Journal of Chromatography A 1684 (2022) 463567

Contents lists available at ScienceDirect

Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Simple and fast determination of tetrodotoxin in human plasma based

on hydrophilic-interaction/ion-exchange mixed-mode solid phase
extraction combined with liquid chromatography-tandem mass
spectroscopy
Liang Xina,b,c,1, Yan Lianga,b,c,1, Shuangshuang Yang a,b,c,1, Fengli Jianga,b,c, Fan Yu a,b,c, Meiwei
Zhanga,b,c, Wei Changd, Wei Wang d, Chen Yu a,b,c, Gangyi Liua,b,c,∗, Youli Lua,b,c,∗
a
Central Laboratory, Shanghai Xuhui Central Hospital/Zhongshan-Xuhui Hospital, Fudan University, Shanghai, China
b
Shanghai Engineering Research Center of Phase I Clinical Research & Quality Consistency Evaluation for Drugs, Shanghai, China
c
Shanghai Institute of Clinical Mass Spectrometry, Shanghai, China
d
Department of Emergency Medicine, Shanghai Xuhui Central Hospital/Zhongshan - Xuhui Hospital, Fudan University, Shanghai, China

A r t i c l e i n f o A B st r ACt

Article history: In this study, we developed and validated a simple, fast and sensitive LC-MS/MS method for the mea-
Received 26 August 2022
surement of tetrodotoxin (TTX) in human plasma. Three HILIC-type solid phase extraction (SPE) carriers
Revised 11 October 2022
(PSA, silica, Siphila i HILIX) with different stationary phase functional groups were compared. The Siphila
Accepted 12 October 2022
Available online 18 October 2022 i HILIX SPE plate containing multi-carboxyl groups was finally selected due to obviously better extraction
recovery of TTX (about 80% of recovery from plasma samples) than the other two and no significant ma-
Keywords: trix effects were observed, which was speculated to have mixed-mode synergistic effects of hydrophilic
Tetrodotoxin interaction and ion exchange. 100μL plasma sample was precipitated rapidly with acetonitrile containing
Hydrophilic-interaction/ion-exchange 1% trichloroacetic acid, and filtrates were loaded onto Siphila i HILIX 96 well SPE plate. After washed
Mixed-mode solid phase extraction
with 95% acetonitrile, TTX was eluted with 200 μL of 50% acetonitrile containing 1% trichloroacetic acid.
LC-MS/MS
2μL of elution solution was directly injected into LC-MS/MS and the total run time on a BEH amide col-
Human plasma
umn was 4.5 min. The method avoids the evaporation and ultrafiltration processes which is simple and
timesaving (<30 min). TTX and internal standard (arginine-15N4) were monitored in positive mode using
m/z 320.3→162.2 (quantification transition for TTX), 320.3→284.1 (confirmation transition for TTX) and
179.2→63.0 (transition for IS), respectively. The method was linear in the range of 0.1–20 ng/mL for TTX
with the low limit of quantification (S/N > 10) of 0.1 ng/mL; the intra- and inter-assay accuracies were in
the range of 98.5%-99.8% (relative standard deviations, RSDs ≤ 5.92%) and 98.8–99.5% (RSDs ≤ 6.23%), re-
spectively. Biases of spiking analysis were ranged from -7.00% to 7.43% for healthy human plasma samples
(RSDs ≤ 8.83%) and from -5.00% to 3.93% for hemolytic, high triglyceride, high cholesterol and high biliru-
bin plasma samples (RSDs ≤ 6.40%), which proved the good anti-interference property of the method. The
results showed that the method is sensitive, accurate, specific, reliable, and can be used to monitor the
concentration of TTX in plasma to meet the needs of clinical research and poisoning screening.
© 2022 The Authors. Published by Elsevier B.V.
This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/)

Introduction
Tetrodotoxin (TTX) poses a serious threat to human health in
coastal areas where marine food is the main food source [1–2]. In-
cidents of food poisoning caused by TTX were reported almost ev-
ery year [3–7]. Compared to other marine toxins, TTX intoxication
∗
Corresponding authors.
E-mail addresses: gyliu@shxh-centerlab.com (G. Liu), yllu@shxh-centerlab.com
(Y. Lu).
1
These authors are considered as co-first authors.
https://doi.org/10.1016/j.chroma.2022.463567
0021-9673/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)
leads to more severe symptoms and higher mortality [8]. The grade
of TTX poisoning depends on the ingested amount, the time since
ingestion, and the health status before poisoning [9–10]. There is a
strong correlation between the symptoms and the levels of TTX in
the blood of victims [11].
As a potent toxin, TTX selectively blocks voltage-gated sodium
(NaV) channels, such as NaV1.1, NaV1.2, NaV1.3, NaV1.6, and NaV1.7
[12–13], which are widely distributed in the human central and pe-
ripheral nervous system [14], making it a very promising drug for
the treatment of pain [15–16]. The therapeutic doses reported in
current clinical trials were 15–45 μg with Cmax of approximately
L. Xin, Y. Liang, S. Yang et al.
1 ng/ml in plasma [17]. While, the lethal dose of TTX in human is
about 1.5–2.0 mg, with a blood concentration of 9 ng/ml [11,18].
Therefore, to ensure drug safety and study the relationship be-
tween drug concentration and efficacy, a highly sensitive and ac-
curate assay is needed to monitor TTX blood or plasma levels in
human.
Although various methods used for the determination of TTX
have been published, such as mouse bioassay [19], immunoas-
say [20], nuclear magnetic resonance (NMR) [21], and liquid chro-
matography with fluorescence detection (LC-FL) [22], most of them
are for shellfish or fish tissue samples. Dealing with human bio-
logical samples is much more challenging because the amount of
TTX in the urine and plasma is generally extremely low. The liquid
chromatography-tandem mass spectroscopy (LC-MS/MS) method
was considered the most popular method for the identification
and quantification of TTX because of its high sensitivity, selectivity,
and specificity [23–24].
Since TTX is a highly polar substance, there would be poor re-
tention on the conventional reverse-phase column and low ioniza-
tion efficiency in mass spectrometry [25]. Hydrophilic interaction
liquid chromatography (HILIC) could help to address these issues
and showed good retention capability and improved ionization ef-
ficiency in LC-MS [26–27]. While ion suppression (one kind of ma-
trix effect) was another challenge in analyzing TTX in biological
samples. Ion suppression usually results from the coelution of non-
detected interferences [28–29]. In general, ion suppression can be
reduced by sample preparation, chromatographic separation, and
MS analysis [30–32].
The most daunting analysis bottleneck is sample preparation.
Because its effective extraction and enrichment from complicated
human biological samples are challenging [33]. The most popu-
lar technique for the cleanup of TTX samples is solid phase ex-
traction (SPE), usually using cation exchange extraction [34–35].
The positively charged guanidinium group of TTX strongly inter-
acts with the sulfonic acid group in the stationary phase, allowing
TTX to be strongly retained on SPE cartridges. Only strong acids
such as hydrochloric acid can elute it, requiring additional solvent
evaporation and residue redissolution steps, which are both time-
consuming and labor-intensive. Therefore, strong cation exchange
extraction techniques are rarely used for the detection of TTX at
present.
Another SPE technique applied to TTX detection is the use of
graphitized carbon carriers. However, the TTX recoveries of urine,
serum, and plasma samples were 43%, 23%, and 20%, respec-
tively [36]. Low recoveries can be attributed to toxin loss and/or
ion suppression during SPE cleanup. The limits of quantification
for serum and plasma samples were 21.8 and 25.4 ng/mL, re-
spectively, which were not sensitive enough for pharmacokinetic
studies. Ochi [37] also used graphitized carbon carriers and opti-
mized the method by ion-pair solid-phase extraction. The differ-
ence was that the use of ion-pair solvent in sample loading condi-
tion improve the retention of TTX on graphitized carbon. While the
method was only applied to the determination of toxins in bivalve
extracts, there was no data to prove its recovery and matrix effect
in human biological samples.
Hydrophilic interaction liquid chromatography-solid phase ex-
traction (HILIC-SPE) can be a third option due to the hydrophilic
nature of TTX. However, few attempts have been made to extract
TTX from plasma using HILIC-SPE technology. One reason is the
limited impurity removal capability of a single HILIC-SPE. Consid-
ering this, double SPE using C18-HILIC had been used to improve
the efficiency of removing ion suppression [38].
In this study, we attempted to use three HILIC-type SPE car-
riers for sample preparation to compare their extraction recover-
ies and influence on matrix effects. A simple and fast SPE pro-
tocol based on hydrophilic-interaction/ion-exchange mixed-mode
2
Journal of Chromatography A 1684 (2022) 463567
was developed to extract TTX from human plasma without evapo-
ration and ultrafiltration. The pretreatment method combined
with LC-MS/MS method was used for the quantitation of TTX in
human plasma with satisfactory sensitivity, recovery, and matrix
effect, which greatly simplified the sample preparation steps and
short- ened the total analytical time.
1. Material and methods
1.1. Reagents and materials
TTX (99.5%) was purchased from Standardpharm Co., Ltd. (New
York, USA). Arginine-15N4 (90.4%) was purchased from TLC Phar-
maceutical Standards Ltd. (Ontario, Canada). Voglibose (98%) was
purchased from Alta Scientific. Ltd. (Tianjin, China). Siphila i HILIX
96 well plates (30 mg/2 mL) were obtained from Siphila Technolo-
gies Co., Ltd. (Tianjin, China). PSA 96 well plates (30 mg/2 mL)
and silica 96 well plates (30 mg/2 mL) were obtained from Tu-
pLabs Co., Ltd. (Tianjin, China). The links for the three SPE plates
were shown in the supplementary materials. Protein precipita-
tion plates (2 mL/well) were obtained from TupLabs Co., Ltd.
(Tianjin, China). Trichloroacetic acid (TCA, ≥99%) was supplied by
Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). HPLC-
grade acetonitrile (ACN) and methanol were supplied by Merck
KGaA (Darmstadt, Germany). HPLC-grade formic acid (FA, 99%)
and ammonium formate (98%) were supplied by ACS (Wilmington,
USA). Water was purified using Simplicity system (Merck KGaA,
Darmstadt, Germany). Blank human plasma was obtained from
healthy donors who signed the Informed Consent Forms.
The standard stock solutions of TTX (100 μg/mL) were prepared
in deionized water and stored at −80 °C until further use. Work-
ing standard solutions were prepared by serial dilution of standard
stock solutions using deionized water at 5 different points (1, 2, 10,
50, 200 ng/mL) before they were added to human blank plasma to
create calibration curves with ranges from 0.1 to 20 ng/mL. The
same way was used to make quality control samples (QCs) at 4
different concentration levels (0.1, 0.5, 2, 10 ng/mL).
1.2. Sample pretreatment
One hundred microliters of human plasma sample (calibration
curves, QCs or real samples) and 10 μL arginine-15N4 (internal
standard, IS, 100 ng/mL) working solution were mixed and trans-
ferred to 96 well protein precipitation plate, then 700 μL of 1%TCA-
ACN was added for protein precipitation. After filtering with a pos-
itive pressure device, all filtrates were transferred to Siphila i HILIX
96 well SPE plate, which was previously activated with 1 mL of
methanol and equilibrated with 1 mL ACN. Wash once with 200 μL
of 95% ACN and elute with 200 μL of 1%TCA-50% ACN. 2 μL of elu-
tion solution was directly injected into LC-MS/MS.
1.3. LC-MS/MS analysis
The LC-MS/MS system consisted of a triple quadrupole mass
spectrometer 6500 system (SCIEX, USA) and an ExionLC system
(SCIEX, USA). Data was acquired and statistically calculated by An-
alyst 1.6.3 (SCIEX, USA).
TTX and IS were monitored using multiple reaction monitoring
(MRM) in positive mode with the transitions of m/z 320.3→162.2
(quantification transition for TTX), 320.3→284.1 (confirmation
transition for TTX) and 179.2→63.0 (transition for IS), respectively.
Ion source parameters were also optimized: 550 °C source temper-
ature, 60 psi ion source gas1, 70 psi ion source gas2, 30 psi curtain
gas, 4000 V ion spray voltage, 120/43 V declustering potential, and
49/27 eV collision energy.
L. Xin, Y. Liang, S. Yang et al.
Fig. 1. Effects of ammonium formate (a) and formic acid (b) concentration in mobile phase for TTX.
Chromatographic separation was performed on an ACQUITY
UPLC BEH Amide column (50×2.1 mm, 1.7 μm, Waters, USA) at
room temperature. The mobile phase consisted of (A) deionized
water and (B) ACN, both containing 10 μmol/L ammonium formate
and 0.01% FA. The following gradient program was used [mobile
phase B]: 85–55% at 0–2 min; held 55% at 2–3 min; 55–85% at 3–
3.01 min; held 85% at 3.01–4.5 min with a flow rate of 0.5 mL/min.
1.4. Method validation
The lower limit of quantification (LLOQ), linearity, inter/intra-
batch accuracy and precision, matrix effect, extraction recovery,
specificity, and interference of the established method were inves-
tigated according to the U.S. food and drug administration (FDA)
bioanalytical method validation guidelines [39].
2. Results and discussion
2.1. Development of chromatography and mass spectrometry method
TTX is difficult to retain on reversed-phase columns [26], and
most recent reports used an amide column with a length of 10 cm
to improve the retention of 10 TTX analogs [37]. Here, an ACQUITY
UPLC BEH C18 Amide column (50×2.1 mm, 1.7 μm, Waters, USA)
was used to separate TTX from its interferences, which performed
better compared with an ACQUITY UPLC BEH C18 HILIC column
(50×2.1 mm, 1.7 μm, Waters, USA). The retention times for TTX
and IS on the amide column were 2.41 min and 2.31 min, respec-
tively, with a total analysis time of 4.5 min.
The pH value and the concentration of ammonium formate in
the mobile phase were considered to have significant effects on
retention time and peak area [37]. Therefore, different FA and am-
monium formate concentrations were tested under the same sepa-
ration conditions. It was observed that the retention time and peak
area of TTX increased with the decrease of FA (from 0.05% to 0.01%,
v: v) (see Fig. 1a) and ammonium formate minimized the retention
time shift between pure solvent and matrix samples but decreased
the peak area of TTX (Fig. 1b). In this study, 10 μmol per liter of
ammonium formate and 0.01%FA (v: v) were used in both mobile
phases to acquire better sensitivity and make the peaks more sta-
ble. The variation of 56 consecutive injections prepared by plasma
samples was tested (see Fig. S1) and the mean retention time was
2.42±0.1 min (2.37–2.47 min), which indicated that the chromato-
graphic separation conditions performed well with excellent repro-
ducibility. MS/MS parameters for TTX and IS were optimized by
direct infusion of individual standard solutions. The quantifier and
confirmation product ion for TTX were m/z 162.2 and 284.1,
respec- tively, although the most abundant product ion was
observed as [M + H–H2O]+ ion (m/z 302.2). The latter has higher
baseline and stronger interference in plasma samples.
3
Journal of Chromatography A 1684 (2022) 463567
2.2. Selection and optimization of SPE
In the beginning, MCX and WCX cation exchange SPE plates
(Waters, USA) were tested to clean up the plasma samples and pu-
rify TTX. It was found that the WCX plate can hardly retain TTX
during loading, and the MCX plate can hardly elute TTX using or-
ganic solvent containing moderate acids like fomic acid or acetic
acid. Strong acid like hydrochloric acid wasn’t tried on MCX plate
due to its damage to liquid chromatography and mass spectrome-
try, even if it can be dried up, poor peak shapes and severe matrix
effect might not be avoided.
In turn, three different HILIC-type SPE plates were attempted
to separate TTX from the plasma matrix. PSA plate containing
polyamide material, silica plate containing silica gel material, and
Siphila i HILIX plate containing multi-carboxyl material were com-
pared. The flow charts of comparison and optimization for three
different HILIC-SPE were shown in Fig. 2. All the loading, washing,
and elution samples were collected and analyzed to calculate the
loss or recovery of TTX.
HILIC-type SPE usually requires high concentrations of organic
solvents as loading solutions and water is used as a strong elu-
ent [40]. In this study, pure ACN and ACN containing 1%TCA,
5%TCA, and 2%FA were investigated respectively as loading solu-
tions, which also acted as protein precipitating agents, denatur-
ing plasma proteins and releasing TTX. 100μL of standard solution
(prepared by deionized water) and plasma samples were precipi-
tated with 700μL of loading solutions in 96-well protein precip-
itation plates respectively and then all filtrates were loaded onto
three different SPE plates, which were previously activated with
methanol and equilibrated with ACN. The results showed TTX was
most strongly retained on the Siphila i HILIX SPE plate during sam-
ple loading. ACN was the optimal loading solvent and facilitated
the retention of TTX on SPE plates, followed by 2%FA-ACN, 1%TCA-
ACN, and 5%TCA-ACN (Fig. 3). The loss of TTX in plasma samples
were less than that in standard solution samples during loading,
which might be due to the poor buffering capacity of standard so-
lution samples compared with plasma.
To further investigate the loss and recovery of TTX during wash-
ing and elution steps, 200μL of 95%ACN was used for washing fol-
lowed by 200μL of 1%TCA-50%ACN for elution. In the 2%FA-ACN
and ACN loading groups, due to the low recoveries after elution,
another 200μL of 5%TCA-ACN was used for further elution to im-
prove the recovery. The results were shown in Fig. 4.
Different retention and elution capabilities of TTX were ob-
served on the same SPE plate under different loading conditions
during washing and elution. Siphila i HILIX was the most distinct
and optimal one (Fig. 4a). In the 1%TCA-ACN loading group, wash-
ing with 95% ACN resulted in a loss of 18% TTX, while elution with
1% TCA-50% ACN resulted in the recovery of 80% TTX in plasma
samples. While during the same loading and washing steps, the
corresponding standard solution samples would lose even more,
with less than 30% TTX recovered by elution. In the 5%TCA-ACN
loading group, both the loading and washing steps resulted in
L. Xin, Y. Liang, S. Yang et al.
Fig. 2. The flow charts of comparison and optimization for three different HILIC-SPE.
more loss of TTX, about 60% for plasma samples and even higher
for standard solution samples (>80%). In the 2%FA-ACN loading
group, no significant TTX loss was observed for plasma samples
after washing with 95%ACN, and the recovery was about 22% after
elution with 1%TCA-ACN. Compared to the two TCA-ACN loading
groups, the total recovery of TTX in plasma samples was reduced
throughout the SPE, so an additional 5%TCA-ACN elution was per-
formed to look for the lost TTX and the total recovery was in-
creased to 60% after the additional elution. This was quite
different from the total recovery of standard solution samples
which was close to 100%. The total recovery of TTX in plasma
samples was sig- nificantly lower in the ACN loading group, no
matter in the load- ing and washing solutions, or the eluted and
twice-eluted samples, while the recovery of standard solution
samples was nearly 100%. In the 2%FA-ACN and ACN loading
groups, the lower recovery of TTX from plasma samples compared
to standard solution samples may be due to the incomplete
release of TTX from plasma proteins upon precipitation of these
two loading solutions. Similar results were observed when using
PSA and silica plates (Fig. 4b and 4c). Strong acids such as TCA in
the precipitating agent may be nec- essary for the release of TTX
from plasma proteins. Excessive TCA resulted in weak retention
of TTX on SPE, which may be related to the decreased polarity of
the TTX-TCA conjugates in the pres- ence of ion pairs solvents, thus
reducing the retention of TTX on HILIC-type SPE plates.
4
Journal of Chromatography A 1684 (2022) 463567
The results showed that the retention capacity among Siphila i
HILIX, PSA, and silica SPE were quite different. Compared to Siphila
i HILIX, PSA and silica had weaker retention of TTX under almost
all loading conditions. It was speculated that both Siphila i HILIX
and silica may have two interaction modes: hydrophilic interac-
tion and ion exchange. Some SiOH-containing silica materials had
been shown to have cation-exchange properties [41]. While in our
study, we found that the ion exchange effect of silica SPE might be
very weak which could be easily inhibited by TCA or FA. In addi-
tion to hydrophilic interaction, Siphila i HILIX may also retain polar
basic compounds through cation-exchange interaction which was
similar or even stronger to that of weak cation-exchange material
with carboxylic acid moieties [42]. It seemed that PSA had only
a pure hydrophilic effect on TTX, and retention was the weakest.
Among the three HILIC materials, Siphila i HILIX was considered
to have better synergistic effect of hydrophilic interaction and ion
exchange, which was suitable for the retention of TTX.
After optimizing the loading conditions, the washing solvents
were selected among 95% ACN, 98% ACN, and ACN. No significant
difference was observed, so 95%ACN was chosen because it had the
strongest cleaning capability. The elution solvent remained 1%TCA-
50%ACN, which had no obvious ion suppression or enhancement
effects on TTX. Since stable isotope labeled TTX is very expen-
sive and difficult to obtain, we used the reported voglibose and
the available arginine-15N4 as candidate internal standards in this
L. Xin, Y. Liang, S. Yang et al. Journal of Chromatography A 1684 (2022) 463567

Fig. 3. The loss of TTX under different sample loading conditions on three HILIC-SPE plates.

Table 1
The major validation parameters for TTX and IS in human plasma.

TTX

Spiked conc. (ng/mL) Intra-assay (n = 6) Inter-assay (n = 6 × 3) Extraction recovery (n = 6) IS-normalized MF (n = 6)

Mean accuracy (%) RSD (%) Mean accuracy (%) RSD (%) Mean (%) RSD (%) Mean (%) RSD (%)

0.1 99.8 5.92 99.5 5.47

0.5 98.7 5.75 99.4 6.23 81.5 5.53 0.979 3.41
2 98.5 2.29 99.1 4.16 80.8 3.58
10 99.2 4.37 98.8 5.26 81.2 6.78 0.968 4.29

Spiked conc. (ng/mL) arginine-15N4 voglibose

Extraction recovery (n = 6) Extraction recovery (n = 6)

Mean (%) RSD (%) Mean (%) RSD (%)

10 74.3 4.26 71.5 3.17

study, both of which showed similar chromatographic behavior to
TTX on the amide column.
The performance of the two candidate internal standards was
further compared in plasma matrix. Post-column injection exper-
iment was used to investigate the different matrix effects of TTX
and the two candidate internal standards in pure solvent and
plasma samples. It was found that TTX was barely enhanced or
inhibited by plasma matrix, arginine-15N4 was slightly enhanced,
while voglibose was severely enhanced (Fig. 5). By comparing the
recoveries, it can be found that both arginine- 15N4 and voglibose
had similar extraction recoveries as TTX. Therefore, arginine- 15N4
was finally selected as IS in this study (see Table 1).
2.3. Method validation
After determining the optimization of SPE conditions, valida-
tion was attempted to investigate the practicability of the present
method. The method displayed excellent linearity of TTX with
1/x as weighting factor, and R value was higher than 0.999 over
a 200-fold linear dynamic range (0.1–20 ng/mL) with the LLOQ
(signal-to-noise ratio, S/N>10) of about 0.1 ng/mL. Moreover, spike-
recovery studies assessed for four different QCs (at 0.1, 0.5, 2, and
10 ng/mL, n = 6) at three consecutive days. The intra- and inter-
assay accuracies (%) were 98.5%−99.8% and 98.8%−99.5%, respec-
tively, calculated by comparing measured concentrations to the-
oretically spiked concentrations. The intra- and inter-assay preci-
sion investigated in terms of relative standard deviations (RSDs%),
were ≤ 5.92% and ≤ 6.23%, respectively. The results were summa-
rized in Table 1, which showed acceptable accuracy and precision
for plasma TTX in the investigated concentration range. The vali-
dation of the method selection proved that samples were prepared
well by Siphila i HILIX. The representative chromatograms of blank
samples and LLOQ samples at concentration of 0.1 ng/mL of TTX
and 10 ng/mL of IS were shown in Fig. 6. No obvious interferences
coeluting with the TTX and the IS was observed and the baselines
of noise were acceptable. The LLOQ was about 0.3 ng/mL for con-
firmation transition (m/z 320.3→284.1) according to the criterion
of S/N>10, indicating that the method in this study would be suit-

5
L. Xin, Y. Liang, S. Yang et al. Journal of Chromatography A 1684 (2022) 463567

Fig. 4. The recoveries of TTX in each step on three HILIC-SPE plates under different sample loading conditions. a. Siphila i HILIX SPE, b. Silica, c. PSA.

6
L. Xin, Y. Liang, S. Yang et al. Journal of Chromatography A 1684 (2022) 463567

Fig. 5. Plasma matrix effect of a. TTX, b. arginine-15 N4 and c. voglibose.

Fig. 6. The representative chromatograms of blank, LLOQ (0.1 ng/mL) of a. TTX and b. IS (10 ng/mL).

7
 L. Xin, Y. Liang, S. Yang et al.
Table 2
Bias and precision of spiked samples from different sources.
0.1 ng/mL (n = 3) 10 ng/mL (n = 3)
sources
Mean (bias%) RSD (%) Mean (bias%)
Plasma1 3.67 8.83 4.23
Plasma2 −3.37 6.16 0.97
Plasma3 1.73 8.59 −0.37
Plasma4 −6.00 2.50 3.20
Plasma5 −1.43 5.90 −7.00
Plasma6 −2.67 1.49 7.43
hemolytic plasma −3.53 4.28 −2.50
high triglyceride plasma 0.90 4.80 −4.67
high cholesterol plasma −3.67 3.10 −2.50
high bilirubin plasma −5.00 4.95 3.93
able for the identification and quantification of TTX at poisoned
concentration levels.
The extraction recovery was assessed by comparing the peak ar-
eas of spiked plasma samples with those of post-extraction spiked
samples at three QC levels. The quantitative matrix factors were
evaluated by comparing the peak areas of post-extraction spiked
samples from six sources at two QC levels with those of neat stan-
dard solutions. The method demonstrated excellent extraction re-
covery for TTX, ranging from 80.8% to 81.5% at three different con-
centrations (RSDs ≤ 6.78%). The mean IS normalized matrix factor
of samples from six sources at two levels were 0.979 and 0.968
respectively, which indicated no significant ion suppression or en-
hancement existed by the optimized method (Table 1). The lost ex-
traction recovery of TTX was mainly in the washing step, as shown
in Fig. 4a, which was speculated to be improved by increasing the
amount of SPE packed material or reducing the washing volume.
Good specificity means that the analytical method can differ-
entiate between the target analyte and interfering substances for
samples from different sources and the bias and precision were
acceptable. Ten blank human plasma samples including samples
from six healthy volunteers and pooled blank human plasma sam-
ples mixing with high concentrations of hemoglobin, triglyceride,
cholesterol, and bilirubin were prepared and investigated by spik-
ing analysis. In detail, working standard solutions of TTX were
spiked to the above 10 blank plasma samples to make QCs at LLOQ
and High concentration levels (0.1, 10 ng/mL). The QCs were pre-
treated and analyzed. The biases and RSDs were listed in Table 2.
The biases of target value were ranged from −7.00% to 7.43% for
healthy human plasma samples (RSDs ≤ 8.83%) and from −5.00%
to 3.93% for hemolytic, high triglyceride, high cholesterol and high
bilirubin plasma samples (RSDs ≤ 6.40%), which proved the good
anti-interference property of the method.
Saxitoxin and its approximately 60 natural analogues, which
are also potent and specific voltage-gated sodium channel block-
ers and share the same binding site as that of TTX [43, 44], also
cause paralysis and even death in extreme cases. In our supple-
mental study, gonyautoxin5 (GTX5) was purchased for interference
test since its retention time was closest to TTX on amide column
[45, 37]. Under the optimized LC and SPE conditions described in
this study, the retention time of GTX5 was 2.53 min which was
close to TTX (see Fig. S2). No interferences were observed
between the TTX and GTX5 MRM transitions. Therefore, we
believe that sax- itoxin and its analogues may not interfere the
detection of TTX. Besides, in a preliminary study, the total recovery
of GTX5 from plasma samples was about 70% according to the
peak area ratio of spiked plasma samples and neat standard
solutions which in- dicated that the described method in our study
may be suitable for quantitation of these compounds after
optimization. In addi- tion, the analogs of TTX like 4,9-anhydro
TTX, 4–epi TTX and so on, usually have similar chemical
structures and lower toxic equiv-
Journal of Chromatography A 1684 (2022) 463567
alency factors [46]. Although their SPE and LC-MS behavior may
be similar to that of TTX, it is likely that they share the same or
similar parent or product ions with TTX, which will interfere the
RSD (%) detection of TTX and require further chromatographic separation.
0.32 More work is needed to prove whether interference exists from
1.73 TTX analogs or whether TTX analogs can also be detected by our
2.99 method or an improved method.
2.51
4.07
1.04 3. Conclusions
3.86
2.92 An LC-MS/MS method had been developed and validated for
6.40
the quantitation of TTX in human plasma. This method used multi-
2.37
carboxyl group SPE materials and did not require evaporation and
ultrafiltration steps. Compared with previous reports, the method
was highly sensitive, simple, and rapid in sample pre-treatment
and had short chromatographic time. The method presented in
this paper showed wide linearity, low LLOQ, good precision and
accu- racy, and high recovery characteristics which well satisfied
the re- quirements of pharmacokinetic study and clinical diagnosis
of TTX.
Credit authorship contribution statement
Liang Xin: Conceptualization, Investigation, Methodology, Writ-
ing - Original Draft. Yan Liang: Data Curation, Investigation, Writ-
ing - Original Draft. Shuangshuang Yang: Methodology, Data
Curation, Visualization. Fengli Jiang: Investigation, Methodology.
Fan Yu: Investigation, Validation. Meiwei Zhang: Data curation,
Methodology. Wei Chang: Funding acquisition. Wei Wang: Data
curation, Validation. Chen Yu: Supervision. Gangyi Liu: Formal
analysis, Project administration, Writing - Review & Editing. Youli
Lu: Funding acquisition, Formal analysis, Project administration,
Writing - Review & Editing.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared to
influence the work reported in this paper.
Data Availability
Data will be made available on request.
Acknowledgments
This research was funded by the Innovation Fund of Science
and Technology Commission of Shanghai Municipality
(20Y11900800) and the General Project of Medical Research Fund
of Xuhui District, Shanghai (SHXH202121).
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.chroma.2022.463567.
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