Tranexamic Acid Analysis as Ethoxycarbonyl-tert-butyldimethylsilyl Derivatives by Gas Chromatography–Tandem Mass Spectrometry

Article abstract of DOI:10.1002/bkcs.11977

Tranexamic acid (TA) as a synthetic derivative of lysine is a potent antifibrinolytic drug used to reduce bleeding during surgeries including dental extraction. The efficacy of TA administration in ovarian cancer and melasma has also been reported in a pr

Full text of DOI:10.1002/bkcs.11977

Article
DOI: 10.1002/bkcs.11977
BULLETIN OF THE
H.-J. Park et al.
KOREAN CHEMICAL SOCIETY
Tranexamic Acid Analysis as Ethoxycarbonyl-tert-butyldimethylsilyl
Derivatives by Gas Chromatography–Tandem Mass Spectrometry
†
,††
‡,††
‡
‡
‡
‡
Hyung-Jin Park,
Hyeon-Seong Lee,
Chan Seo, Jeuk Min, Moongi Ji, Youngbae Kim,
§
‡,¶
∥
∥
**
Subin Choi, Doo-Young Kim, Jung Dong Kim, Do Hyeon Jeong, Wonjae Lee, and Man-
‡
,
*
Jeong Paik
†Department of Physiology and Department of Biomedical Sciences, Ajou University School of Medicine,
Suwon, Republic of Korea
‡
College of Pharmacy, Sunchon National University, Suncheon, Republic of Korea.
*
E-mail: paik815@sunchon.ac.kr
§
Redone Tech, Laboratories of Marine New Drugs, Seoul, Republic of Korea
¶
Hyundai Pharm, New Drug Discovery Lab, Yongin, Republic of Korea
Raphas, Avison Biomedical Research Center, Seoul, Republic of Korea
College of Pharmacy, Chosun University, Gwangju, Republic of Korea
∥
*
*
†
†
These authors contributed equally to this work.
Received October 22, 2019, Accepted January 5, 2020
Tranexamic acid (TA) as a synthetic derivative of lysine is a potent antifibrinolytic drug used to reduce
bleeding during surgeries including dental extraction. The efficacy of TA administration in ovarian cancer
and melasma has also been reported in a previous study. In this study, the mass spectral data set of TA as
ethoxycarbonyl-tert-butyldimethylsilyl derivatives was newly constructed by gas chromatography-tandem
mass spectrometry in multiple reaction-monitoring mode. This analytical method was validated with good
linearity (r > 0.9988), limit of detection (LOD, 0.22 ng/mL), limit of quantification (LOQ, 0.72 ng/mL),
relative standard deviation (1.3 to 4.8%), and relative error (−6.8 to 3.5%) under optimal conditions. The
LOD and LOQ were assessed to be lower than other reported methods. Therefore, the present method will
be useful for monitoring of TA in the pharmacological and cosmetic fields.
Keywords: Tranexamic acid, Ethoxycarbonyl-tert-butyldimethylsilyl derivatives, Gas chromatography-
tandem mass spectrometry
Introduction
most methods have been performed by liquid chromatogra-
phy equipped with mass spectrometry. Some derivatization
methods using ethyl chloroformate (ECF) and
heptafluorobutyric acid (HFBA) have also been reported
for TA analysis by gas chromatography–mass spectrometry
Tranexamic acid (TA, Figure 1) as a synthetic derivative of
1
–4
lysine is known as potent antifibrinolytic drug. TA inter-
acts at the lysine-binding site of plasminogen molecules
during fibrinolysis, which inhibit plasminogen activators
24,25
(GC–MS).
However, ethoxycarbonylation (EOC) com-
5
,6
from converting plasminogen to plasmin. It was reported
that regulated TA administration reduces the bleeding dur-
bined with tert-butyldimethylsilylation (TBDMS) for
quantitative analysis of TA has not been attempted by
GC-tandem mass spectrometry (GC–MS/MS). The GC–
MS/MS analysis requires pre-derivatization process of
analytes to block all these active protons. In this study,
EOC-TBDMS has a good advantage due to its enhanced
MS property compared with that of single EOC derivative.
Also, GC–MS/MS in multiple reaction monitoring (MRM)
mode is selective, sensitive, and accurate. Previously, we
reported amino acid (AA) profiling analysis as EOC-
7
ing surgery and dental extraction. Early administration of
TA was reported to decrease morbidities in women with
8
post-partum hemorrhage. In ovarian cancer patients, treat-
ment with TA showed inhibition of malignant cells and
9
marked reduction in the ascites. Furthermore, TA plays an
1
0
important role in the treatment of melasma where plasmin
1
1
indirectly inhibits melanin synthesis. Topical application
of TA showed a dose-dependent preventative effect in UV-
exposed skin and reduction of arachidonic acid-induced
TBDMS derivatives, which was effective for biological
12
26–28
pigmentation. In previous reports, quantitative analysis of
TA has been performed using various UV-active deriva-
tives by high-performance liquid chromatography (HPLC)
fluids by GC and GC–MS.
Therefore, as a new
approach in the current study, we developed a method for
TA analysis as EOC-TBDMS derivatives in GC–MRM–
MS/MS. A new mass spectral data of TA was established
as EOC-TBDMS derivatives. Finally, we validated the
1
3–23
for various biological matrices.
Since TA analysis
without any derivatization is difficult for UV-detection,
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Gas Chromatography-Tandem Mass Spectrometry.
The GC-MRM-MS/MS analysis was performed with a
GC–MS-TQ8040 interfaced to triple quadrupoles mass
spectrometer with electron impact mode in 70 eV, equipped
with Ultra-2 (5% phenyl–95% methylpolysiloxane bonded
phase; 25 m × 0.20 mm i.d., 0.11 μm film thickness) cross-
linked capillary column (Agilent Technologies, Palo Alto,
CA, USA). Injector, interface, and ion source temperatures
Figure 1 Chemical structure of TA.
current method under optimal conditions, which will be
useful for TA monitoring and quality control in the pharma-
cological and cosmetic fields.
ꢀ
were maintained at 260, 300, and 230 C, respectively.
Helium was used as the carrier gas at a flow rate of
0
.5 mL/min in a constant flow mode. Samples (ca 1.0 μL)
were introduced by AOC-20i auto-injector and AOC-20s
auto-sampler in the split-injection mode (10:1). Oven tem-
Experimental
ꢀ
perature was initially set at 180 C for 1 min, and
Chemical and Reagents.
-aminocaproic acid as an internal standard (IS) and ethyl
chloroformate (ECF) were purchased from Sigma-Aldrich
St. Louis, MO, USA). Mouse balb/C serum was purchased
from Innovative Research (Novi, MI, USA). N-Methyl-N-
tert-butyldimethylsilyl trifluoroacetamide (MTBSTFA) was
obtained from Pierce (Rockford, IL, USA). Toluene, diethyl
ether, ethyl acetate, and dichloromethane were purchased as
pesticide-grade from Kanto Chemical (Tokyo, Japan).
Syringe-Driven Filter Unit (0.45 μm pore size) was pur-
chased from Merck Millipore (Darmstadt, Germany). All
other chemicals used were of analytical grade.
The TA standard,
ꢀ
ꢀ
programmed to 300 C, at a rate of 30 C/min with holding
time for 5 min. The mass range scanned was 45–600 u at a
rate of 3333 u/s. In the MRM mode, one precursor ion of
TA and IS, and three characteristic ions of each TA and IS,
were used for peak identification and quantification. All
GC-MRM-MS/MS runs were performed in triplicate.
6
(
Sequential EOC-TBDMS Derivatives. TA and IS work-
ing standard solutions were used for optimization in the reac-
tion. A two-phase extractive EOC reaction was immediately
performed by mixing for 10 min with ECF (20 μL) in the
dichloromethane phase (1 mL) after adjusting pH ≥ 12 with
5
.0 M sodium hydroxide in aqueous phase. The aqueous
Preparation of Standard Solutions. Stock solutions of
TA and IS standards were prepared at 10 μg/μL in 0.1 M
HCl. Then, they used to prepare TA and IS working solu-
tion (0.1 μg/μL) with 0.1 M HCl. All standard solutions
phase solution was then acidified with 10% sulfuric acid,
followed by saturation with sodium chloride. The EOC
derivatives were sequentially extracted with diethyl ether
(3.0 mL) and ethyl acetate (2.0 mL); the combined extracts
ꢀ
ꢀ
prepared were stored at 4 C.
were evaporated under a gentle nitrogen stream at 40 C. For
Figure 2 Electron-impact mass spectrum of TA as EOC-TBDMS derivatives.
Table 1. Mass spectral data of TA as EOC/TBDMS derivatives.
a
Mass spectral data set
Base
Molecular
ion [M]
ion
[M-57]
+
+
+
+
Analyte
[M-15]
328 (2)
[M-103]
Other characteristic ions
Tranexamic
acid
343 (1)
286 (100)
240 (32) 75 (59), 95 (35), 212 (27), 93 (27), 73 (25), 121 (25), 287 (19), 103
(18), 197 (12), 129 (12), 67 (10), 138 (10), 258 (10), 118(9), 104
(
7
8), 74 (7), 81 (6), 241 (6) 166 (6), 155 (6), 288 (6), 79 (5), 102 (5),
7 (5), 105 (5), 76 (5), 213 (5), 59 (4), 168 (4), 82 (4)
a
m/z values with relative abundances of ions (%) in parentheses.
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Method Development for Tranexamic Acid as EOC-TBDMS Derivatives by
GC-MRM-MS/MS
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TBDMS derivatization, the prepared EOC derivatives were
reacted with MTBSTFA (20 μL) in toluene (20 μL) at 60 C
for 30 min for the GC–MRM–MS/MS analysis.
the range of 0.1250 μg to 1.50 μg/mL, and each calibration
sample was combined with IS of 0.5 μg/mL in serum of
20 μL. The values of slope, intercept, and correlation coef-
ficient (r) were determined to linearity evaluation using
least-squares regression analysis for the calibration curve
constructed, which based on peak area ratios relative to
IS. The LOD and LOQ for TA were calculated at three-
and 10-times value of the standard deviation of blank
divided by slope of calibration curve, respectively. The
repeatability of intra- and inter-day were obtained in tripli-
cates under optimal conditions. Accuracy was evaluated by
comparing the spiked amount value and calculated amount
value. Repeatability and accuracy were performed in tripli-
cate under optimal conditions at three different concentra-
tions of 0.25, 0.75, and 1.25 μg/mL.
ꢀ
Method Validation and Application for TA Analysis in
Serum Sample as EOC-TBDMS Derivatives. Mouse
balb/C serum was used for matrix validation and applica-
tion in this study. A serum sample (20 μL) containing
0.5 μg of IS was vortex-mixed with acetonitrile (0.1 mL)
for 3 min. Then, mixture was diluted with 0.88 mL of dis-
tilled water and centrifuged (15 000 rpm, 15 min) to precip-
itate proteins. The supernatant phase was subjected to the
aforementioned EOC-TBDMS reactions prior to GC–
MRM–MS/MS analysis. Linearity, repeatability (%RSD,
relative standard deviation), accuracy (%RE, relative error),
limit of detection (LOD), and limit of quantification (LOQ)
were evaluated for method validation in serum sample with
triplicate runs. Seven calibration points were performed in
Results and Discussion
Optimal Conditions for TA Analysis in Serum by GC–
MRM–MS/MS. The TA analysis as EOC-TBDMS deriva-
tives was performed under optimal conditions. The EI mass
spectral data of TA as EOC-TBDMS derivatives was pres-
ented in Figure 2 and Table 1. We identified the molecular
ion as m/z 343. By applying scan mode, the characteristic
fragment ions of EOC-TBDMS were identified as m/z
Table 2. Ion transitions of TA and IS as EOC-TBDMS
derivatives.
Ion transitions
Precursor
Analyte
ion (m/z)
Product ions (m/z)
CE (V)
a
b
b
Tranexamic acid
286
260
75 , 121 , 93
24, 9, 15
3, 9, 24
a
b
b
+
+
+
6
-Aminocaproic
acid (IS)
214 , 171 , 75
328 [M-15] , m/z 286 [M-57] , and m/z 240 [M-103]
Table 1 and Figure 2).
The ion m/z 286 [M-57] was selected as the precursor
ion of TA due to characteristic ion with major intensity as
(
+
a
b
Quantification ion (Underline).
Identification ion.
Figure 3 MRM chromatograms of TA and IS in blank serum (a), and serum spiked with standards (b) as EOC-TBDMS derivatives by
GC–MS/MS.
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Method Development for Tranexamic Acid as EOC-TBDMS Derivatives by
GC-MRM-MS/MS
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base peak, which was found to be very selective and stable
in the detection of TA analysis. In GC–MS/MS, the precur-
sor ion m/z 286 was fragmented to produce product ions in
ranging from 3 to 45 V of collision energy (CE). Three
product ions with the maximum intensity were selected for
identification and quantification: m/z 75, 121, and 93, with
+
a CE range of 9 to 24 V. The ion m/z 260 [M-57] as pre-
cursor ion, m/z 214, 171 and 75 as product ions were
selected for identification and quantification of IS. The
quantification product ion was selected based on the criteria
of ion intensity and potential interferences from other com-
pounds; the selected product ion was denoted underlined in
Table 2. MRM chromatograms of TA and IS in blank
serum (3A) and serum spiked with standards (3B) showed
as EOC-TBDMS derivatives by GC–MS/MS in Figure 3.
In the general EOC derivatization, excess ECF is
required due to its instability in basic aqueous solution.
However, this two-phase EOC derivatization in the dic-
hloromethane phase is not needed excess ECF. Especially,
EOC-TBDMS derivatives have a good advantage than
EOC derivatives due to their enhanced MS property. In
addition, this method by using tandem MS is much more
accurate and selective in the presence of interference than
24
previously reported method.
Method Validation for TA Analysis by GC–MS/MS.
The linearity, repeatability, accuracy, LOD, and LOQ were
measured for method validation of TA analysis by GC–
MRM–MS/MS. The correlation coefficient (r) is better than
0
1
.9988 in the calibration curve ranging from 0.125 to
.5 μg/mL. The repeatability and accuracy were measured
with three different concentrations (0.25, 0.75, and 1.25 μg/
mL), and was performed in triplicate on different three
days. Intra-day repeatability (%RSD) and accuracy (%RE)
varied from 1.3 to 1.8% and 2.3 to 4.8%, respectively,
whereas the inter-day repeatability (%RSD) and accuracy
(%RE) varied from 2.3 to 4.8% and −6.8 to 3.5%, respec-
tively (Table 3). The LOD and LOQ were assessed as 0.22
and 0.72 ng/mL, respectively. The LOD and LOQ were
24
assessed to be lower than other reported method. When
applied to serum samples with spiked of TA, TA was iden-
tified and quantified without interferences. Therefore, the
present method will be reliable for the quantification of TA
in serum samples.
Conclusion
We developed a method for TA analysis in serum as EOC-
TBDMS derivatives by GC–MS/MS with MRM mode. In
this study, the mass spectral data set of TA was newly con-
structed as EOC-TBDMS derivatives. This method was val-
idated with good linearity (r ≥ 0.9988), LOD of 0.22 ng/
mL, LOQ of 0.72 ng/mL, intra-day repeatability (%
RSD = 1.3 to 1.8), and accuracy (%RE = −4.0 to 3.5), as
well as inter-day repeatability (%RSD = 2.3 to 4.8) and
accuracy (%RE = −6.8 to 3.5) under optimal conditions.
Thus, the present method will be useful for the
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Method Development for Tranexamic Acid as EOC-TBDMS Derivatives by
GC-MRM-MS/MS
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measurement and quality control of TA in the pharmaco-
logical and cosmetic fields.
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