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Determination of 17 Alpha-Methyltestosterone in Soil.

Poster · August 2017

DOI: 10.13140/RG.2.2.29461.58083

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 Determination of 17 Alpha-Methyltestosterone in Soil
Lita Chheang*1, Chim Math2, Sudtida Pliankarom Thanasupsin3
1
Internship Student, Department of Chemistry, Royal University of Phnom Penh, Phnom Penh, Cambodia
2
Master Candidate, King Mongkut’s University of Technology Thonburi, Bangkok, Thailand
3
Advisor, King Mongkut’s University of Technology Thonburi, Bangkok, Thailand
1
Tel: (+885) 81 340 825/11 321 796
Abstract—17 17 alpha-Methyltestosterone (MT) is a synthetic
anabolic and androgenic steroid hormone. It is commonly used
to promote both muscle growth and the development of male
sexual characters in both humans and animals. In Thailand, MT
is widely used in aquaculture practices. Treatment of tilapia fry
with MT-impregnated food to produce all-male populations has
become a common aquaculture practice. The left over of the food
becomes major environmental concerns as they can affect the
biological activities of non-target organisms (plants, water,
soil,..). In recent years, There are many researcher try to find
and develop new analytical methodologies to achieve lower
detection levels. This study aim to develop a new method to
determine MT only in soil and water samples by using HPLC
that took from fish farm in Bang Bo, Bang Bo District, Samut
Prakan. It was found that the optimal condition for MT
detection. According to the results, the validation of water and
sediment analytical methods recoveries ranged from 95.11 to
96.81% and 94.41 to 94.85%, respectively. Average of MT
concentration in water and sediment were found
0.001159692±0.001418 to 0.004424164±0.001418 mg/L and
0.15711798±0.0529 to 0.262956±050.0529 mg/L So, it mean that
MT can behave in both of sediments and water but it's likely to
amount in sediment.
I. Introduction
MT is a hormone that is synthesized by the Androgen
hormone. In addition to being useful. Tilapia sex
transformation in medical MT is used in conjunction with
estrogenic hormones for the treatment of various conditions,
including air, lack of testosterone. Hot air flushes in females,
but on the other hand, if hormones MT enter into the body in
large amounts, it will cause side effects to the work of the
organs. And the internal system such as swelling, headache,
acne, hair loss and so on. [1]
Formula: C20H30O2
Molar mass: 302.451 g/mol
FIG. 1.1 PICTURE OF 17 ALPHA-METHYLTESTOSTERONE
Tilapia culture can be used in ponds, cement ponds, and
nylon tentacles. Tilapia culture using sex hormone MT in
ponds can result in the accumulation of MT within the ponds.
The study found that after the feeding of fish, the hormone
mixture was able to detect MT concentrations. In the Tilapia
culture pond. [2] Soil sediments within the pond are capable
of absorbing MT well, which results in the accumulation of
MT in sediment [3] and will affect the organisms that inhabit
it when receiving MT. Therefore, the research aims to study
the analytical method to determine the accumulation of MT in
the Nile Tilapia pond. And study the relationship between MT
content accumulated in sediment and water. This may have an
impact on the environment.
II. Experimental
A. Reagents and materials
Table 2.1 Chemicals and Materials for experiment
Chemicals Materials
Standard MT power Vial bottles (2mL)
Methanol Filtering equipment
Ethanol Volumetric flask (5 mL, 10 mL)
Acetonitrile Beaker glass (25 mL)
Hexane Filter membrane (0.2 µm)
Dichloromethane Solid phase extraction (SPE)
Ethyl Acetate Nylon syringe filter (0.020 µm)
Deionized Water Rotational evaporator, HPLC
Samples Sampling Equipments
B. Sample Collection
Soil sampling on ponds Define points to keep all sides of
the pond at least 1 point. If any of the ponds are very long.
Define every 30-40 meters of every side, collecting all soil
samples. The depths 0-15 cm brings the soil collected from
these points into one sample. Soil sampling on the
embankment is inside the pond. The principle is to set the
number of points to store as well. And set up the soil at the
reservoir and keep the soil 0-15 cm deep. Bring all the points
together and combine them into one sample.
Soil sampling is at the bottom of the pond. Define is the
distribution points for the bottom of the pond. 10-15 points
(the pond size is not more than 10 rai) if ponds. The bottom of
the pond looks like. Groove should be set point Keep it
covered with grooves. Keep soil at all designated points. With
a depth of 0-15 cm. Bring together one sample.
At least 6 types of water samples and 6 types of sediment
samples were collected from fish farm at Bang Bo, Bang Bo
District, Samut Prakan. w C) during
transportation, and transfer as soon as possible to Chemistry
Building department of Chemistry, KMUTT.
Table 2.2 The number of water and sediment samples collected from Fish Farm
Site map Water samples Sediment samples
1 1 1
2 1 1
3 1 1
4 1 1
5 1 1
6 1 1
Total 6 6
C. Standard graphing experiments

Standard plot of MT solution is for MT analysis in water and

Soil sedimentation from ponds of tilapia. The results are
shown in Table 1, and the data were mapped between the sea Fig. 3.1 Graph of standard MT in the range of 0.01-10 mg/ L
and the standard solution concentrations in the range of 0.01-
10 mg/ L and 0.01-20mg / L. The results of the experiments
are shown in Fig. 1, 2, and 3 respectively.
D. Spiked sample preparation
In this study, except for the experiments mentioned in real
samples, all other experiments were carried out with Sample.
The spiked samples containing the acetonitrile at 5 mL
concentration standard levels 5 ppm and 10 ppm of 17 alpha-
Methyltestosterone were prepared by spiking the working
standard solutions into 2 g of sediment samples and water also
spiked samples 5 mL of concentration standard levels 5 ppm
and 10 ppm.

E. Preparation of sample solution

Fig. 1.2 Graph of standard MT in the range of 0.01-20 mg/ L

2g of sediment samples powder were weighed and transferred
into a 100 mL Erlenmeyer flask. 10 mL of Hexane and Also, in this study the calibration curve was conducted
Acetonitrile (1:1) were added into the Erlenmeyer flask. The without using internal standard. Eleven different MT
samples were subjected to ultrasonic treatment for 15 min.
After the ultrasonic extraction was completed, the samples concentration 0f 0.01, 0.025, 0.05, 0.10, 0.25, 0.5, 1.0, 2.5,
were added Dichloromethane and Acetonitrile (3:1) 10 mL 5.0, 10.0, and 20.0 mg/L were prepare from working solution
and then sonicated 5 min again. After it was finished, Ethyl as described in Table 3.1
Acetate 10 mL was added into each sediment samples and
started sonicated 5 min again. After the sample was extracted
three times, Filtrate each sediment samples by filter paper, Table 3.1 Peak area of standard MT from HPLC
110 mm into round-bottom flask. The extracts were combined Num. of Concentration Peak Area Peak Area Av. (Peak
and evaporated to volume about 1 mL 60 conc. (mg/L) (1) (2) Area)
flow with N2 gas until dryness and then 4 mL of Acetonitrile 1 0.01 958 718 838
and shake them about 3-5 min. Next, Filtrate by nylon syringe 2 0.025 1412 1488 1450
filter 0, 20 µm into Vial bottles (2 mL) and analysis with 3 0.05 2084 2084 2084
HPLC.
4 0.10 2655 2959 2807
5 0.25 6558 6520 6539
F. Solid-phase extraction 6 0.50 11741 12685 12213
7 1.0 24592 25049 24821
The water samples solution were passed through plastic
syringe. Then Methanol 6 mL, Ethanol 3 mL, and Deionized 8 5.0 120284 119849 120067
water 6 mL, which were used as elution solvent, was passed 9 10.0 246918 247919 247419
through the plastic syringe. Next is loading with 400 mL of
water samples solution and wash them with Deionized water The calibration curve was obtained by plotting of
10 mL. After this, elute with Acetonitrile 20 mL and filtrate MT against MT concentrations (mg/L). Figure 3.2 (0.01-10
by nylon syringe filter 0, 20 µm and go to analysis with
HPLC. The elute was referred to as the extraction solution. mg/L) shows the calibration curve of MT without internal
standard. The linearity of MT peak area was correlated with
III. Results and discussion MT concentrations based on the following equation: y=
25205x – 696.14 (R2 = 0.9998) and Figure 3.1 (0.01- 20 mg/L)
A. Standard curve also has y = 24585x + 14134 ( R2 = 0.9997).
standard solution concentrations in the range of 0.01-10 mg/ L
and 0.01-20mg / L
 B. LOD and LOQ determination c. Spike sediment
Table 3. 5 Results of Spike sediment
Spike N
Rt Peak Con of MT Av of Re
con. Re . % Recovery
(min) Area (mg/L) (%)
(mg/L) I
The limit of detection (LOD) was calculated from the
standard and slope of the calibration curve without internal 1 6.816 140248 5.592 89.471

standard. The standard blank was measured by average peak 1 2 6.821 140047 5.584 89.343
area of acetonitrile as mobile phase without MT. The
calculated LOD of this method was 0.01446185 mg/L, as 3 6.814 138612 5.527 88.432

shown in the calculation below (Kusha Kumar Naik, 2015) 1 6.814 167723 6.682 106.911
LOD = 3 (SD) / slope [13]
LOD = 3 (118.307556) / 24542 5 2 2 6.818 168638 6.718 107.492 94.41
= 0.01446185 mg/L 3 6.799 143132 5.706 91.301
The limit of quantification (LOQ) was calculated from the
standard deviation of blank (acetonitrile) and slope of the 1 6.791 140902 5.618 89.920
curve without internal standard. The calculated LOQ of this 3 2 6.799 145039 5.782 92.512
method was 0.04820616 mg/L, as shown in the calculation
below. 3 6.807 147831 5.893 94.284
LOQ = 10 (SD) / slope [13] 1 6.819 272502 10.839 86.712
LOQ = 10 (118.307556) / 24542
= 0.04820616 mg/L 1 2 6.811 276176 10.983 87.878
Table 3. 2 Table of LOD and LOQ determination
3 6.823 272132 10.824 86.595
MT Retention
Num. of Peak LOD LOQ 10 94.85
concentratio Time SD
injection Area (mg/L) (mg/L)
n mg/L (min) 1 6.799 322967 12.841 102.730
1 6.791 594
2 2 6.829 326670 12.988 103.905
2 6.782 707
3 6.791 732 3 6.818 318370 12.659 101.271
4 6.813 990
5 6.789 838 According to the results in Table 3.3 and Table 3.5, the
6 6.786 846
118.31 0.014 0.048 validation of water and sediment analytical methods
0.01 7 6.799 955 recoveries ranged from 95.11 to 96.81% and 94.41 to 94.85%,
8 6.799 746
respectively.
9 6.799 859
10 6.816 780 D. Water samples and Sediment samples.
a. Water Sample
C. Validation Method Table 3. 6 Results of water sample
a. Spike water Re N. I
Rt Peak Conc. of MT
Av. Of MT Conc.
(min) Area (mg/L)
Table 3. 3 Results of Spike water (mg/L)
Spike Con. of Av. 6.843 1350 0.002458
N. Rt Peak
con. Re
I (min) Area
MT % Recovery of Re 1 2 6.909 1664 0.003097 0.002904
(mg/L) (mg/L) (%) 3 6.841 1204 0.002161
1 6.710 121506 4.848 96.967
1 1 6.851 2231 0.004250
2 6.677 122824 4.901 98.012
2 2 6.834 1909 0.003594 0.004424
3 6.641 119011 4.749 94.987
5 96.81 3 6.784 2025 0.003831
1 6.682 123503 4.928 98.551
2 2 6.688 120133 4.794 95.877 1 6.963 650 0.001034
3 6.688 120878 4.823 96.468 3 2 6.859 913 0.001569 0.001160
1 6.739 239200 9.518 95.178 3 6.831 422 0.000571
1
2 6.651 231581 9.216 92.155
3 6.684 235765 9.382 93.815
10 95.11 b. Sediment Sample
1 6.729 242292 9.640 96.405
2 2 6.702 244110 9.713 97.126 Table 3. 7 Results of Spike water
3 6.781 241277 9.600 96.002
Rt Peak Conc. of MT Av. Of MT Conc.
Re N. I
(min) Area (mg/L)
b. Spike natural water (mg/L)
6.941 3134 0.243
Table 3.4 Results of Spike natural water 0.263
1 2 6.886 4053 0.318
Spike N Con. of Av. 3 6.774 2940 0.228
R. T Peak
con. Re . MT % Recovery of Re
(min) Area 1 6.936 2197 0.160
(mg/L) I (mg/L) (%) 0.157
1 6.706 117704 4.697 93.95 2 2 6.943 2118 0.154
1 3 - -
2 6.721 116733 4.659 93.18
3 6.716 113790 4.542 90.85 1 6.861 2802 0.212
5 92.86 0.212
1 6.719 116419 4.647 92.93 3 2 - -
2 2 6.713 116443 4.647 92.95 3 - -
3 6.729 116890 4.665 93.30 Re = Repetition, Rt = Retention time, N.I = Number of Injection,
Av = Average, SD = Standard.
Conc. = Concentration.
As shown in Table 3.6 and Table 3.7 MT average
concentration in water and sediment were found
0.001160±0.001418 to 0.004424±0.001418 mg/L and
0.157±0.0529 to 0.263±0.0529 mg/L So, MT was preferred to
sorbed onto sediment.
IV. CONCLUSIONS
According to the results above, the calibration curve was
obtained by plotting of MT against MT concentrations
(mg/L). Figure 3.2 (0.01-10 mg/L) is y= 25205x – 696.14 (R2
= 0.9998) and Figure 3.1 (0.01- 20 mg/L) also has y = 24585x
+ 14134 (R2 = 0.9997). LOD and LOQ are 0.01446185 mg/L,
0.04820616 mg/L in respectively. And For the Table 3.3 and
Table 3.5, the validation of water and sediment analytical
methods recoveries ranged from 95.11 to 96.81% and 94.41 to
94.85%, respectively. Last is about real sample in the Table
3.6 and Table 3.7 MT average concentration in water and
sediment were found 0.001159692±0.001418 to
0.004424164±0.001418 mg/L and 0.15711798±0.0529 to
0.26295605±0.0529 mg/L So, it mean that MT can behave in
both of sediments and water but it's likely to amount in
sediment.
[7] Froehner, S., Machado, K.S., Stefen, E., and Nolasco, M., 2011,
"Occurrence of Sexual Hormones in Sediments of Mangrove in Brazil",
Water, Air, & Soil Pollution, Vol. 219, No. 1, pp. 591-599
[8] Marwah, A., Marwah, P., and Lardy, H., 2005, "Development and
Validation of a High Performance Liquid Chromatography Assay for
17α-Methyltestosterone in Fish Feed", Journal of Chromatography B,
Vol.824,No.1–2,pp.107-115
[9] [9] Salvia, M.-V., Vulliet, E., Wiest, L., Baudot, R., and Cren-Olivé,
C., 2012, "Development of a Multi-Residue Method Using
Acetonitrile-Based Extraction Followed by Liquid Chromatography–
Tandem Mass Spectrometry for the Analysis of Steroids and
Veterinary and Human Drugs at Trace Levels in Soil", Journal of
Chromatography.A,Vol.1245,pp.122-133.
[10] [10] Snyder, L.R., Kirkland, J.J., 1979, Introduction to Modern Liquid
Chromatography, 2nd ed., John Wiley and Son, Canada, p.162.
[11] McMaster, M.C., 2007, HPLC, A Practical User's Guide, 2nd ed., John
Wiley and Son,Canada, pp.45-53.
[12] Rezk, N.L., Crutchley, R.D., and Kashuba, A.D.M., 2005,
"Simultaneous Quantification of Emtricitabine and Tenofovir in
Human Plasma Using High-Performance Liquid Chromatography after
Solid Phase Extraction", Journal of Chromatography B, Vol. 822,No.
1–2, pp. 201-208.
[13] Kusha Kumar Naik, S. K. (2015). Electrodeposited Spinel NiCo2O4
Nanosheet Arrays for Glucose Sensing Application. RSC Advances, 8.

Recommendation
Due to limitations of this research; for the next researcher, I
would like to suggest them should be study and develop new
method which is fast and save more solvent. e.g. HPLC-
electrochem and one more should be design sampling technic
or method. e.g. more samples, different time take samples.

Acknowledgment
This research work was supported by International Internship
Program 2017. I would like to express my sincere gratitude to
Dr. Sudtida Pliankarom Thanasupsin (Advisor), Miss
Math Chim (Lab Buddy), Miss Warunee Khirirat (Co-lab
Buddy)

REFERENCES
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Methyltestosterone, Methandrostenolone, and Nandrolone Decanoate.
Physiology & Behavior,, II(61), 265–272.
[3] Ong, S.K., Chotisukarn, P., and Limpiyakorn, T., 2012, "Sorption of
1 α-Methyltestosterone onto Soils and Sediment", Water Air & Soil
Pollution, Vol. 223, No. 7, pp. 3869-3875.
[4] . Zh g . Zhu J. K. O. i P. K. T. M “A v u hi
elevated channel low-temperature poly- i TFT ” IEEE Electron
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[5] M. W g u J. P. v W i P. Ob N. Gi i “High
u i fib i ibu u wi h h OFDR ” i
Proc. ECOC’00, 2000, paper 11.3.4, p. 109.
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APPENDIX

I. PEAK SPECIFICATION

1. Acetonitrile Peak

Acetonitrile

2. MT Peak

MT

Acetonitrile
3. 0.01 mg/L Peak

Acetonitrile

MT

4. 0.01-10 mg/L Peak

MT

Acetonitrile
II. Accuracy Peak
A. Spike water
 5 ppm spike water sample with 5 ppm of MT standard

Acetonitrile

 10 ppm spike water sample with 10 ppm MT standard

MT

Acetonitrile
 B. Spike natural water

 Spike natural water with 5 ppm standard MT Peak

MT

Acetonitrile

C. Spike sediment
 Spike sediment Peak (5 ppm spike MT and 5 ppm standard)

MT

Acetonitrile
  Spike sediment Peak ( 10 ppm spike MT and 10 ppm standard)

MT

Acetonitrile

III. Water sample

Acetonitrile
MT

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