs with
primary amine groups were reacted with isotopic formaldehydes to synthesize ISs and standards
primary amines (SMC, SEC, SAC, and alliin) in garlic. The four OSCs were reacted with
isotopic formaldehydes to produce ISs using D2-formaldehyde and standards using H2-
formaldehyde.

Quantitative methods for four OSCs (SMC, SEC, SAC, and alliin) was developed and
compared the contents of these OSCs in garlic samples processed with different pretreatment and
extraction methods (GEW, GEM, PGEW, and PGEM). According to the MS spectra and
statistical results, we concluded that heat degrades OSCs in garlic. However, the high
temperature would also loosen the bulbs of garlic facilitation the extraction of OSCs by water.

Raw garlic was found to contain far more alliin than cooked garlic. We also observed that
more OSCs could be extracted from uncooked garlic than from cooked garlic, regardless of
whether the extract solvent was water or methanol. For example, more S-allylcysteine and S-
allylcysteinine sulfoxide was extracted with methanol than with water. However, water extracted
more Smethylcysteine and S-ethylcysteine than methanol.