Journal of Chromatography B 1093–1094 (2018) 60–65

Contents lists available at ScienceDirect

Journal of Chromatography B
journal homepage: www.elsevier.com/locate/jchromb

Dry Blood Spot sample collection for post-exposure monitoring of chemical T

warfare agents – In vivo determination of phosphonic acids using LC-MS/MS
⁎
Lilach Yishai Aviram , Miriam Magen, Shira Chapman, Adi Neufeld Cohen, Shlomi Lazar,
Shai Dagan
Israel Institute for Biological Research (IIBR), P.O.B. 19, Ness Ziona, Israel

A R T I C LE I N FO A B S T R A C T

Keywords: Phosphonic acids are the direct and immediate metabolites of organophosphorus chemical warfare agents (OP-
Phosphonic acids CWAs). Accordingly, their detection serves for evaluating exposure to OP-CWAs in a terror or war scenario. After
Isopropyl methylphsophonic acid exposure, phosphonic acids are present in the blood; however, blood drawing must be carried out by medical
LC-MS personnel, hence the number of samples that can be drawn in a mass-casualty event is limited. Herein, we
Dry Blood Spot
describe a new approach developed for the determination of phosphonic acids in blood using Dry Blood Spots
(DBSs) on a filter paper. The method is based on a simple sample preparation protocol, followed by LC-MS-MS
targeted (MRM) analysis. The detection limits of Soman (GD), Cyclosarin (GF) and VX metabolites in whole
blood were as low as 1 ng/ml, while the detection limits were 0.3 ng/ml for the GF metabolite and 0.5 ng/ml for
the Sarin (GB) metabolite. Good recoveries were obtained in the range of 1–100 ng/ml for GB and GD meta-
bolites, and 3–100 ng/ml for GF, VX and RVX metabolites, with a linear response (R2 = 0.99). The method has
proven to be reliable even with DBS samples stored up to 35 days at room temperature before analysis. This
method was implemented in a 24 h time-course determination of the Sarin metabolite in an in - vivo experiment,
after rat exposure to 1 LD50 of Sarin. This technique is simple, rapid, sensitive, robust, long lasting and com-
patible with field collection and storage; hence, it can serve for large-scale sampling and reliable monitoring of
potential OP-CWAs casualties. Since DBS sampling is amenable to nonprofessionals, including self-sampling, this
technique is highly suitable for mass-casualty incidents.

1. Introduction
Chemical warfare agents (CWAs) were first used on a large scale in
World War I, mainly chlorine gas and sulfur mustard [1]. Organopho-
sphorus (OP) CWAs, which are far more potent, were developed later;
the G-type agents (Tabun (GA), Sarin (GB), Soman (GD) and Cyclosarin
(GF)) were developed in the late 1930s through the 1940s, and the V-
type agents were first synthesized in the early 1950s [2]. VX was used
in the terror attack in Osaka Japan in 1994, whereas Sarin was used in
the terror attacks in Mutsumoto in 1994 and in Tokyo in 1995. Re-
cently, it has been widely used in the ongoing civil war in Syria with
thousands of casualties [3]. Such events require the monitoring of ca-
sualty exposure to the CWA.
OP-CWAs have very short lifetimes in the human body [4], and they
are hydrolyzed, metabolized or adducted to nucleophilic sites on
macromolecules such as proteins and DNA [5]. A major and straight-
forward route for the detection of exposure to G- and V-type agents is to
monitor the hydrolysis products, methylphosphonic acids (pKa
~2.2–2.3) [6,7], since these products are indicative as they are present
in the body only after exposure to CWA. Phosphonic acids can be de-
tected at concentrations as low as sub ng/ml in body fluids using LC-MS
[8–10], and they are effective markers after exposure. However, using
conventional extraction methods requires relatively large volumes of
body fluids such as urine or blood.
In the Tokyo Sarin terror attack in 1995, concentrations of
2–135 ng/ml isopropyl methyl phosphonic acid (IMPA) were found in
blood samples of the casualties drawn 1.5–2.5 h after hospitalization
[11]. The pharmacokinetics of the hydrolyzed metabolites of Soman,
Sarin and GF in rats (urine, blood and lung) after injection of the agent
at a dose of 75 μg/kg were reported by Shih [4]. The concentrations
that were found in whole blood for GF and Soman and in plasma for
Sarin (at t = 0) were 200–500 ng/ml. The Sarin metabolite could not be
detected after 10 h.
The use of the Dry Blood Spot (DBS) technique is common for the
collection of small amounts of blood, especially from newborns for
screening applications [12]. This method was first published by Robert

⁎
Corresponding author.
E-mail address: lilachy@iibr.gov.il (L. Yishai Aviram).

https://doi.org/10.1016/j.jchromb.2018.06.035
Received 12 April 2018; Received in revised form 7 June 2018; Accepted 17 June 2018
Available online 19 June 2018
1570-0232/ © 2018 Elsevier B.V. All rights reserved.
L. Yishai Aviram et al. Journal of Chromatography B 1093–1094 (2018) 60–65

Table 1
Optimized mass spectrometry detection parameters.
Metabolite structure Negative ion formula tR (min) MRM (ESI−) DP EP CE CXP
Declustering potential (V) Entrance potential (V) Collision energy (eV) Collision exit potential (V)

EMPA (VX acid) C3H9PO3− 3.10 123.0 > 95.0 −40 −10 −18 −7
3.10 123.0 > 79.0 −40 −10 −40 −7

IMPA (GB acid) C4H11PO3− 4.15 137.0 > 95.0 −35 −10 −20 −9
4.15 137.0 > 79.0 −35 −10 −38 −13

iBuMPA (RVX acid) C5H13PO3− 6.40 151.1 > 77.0 −50 −10 −32 −15
6.40 151.1 > 79.0 −50 −10 −44 −7

CMPA (GF acid) C7H15PO3− 7.50 177.1 > 95.0 −120 −10 −30 −10
7.50 177.1 > 79.0 −120 −5 −60 −10

TPMPA (GD acid) C7H17PO3− 8.20 179.1 > 95.0 −90 −10 −26 −7
8.20 179.1 > 79.0 −90 −10 −50 −13

Guthrie in the early 1960s and is based on sampling a small blood spot
onto a filter paper instead of drawing a few ml of blood into a vial. With
the DBS technique, sampling is easy and fast without the need for
trained professionals. However, the small amounts of blood drawn
(~20–40 μl) make this approach more analytically challenging in terms
of sample preparation and sensitivity compared with traditional blood
sampling. In the last decade, the use of DBS sampling has grown and
gained popularity in pharmaceutical, clinical, toxicological and forensic
research [13–19]. This is attributed to the fact that the sensitivity and
selectivity of analytical methods has improved, especially with mass
spectrometry [20]. In addition to the convenient minimally invasive
sampling, the DBS technique has several additional advantages [21]
including the following: it can be easily shipped on the filter paper card
without the risk of infection since pathogenic agents are deactivated on
the paper; in numerous studies it was found that many drugs are more
stable in DBS samples than in refrigerated or frozen liquid blood
[22,23]; and in some cases it is even easier to sample a victim casualty
by pricking his/her finger instead of asking him/her to give a urine
sample at a forensic scene. Additionally, this method is ideal for
pharmacokinetics research, especially in small animals [24].
In recent years, a few papers have been published that monitored
exposure to CWA by measuring the activity of butyrylcholinesterase
from a DBS [25]. However, this method is not specific to a certain toxic
compound. Recently, Hamelin et al. reported the use of DBSs for
monitoring exposure to nerve agents through analyzing the protein
adduct and metabolites [26]. McKenna et al. also published a paper
describing the use of paper spray mass spectrometry to detect the hy-
drolysis products of chemical warfare agents in body fluids [27].
Herein, we report here the use of DBSs for monitoring exposure to
chemical warfare agents through analyzing the immediate metabolites,
phosphonic acids, by LC-MS/MS. To the best of our knowledge, this is
the first report that implemented this method in an in vivo experiment
in rats, monitoring their low levels of isopropyl methylphosphonic acid
(IMPA) after being exposed to Sarin using DBSs.
2. Material and methods
2.1. Chemicals and reagents
Sarin (isopropyl methylphosphonofluoridate) was supplied by the
Department of Organic Chemistry of IIBR. It was dissolved in propylene
glycol (30 mg/ml) and kept frozen. Fresh dilute solutions in saline
(300 μg/ml) were prepared for the experiment.
All of the isopropyl methylphosphonic acids; ethyl methylpho-
sphonic acid, pinacolyl methylphosphonic acid, cyclohexyl methyl-
phosphonic acid, tripropyl methyl phosphonic acid and isobutylmethyl
phosphonic acid (IMPA, EMPA, MPA, CPMPA, TPMPA and ibuMPA
acid), were synthesized in house. MeOH and LC-water (both LC-MS
grade) were purchased from Bio-Lab Ltd. (Israel). Ammonium formate
(LC-MS grade) was purchased from Sigma (St. Louis, MO, USA). The
filter paper (number 1) was purchased from Whatman. Human blood
was purchased from the Israeli Blood Bank.
2.2. Preparation of standard solutions
Stock solutions of the acids were prepared to obtain a concentration
of 2000 μg/ml in MeOH. The working standard solutions were prepared
by diluting the stock solutions with TDW (LC-MS grade) to final con-
centrations ranging from 0.5 ng/ml to 2000 ng/ml. The stock and
standard solutions were stored at −20 °C and brought to room tem-
perature before use. The working standard solutions were spiked in
human whole blood and rat blood, and they were used immediately
after spiking. All of the spiking volumes were always lower than 3% (v/
v).
2.3. LC-MS/MS instrument and conditions
Chromatographic separation was achieved using an Agilent 1290
HPLC (Palo Alto, CA, USA) equipped with a C18 Gemini column (ID 3
μm, 150 × 2 mm, Phenomenex) at 40 °C. All of the injections were
10 μl. The mobile phase consisted of 1 mM ammonium formate in LC-
water (+4.5% methanol) at a pH = 6.4 (A) and in methanol (B). The
LC flow rate was 0.3 ml/min. The gradient profile was as follows: 100%
A, decreased linearly to 5% between 0 and 8.00 min, and then de-
creased further to 0% between 8.00 and 13 min. After 20 min, it re-
turned to 100% equilibrating for 10 min up to 30 min. This LC method
is a general method serving for multiple applications in our lab. It can
definitely be shortened if specified for OP acids. A 5500 Qtrap LIT
quadrupole MS (AB Sciex, Foster City, CA, USA) was used for analysis.
The MS was operated in the MRM mode in the negative ESI mode. The
ion spray voltage was 4500 V, the ion source heater temperature was
600 °C, the ion source gas 1 (N2) pressure was 40 psi, the ion source gas
2 (N2) pressure was 70 psi, and the dwell time was 10 ms. The opti-
mized MRM conditions as well as the retention times for all 5 acids are
summarized in Table 1. Most of the products are common to all of the
acids: 95 is CH5PO3− and 79 is PO3−.

61
L. Yishai Aviram et al.
2.4. Sample preparation
Human blood (20 μl spiked with OP-acids solution) was spotted on a
Whatman filter paper (number 1). After approximately 2 h, when the
spot dried at room temperature, the blood spot was cut, put in a 4 ml
glass vial with 400 μl of MeOH, and vortexed for 10 min. The extract
was then transferred to a glass vial and dried under nitrogen by an
automatic evaporator (Turbo Vap LV manufactured by Calipar) at 40 °C
for 15 min. An amount of 100 μl of LC-MS water was added to the vial,
followed by vortexing for 2 min. The final dilution factor was therefore
1:5. The reconstituted extract (in water) was filtered and poured into a
LC-vial for LC-MS analysis. In the in-vivo experiment in 2 out of the 4
samples, an internal control (EMPA, 25 ng/ml) was spiked in the ex-
tract. EMPA was chosen to be the internal control because of its che-
mical similarity to IMPA with regard to its chemical structure, retention
time, relatively close masses and compatibility with negative ioniza-
tion.
2.5. Animals
Male albino Sprague-Dawley rats weighing 300–350 g at the be-
ginning of the experiment were purchased from Envigo (Israel). All
procedures involving animals were in accordance with the Guide for the
Care and Use of Laboratory Animals, National Academy Press,
Washington, DC, 1996 and were approved by the Institutional Animal
Care and Use Committee. The rats were housed three per cage in a
controlled environment with a constant temperature of 21 ± 2 °C and
a 12-h light/dark cycle with the lights turned on at 7 am. Food and
water were available ad lib. The in vivo experimental outline was as
follows: 5 rats were exposed to sarin (100 μg/kg im., 1 LD50) and
monitored for signs of toxicity for up to 24 h. Blood samples of 20 μl
were drawn from the tails of the rats before the introduction of Sarin
and at the following time course: 15, 30, 60, 120, 240 min and 24 h post
intoxication. The DBS samples were put directly on the Whatman filter
paper and dried for 2.5 h at room temperature. In parallel, blood
samples of 80–100 μl were drawn from the tail of two rats directly into
a heparin test tube for additional analysis.
2.6. Method validation
This method is suitable for unique forensic cases, and therefore, a
“limited validation” [29] was performed. It was validated for se-
lectivity, standard curve linearity, limit of quantitation, limit of detec-
tion, accuracy, precision, matrix effects and stability. The absence of
inferences is necessary to guarantee proper detection of the analyte. To
check for endogenous peaks, 6 different human whole blood and 5
different rat whole blood blanks were analyzed. Calibration curves of
all five acids in water and in human blood were obtained. We followed
two MRM transitions for each acid (Table 2). The dominant MRM
transition serves for quantitation and the additional one, for identifi-
cation (according to the European guidelines). The concentrations
spiked in whole blood were in the range of 0.3–100 ng/ml for each acid.
The calibration curves of IMPA and EMPA were also conducted in rat
blood, in the range of 0.5–50 ng/ml. These concentration levels were
chosen as they represent the estimated concentrations in blood after
Table 2
Linearity, LOD and LOQ of the 5 acids in blood (DBS sample preparation).
CWA metabolite R2 LOD (ng/ LOQ (ng/ Linear dynamic range
ml) ml) (ng/ml)
IMPA 0.992 0.5 1 1–100
EMPA 0.990 1 3 3–100
iBuMPA 0.996 1 3 3–100
CMPA 0.997 1 3 3–100
TPMPA 0.996 0.3 1 1–100
62
Journal of Chromatography B 1093–1094 (2018) 60–65
exposure to 0.5 LD50–1 LD50 of the CWA [7]. The LOD was evaluated
based on signal to noise ratio ≥3. The LOQ was evaluated based on an
S/N ≥10 ratio threshold. The precision and accuracy were measured at
two concentrations, 10 ng/ml and 100 ng/ml, using 6 different human
blood. Sample matrix effects such as ion suppression and/or enhance-
ment were examined with six different human blood samples and five
rat blood samples. The acid standards are known to be stable and un-
reactive. However, the stability of the acids on the paper was unknown;
hence this was examined too.
3. Results and discussion
3.1. Optimization of extraction procedures
The method was developed using human blood, where the sample
preparation stage was optimized. In DBSs, volumes of 10–50 μl are
usually reported. We decided to work with 20 μl since it is a common
volume in a standard diabetic kit. Extraction was carried out only after
2 h, when the blood had completely dried on the paper. We examined
different drying periods (2, 2.5, 3, and 4 h) and determined that there
were no major changes. We also examined four different polar extrac-
tion solvents compatible with the polar acids: MeOH:H2O (50:50),
MeOH + 1% formic acid (pH = ~4.5), MeOH + 0.2 M HCl (pH ~1)
and MeOH. The best extraction recoveries were obtained when using
MeOH and MeOH:H2O (50:50). We decided to work with MeOH since it
is a universal solvent that could also extract drugs of abuse and pesti-
cides for future work. The volume of the extraction solvent
(400–800 μl) did not have a major effect on the extraction yield. Hence,
a 400 μl volume was chosen since it was the minimum volume that
covered the entire spot area in the vial. The effect of the extraction time
with vortex mixing (10 min – 3 h) was also examined. It was found that
all acids could be extracted in 10 min with vortex mixing without sig-
nificant changes in the peak heights or areas. The use of an ultrasonic
bath in the extraction step was also studied and did not make any im-
provement. After drying the extract to complete dryness using an eva-
porator, water was added to the extract to allow better compatibility
with the LC mobile phase. We compared different volumes of water at
the end of the sample preparation process including 50 μl, 100 μl, 200 μl
and 400 μl. We decided to work with 100 μl in order to minimize di-
lution but yet be able to run the sample at least 5 times (such as in cases
when we will need to spike the original extraction). We evaluated the
method by comparing blood spots vs. water spots. No major changes in
the recoveries were found.
3.2. Validation of the proposed methods
3.2.1. Selectivity (interferences)
The selectivity of the method was determined using six different
human blood samples. No differences were apparent. No interfering
peaks from endogenous compounds were observed at the same reten-
tion times of the MRM transitions for the five phosphonic acids. MRM
chromatographs of blanks vs. blanks with spiked acids are shown in
Fig. 1.
3.2.2. Linearity, LOD and LOQ
Calibration curves of the 5 acids in human blood were obtained. The
concentrations in whole blood were 1, 5, 10, 15, 20, 50, and 100 ng/ml,
as they are in the estimated blood concentration range after exposure to
0.5 LD50–1 LD50 of the CWA [7]. The regression coefficients (R2)
were > 0.990.
The limit of detection (LOD) was calculated for a signal to noise
ratio > 3. The limit of quantitation (LOQ) was estimated for S/N > 10.
Both were determined by spiking various concentrations into a DBS
blank. The LODs for all of the acids are summarized in Table 2.
L. Yishai Aviram et al. Journal of Chromatography B 1093–1094 (2018) 60–65

Table 3
Accuracy and precision (n = 6) in DBS spiked with 10 ng/ml and 100 ng/ml of
the five OP acids in blood.
CWA product MRM ESI(−) Nominal concentration 10 ng/mL
transition n=6

Mean measured Precision Accuracy

concentration (%CV) (bias %)
(ng/ml)

EMPA (VX acid) 123.0 > 95.0 10 12.6 +2.6

123.0 > 79.0 10 10.5 +1.7
IMPA (GB acid) 137.0 > 95.0 11 11.6 +9.4
137.0 > 79.0 12 13.6 +14.7
iBuMPA (RVX acid) 151.1 > 77.0 10 14.0 +2.9
151.1 > 79.0 11 15.4 +5.2
CMPA (GF acid) 177.1 > 95.0 10 12.8 +2.4
177.1 > 79.0 9 9.5 −11.0
TPMPA (GD acid) 179.1 > 95.0 10 13.5 +0.3
179.1 > 79.0 10 10.1 +1.1

CWA product MRM ESI(−) Nominal concentration 100 ng/ml

transition n=6

Mean measured Precision Accuracy

concentration (%CV) (bias %)
(ng/ml)

EMPA (VX acid) 123.0 > 95.0 106 8.2 +5.7

123.0 > 79.0 106 8.2 +5.5
IMPA (GB acid) 137.0 > 95.0 105 7.5 +5.2
137.0 > 79.0 91 7.0 −8.9
iBuMPA (RVX acid) 151.1 > 77.0 97 7.5 −2.8
151.1 > 79.0 99 8.1 −1.0
CMPA (GF acid) 177.1 > 95.0 108 7.4 +8.0
177.1 > 79.0 92 7.8 −8.5
TPMPA (GD acid) 179.1 > 95.0 92 2.0 −7.6
179.1 > 79.0 87 6.8 −13.1

3.2.3. Accuracy and precision

The concentrations were determined from the appropriate DBS ca-
libration plot that was obtained on the same day as the analytical run,
as described before. The precision (%CV) and accuracy (%) were de-
termined at two concentrations, 10 ng/ml and 100 ng/ml. For each
concentration, the sample preparation procedure was repeated 6 times
by the same person. The accuracy and precision results are summarized
in Table 3. The quantitation was calculated by using the dominant
MRM transition for each acid, usually with a better S/N ratio.

3.2.4. Recoveries
The absolute recoveries were calculated. All acids were spiked at
10 ng/ml in whole blood and went through the sample preparation
procedure. The results were compared to standards (2 ng/ml) in water.
The average recoveries ranged between 51% and 100% with a standard
deviation smaller than 14% for each acid. Recoveries for the four larger
acids were between 78% and 100%. Lower recoveries (65% for the
dominant MRM transition, and 51% for the second transition) belong to
EMPA which exhibits a low signal with a lower S/N ratio.

Fig. 1. MRM chromatograms of an extract of a DBS of human blood spiked with
1 ng/ml acids (red), with the same MRM transitions for a DBS blank (blue). (For
interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
3.2.5. Stability
The stability of the acids in the standard solution (in water) is
known to be good, as the methyl phosphonic acids are unreactive and
relatively stable. We did not observe any deterioration while keeping
the acids at room temperature, under refrigeration and in the freezer, as
reported in previous publications [8].
Stability tests of the acids on the paper were conducted. A drop of
20 μl blood with 50 ng/ml acid were spiked on the filter paper and left
in the fume hood at room temperature for up to 840 h (35 days). At
various time courses, the spots (n = 3) were extracted and analyzed as
described above. The results are shown in Fig. 2. There is a modest

63
L. Yishai Aviram et al. Journal of Chromatography B 1093–1094 (2018) 60–65

Fig. 2. Sample preservation on DBS. 50 ng/ml of the OP-acids in human blood, aging at room temperature for up to 35 days.

Fig. 3. In vivo 24 h time course for IMPA, using DBS. IMPA levels in rat blood, using DBS, after rat exposure to Sarin, 1LD50 im. The red and yellow lines represent
the proposed biphasic decay. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

decrease for all the acids after ~400 h (> 15 days). A decrease of up to
45% was observed for TPMPA after 840 h (35 days), while a decrease of
35% was observed for CMPA and a decrease of 30% was observed for
iBuMPA. The more pronounced decreases were observed in the largest
acids. We believe that this is due to binding of the hydrophobic chains
of the larger acids to the cellulose of the DBS paper [28]. However,
based on the LOD of 0.3 ng/ml, it will probably be possible to detect
TPMPA at the very low concentration of 1 ng/ml at t = 0, even 35 days
after sampling (while keeping the paper at room temperature).
3.2.6. Matrix effects in LC-MS/MS
Ion suppression or enhancement is common in LC-MS with complex
matrices [29]. In this research, for all of the acids, no ion suppression or
enhancement was evident. This was checked in 5 different human blood
samples and in 5 different rat blood samples. After analyzing the DBSs
of blank samples, the same blood blanks were spiked at known con-
centrations (1 ng/ml, 5 ng/ml, and 15 ng/ml) and went through the
DBS preparation procedure (as described above). Both samples (blank
and spiked) were compared to make sure there are no matrix effects. In
Fig. 1, an example of blank blood vs. spiked blood is shown.
3.3. Rat exposure to GB-in vivo experiment
The new method was used to analyze IMPA obtained after rat ex-
posure to Sarin. Five rats were exposed to 1 LD50 GB (90 μg/kg) I.M.
20 μl (one drop) of blood from the rat tail was drawn and dried on the
filter paper. The first sample was collected before exposure and the rest
in the following time intervals after exposure: 15 min, 30 min, 60 min,
120 min, 240 min and 1440 min (24 h). In 2 out of the 4 samples, an
internal control (EMPA, 25 ng/ml) was spiked in the extract, serving as
a control for solution and instrument stability. Most of the validation
obtained for human blood is suitable for this experiment except for
matrix effects, which need to be evaluated separately. For that purpose,
five rat blanks were examined, and no interferences at the retention
times of the internal control and IMPA were found.
Quenching (electrospray ion suppression) was also examined by

64
L. Yishai Aviram et al.
spiking 5 ng/ml of IMPA and EMPA into the final extract (water). The
results were compared to standard solutions of the acids in water and
found to be similar. Hence, there was no apparent quenching in this
matrix. Fig. 3 represents the acid concentration in the rat blood over
time. The concentration was calculated according to a calibration curve
of IMPA spiked in rat blood blank as in the method described above
(DBS sampling). We obtained a biphasic elimination curve with esti-
mated half-lives of t1/2 (1) = ~50–60 min (primary) and t1/2
(2) = ~600 min (secondary).
The average body weight of a rat is approximately 300 g, and thus 1
LD50 of Sarin (IV) of 90 μg/kg is approximately 30 μg of Sarin. The
estimated total volume of rat blood is approximately 23 ml. Hence, the
concentration of GB in the rat blood immediately after the exposure
should be 30 μg/23 ml = 1.3 μg/ml. The hydrolysis of GB to IMPA in
the body is fast and occurs immediately after the exposure [6]. We
detected ~100 ng/ml IMPA at t = 15 min, which is a reasonable por-
tion originating from of the initial Sarin amount. These amounts are
similar to other findings in the literature [4,11].
4. Conclusions
A new method for the detection of hydrolyzed OP-CWAs using DBS
was developed. This method is suitable for unique forensic cases where
the question is whether there was exposure to specific CWA. We believe
that the DBS approach is highly adequate for that demand since it can
be performed in the field, by a non-professional, immediately after
exposure. Although the collected blood volume is very small, the sen-
sitivity obtained was sufficient to detect OP acids as low as 1 ng/ml in
the blood, which is within the low limit concentration in a real case
scenario. For operational purposes, most important are the qualitative
results. However, quantitative data would be also beneficial, especially
in statistical studies such as in clinical correlations. We are the first to
implement a method that determines phosphonic acids using a DBS in
an in vivo experiment. In the in–vivo experiment, we evaluated the
IMPA time course concentrations up to 24 h. In a real scenario, a single
point confirmed detection may be sufficient. This method showed good
results both in human blood and in the in vivo experiment. The ability
to sample in the field immediately after exposure is also crucial in cases
of exposure to various drugs of abuse/pesticides/CWAs that have a fast
route of disposal from the body, especially at low doses.
Funding
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
Declarations of interest
None.
References
[1] D. Noort, H.P. Benschop, R.M. Black, Biomonitoring of exposure to chemical war-
fare agents: a review, Toxicol. Appl. Pharmacol. 184 (2002) 116–126.
[2] K. Kim, O.G. Tsay, D.A. Atwood, D.G. Churchill, Destruction and detection of
chemical warfare agents, Chem. Rev. 111 (2011) 5345–5403.
[3] H. John, M.J. van der Schans, M. Koller, H.E.T. Spruit, F. Worek, H. Thiermann,
D. Noort, Fatal sarin poisoning in Syria 2013: forensic verification within an in-
ternational laboratory network, Forensic Toxicol. 36 (2017) 61–71.
[4] M.L. Shih, J.D. McMonagle, T.W. Dolzine, V.C. Gresham, Metabolite pharmacoki-
netics of soman, sarin and GF in rats and biological monitoring of exposure to toxic
65
Journal of Chromatography B 1093–1094 (2018) 60–65
organophosphorus agents, J. Appl. Toxicol. 14 (1994) 195–199.
[5] R.M. Black, History and perspectives of bioanalytical methods for chemical warfare
agent detection, J. Chromatogr. B 878 (2010) 1207–1215.
[6] B.T. Røen, S.R. Sellevåg, E. Lundanes, Quantification of nerve agent biomarkers in
human serum and urine, Anal. Chem. 86 (2014) 11833–11840.
[7] R.M. Black, R.W. Read, Biological markers of exposure to organophosphorus nerve
agents, Arch. Toxicol. 87 (2013) 421–437.
[8] B.T. Røen, S.R. Sellevåg, E. Lundanes, On-line solid phase extraction-liquid chro-
matography–mass spectrometry for trace determination of nerve agent degradation
products in water samples, Anal. Chim. Acta 761 (2013) 109–116.
[9] R.M. Black, R.W. Read, Analysis of degradation products of organophosphorus
chemical warfare agents and related compounds by liquid chromatography–mass
spectrometry using electrospray and atmospheric pressure chemical ionisation, J.
Chromatogr. A 794 (1998) 233–244.
[10] R.W. Read, R.M. Black, Rapid screening procedures for the hydrolysis products of
chemical warfare agents using positive and negative ion liquid chromato-
graphy–mass spectrometry with atmospheric pressure chemical ionisation, J.
Chromatogr. A 862 (1999) 169–177.
[11] D. Noort, A.G. Hulst, D.H.J.M. Platenburg, M. Polhuijs, H.P. Benschop, Quantitative
analysis of O-isopropyl methylphosphonic acid in serum samples of Japanese citi-
zens allegedly exposed to sarin: estimation of internal dosage, Arch. Toxicol. 72
(1998) 671–675.
[12] P.A. Demirev, Dried blood spots: analysis and applications, Anal. Chem. 85 (2012)
779–789.
[13] S.S. Simões, A.C. Ajenjo, M.J. Dias, Dried blood spots combined to an UPLC–MS/MS
method for the simultaneous determination of drugs of abuse in forensic toxicology,
J. Pharm. Biomed. Anal. 147 (2018) 634–644.
[14] K. Heussner, M. Rauh, N. Cordasic, C. Menendez-Castro, H. Huebner, M. Ruebner,
M. Schmidt, A. Hartner, W. Rascher, F.B. Fahlbusch, Adhesive blood microsampling
systems for steroid measurement via LC–MS/MS in the rat, Steroids 120 (2017) 1–6.
[15] L.C. Martial, R.E. Aarnoutse, M. Mulder, A. Schellekens, R.J.M. Brüggemann,
D.M. Burger, A.H. Schene, A. Batalla, Dried blood spot sampling in psychiatry:
perspectives for improving therapeutic drug monitoring, Eur.
Neuropsychopharmacol. 27 (2017) 205–216.
[16] P. Beaudette, K.P. Bateman, Discovery stage pharmacokinetics using dried blood
spots, J. Chromatogr. B 809 (2004) 153–158.
[17] N. Spooner, R. Lad, M. Barfield, Dried blood spots as a sample collection technique
for the determination of pharmacokinetics in clinical studies: considerations for the
validation of a quantitative bioanalytical method, Anal. Chem. 81 (2009)
1557–1563.
[18] M. Barfield, N. Spooner, R. Lad, S. Parry, S. Fowles, Application of dried blood spots
combined with HPLC-MS/MS for the quantification of acetaminophen in tox-
icokinetic studies, J. Chromatogr. B 870 (2008) 32–37.
[19] W. Li, F.L.S. Tse, Dried blood spot sampling in combination with LC-MS/MS for
quantitative analysis of small molecules, Biomed. Chromatogr. 24 (2010) 49–65.
[20] A. Thomas, H. Geyer, W. Schanzer, C. Crone, M. Kellmann, T. Moehring, M. Thevis,
Sensitive determination of prohibited drugs in dried blood spots (DBS) for doping
controls by means of a benchtop quadrupole/Orbitrap mass spectrometer, Anal.
Bioanal. Chem. 403 (2012) 1279–1289.
[21] J. Déglon, A. Thomas, P. Mangin, C. Staub, Direct analysis of dried blood spots
coupled with mass spectrometry: concepts and biomedical applications, Anal.
Bioanal. Chem. 402 (2011) 2485–2498.
[22] K.C. Waterman, R.C. Adami, Accelerated aging: prediction of chemical stability of
pharmaceuticals, Int. J. Pharm. 293 (2005) 101–125.
[23] A.A. Alfazil, R.A. Anderson, Stability of benzodiazepines and cocaine in blood spots
stored on filter paper, J. Anal. Toxicol. 32 (2008) 511–515.
[24] A.F. Lehner, W. Rumbeiha, A. Shlosberg, K. Stuart, M. Johnson, R. Domenech,
H. Langner, Diagnostic analysis of veterinary dried blood spots for toxic heavy
metals exposure, J. Anal. Toxicol. 37 (2013) 406–422.
[25] J.W. Perez, B.G. Pantazides, C.M. Watson, J.D. Thomas, T.A. Blake, R.C. Johnson,
Enhanced stability of blood matrices using a dried sample spot assay to measure
human butyrylcholinesterase activity and nerve agent adducts, Anal. Chem. 87
(2015) 5723–5729.
[26] E.I. Hamelin, T.A. Blake, J.W. Perez, B.S. Crow, R.L. Shaner, R.M. Coleman,
R.C. Johnson, Bridging the gap between sample collection and laboratory analysis:
using dried blood spots to identify human exposure to chemical agents, Proc. SPIE
Int. Soc. Opt. Eng. (2016), http://dx.doi.org/10.1117/12.2223796.
[27] J. McKenna, E.S. Dhummakupt, T. Connell, P.S. Demond, D.B. Miller, J.M. Nilles,
N.E. Manicke, T. Glaros, Detection of chemical warfare agent simulants and hy-
drolysis products in biological samples by paper spray mass spectrometry, Analyst
142 (2017) 1442–1451.
[28] R.A. Koster, R. Botma, B. Greijdanus, D.R.A. Uges, J.G.W. Kosterink, J.-
W.C. Alffenaar, D.J. Touw, The influence of the dried blood spot drying time on the
recoveries of six immunosuppressants, J. Appl. Bioanal. (2015) 116–122.
[29] F.T. Peters, O.H. Drummer, F. Musshoff, Validation of new methods, Forensic Sci.
Int. 165 (2007) 216–224.