Journal of Chromatography B 1234 (2024) 124028

Contents lists available at ScienceDirect

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

A liquid chromatographic-mass spectrometric procedure for analysis of

pentaerythrityl tetranitrate metabolites – Development, validation and
application to ovine serum and human plasma samples
Mariann Städtler a, 1, Daniela Wissenbach b, 1, Dirk K. Wissenbach b, Laura Franke b,
Jana Pastuschek c, Ekkehard Schleussner c, Beth Allison d, Frank T. Peters b, *, Tanja Groten c
a
Jena University Hospital, Center for Clinical Trials, Friedrich Schiller University Jena, Germany
b
Jena University Hospital, Institute for Forensic Medicine, Friedrich Schiller University Jena, Germany
c
Jena University Hospital, Department of Obstetrics, Friedrich Schiller University Jena, Germany
d
The Ritchie Centre Hudson Institute of Medical Research, Clayton, VIC, Australia

A R T I C L E I N F O A B S T R A C T

Keywords: Pentaerythrityl tetranitrate (PETN) is an established drug in the treatment of coronary heart disease and heart
Liquid chromatography- high resolution mass failure. It is assumed, that the vasodilative and vasoprotective effects of PETN also have a positive impact on
spectrometry pregnant patients with impaired placental perfusion and studies evaluating the effect of PETN in risk pregnancies
Fetal growth retardation
have been carried out. In the context of these clinical trials, measuring of serum levels of PETN and its me­
Pentaerythrityl tetranitrate
Human
tabolites pentaerythrityl trinitrate (PETriN), pentaerythrityl dinitrate (PEDN), pentaerythrityl mononitrate
Ovine (PEMN) and pentaerythritol (PE) were required. To evaluate the transfer of PETN and its metabolites (PEXN)
Pregnant from the mother to the fetus using samples from a human clinical trial and animal study, the present work aimed
to develop a rapid and simple method to simultaneously analyze PEXN in human and ovine samples.
A method employing a rapid and simple liquid–liquid extraction followed by reversed-phase (C18) liquid
chromatography coupled to high-resolution mass spectrometry with negative electrospray ionization was
developed and validated for the detection of PETN and PEXN in human and ovine samples. PE could only be
qualitatively detected at higher concenrations. Method validation requirements, including accuracy, repeat­
ability and intermediate precision were fulfilled in ovine and human samples for all other PEXN with exception
PETriN in human samples. The recovery (RE) in ovine samples was 76.7 % ± 12 % for PEMN, 98 % ± 23 % for
PEDN, 94 % ± 22 % for PETriN, in human samples RE was 59 % ± 16 % for PEMN, 67 % ± 19 % for PEDN, 71 %
± 17 %. The matrix effects (ME) in ovine samples were 90 % ± 11 % for PEMN, 70 % ± 30 % for PEDN, 107 % ±
17 % for PETriN, in human samples the ME were 93 % ± 13 % for PEMN, 84 % ± 17 % for PEDN, 98 % ± 16 %
for PETriN. The limits of quantification (LOQ) in ovine samples were 1.0 ng/mL for PETriN and 0.1 ng/mL for
PEMN and PEDN. The LOQs in human samples were 5.0 ng/mL for PETriN and 0.3 ng/mL for PEMN und PEDN.
The newly developed method was used to analyze 184 ovine serum samples and 18 human plasma samples.
In ovine maternal samples, the highest observed PEDN concentration was 3.5 ng/mL and the highest PEMN
concentration was 10 ng/mL, the respective concentrations in fetal serum samples were 4.9 ng/mL for PEDN and
5.4 ng/mL for PEMN. PETriN was only detected in traces in maternal and fetal samples, whereas PETN could not
be detected at all. In human maternal samples, the highest concentration for PEDN was 27 ng/mL and for PEMN
150 ng/mL. In umbilical cord plasma, concentrations of 2.3 ng/mL for PEDN and 73 ng/mL for PEMN were
detected. Although the PEMN and PEDN concentrations in the human samples were several times higher than in

Abbreviations: PETN, pentaerythrityl tetranitrate; PETriN, pentaerythrityl trinitrate; PEDN, pentaerythrityl dinitrate; PEMN, pentaerythrityl mononitrate; PE,
pentaerythritol; NO, nitric oxide; FGR, fetal growth restriction; PEXN, PETN and/or its metabolites; GC, gas chromatography; MS, mass spectrometry; BSTFA, N,O-bis
[trimethylsilyl]trifluoro-acetamide; LLOQ, lower limit of quantification; LC, liquid chromatography; 1,3-DNG, 1,3-Dinitroglycerine; 2-MNG, 2-Mononitroglycerine;
IS, internal standard; Cal, calibration standards; QC, quality control; ME, matrix effects; RE, recovery; RSD, relative standard deviations; SUAL, single umbilical artery
ligation.
* Corresponding author at: Jena University Hospital, Institute for Forensic Medicine, Building F2, Am Klinikum 1 07747, Jena, Germany.
E-mail address: frank.peters@med.uni-jena.de (F.T. Peters).
1
authors contributed equally.

https://doi.org/10.1016/j.jchromb.2024.124028
Received 23 August 2023; Received in revised form 12 January 2024; Accepted 20 January 2024
Available online 24 January 2024
1570-0232/© 2024 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
M. Städtler et al. Journal of Chromatography B 1234 (2024) 124028

ovine samples, neither PETN nor PETriN signals could be detected. These results demonstrated that the me­
tabolites were transferred from mother to fetus with a slight time delay.

1. Indroduction pass to the fetus during pregnancy. Furthermore, animal models are
included to get information on the placental transfer of PEXN.
Pentaerythrityl tetranitrate (PETN) is an organic nitro compound So far, pharmacokinetics and metabolism of PETriN have been
and is classified according to its duration of action as long-term nitrate studied following sublingual and oral administration of radiolabeled
14
[1]. Due to its vasodilative effect, PETN has been used in clinical ap­ C-PETriN in men. PETriN could be detected in urine and blood minutes
proaches in patients suffering from ischemic heart disease. It was after administration. PE and PEMN, were detected in urine with elimi­
approved in the USA as “Perinitrate” in 1953 and as “Dilcoran®” in 1959 nation half-lives of 10.5 h and 7.3 h, respectively [17]. In a study
in Germany. Since 1964, PETN was used under the name of “Penta­ including 24 male volunteers only PEMN and PEDN could be detected in
long®” in the former east Germany GDR (German Democratic Republic) plasma reaching concentrations above the limit of quantification (50
for the prophylaxis and long-term treatment of angina pectoris and pg/mL). For PEDN, a maximum concentration of 17.4 ng/mL was
chronic heart failure [2]. PETN is rapidly metabolized to three active determined 2.5 h after administration, and for PEMN, a maximum
substances, namely pentaerythrityl trinitrate (PETriN), pentaerythrityl
dinitrate (PEDN) and pentaerythrityl mononitrate (PEMN). The final
metabolite is the pharmacologically inactive pentaerythritol (PE)
(Fig. 1) [1].
Although organic nitrates have been used therapeutically on a large
scale worldwide for decades, the molecular principle of action was not
recognized before the 1980s.
An “Endothelium-Derived-Relaxing-Factor” was discovered in 1980
as a signal molecule involved in blood vessel relaxation [3]. In 1987,
“Endothelium-Derived-Relaxing-Factor” was identified as identical to
nitric oxide (NO) [4], which is formed by the intracellular NO synthase
from arginine and oxygen [5]. The biological action of NO is caused by
reaction with NO acceptors (soluble guanylate cyclase, hemoglobin,
myoglobin) [6] and thiols, forming nitrosothiols. These processes affect
key biological functions, like protection from oxidative stress and inhi­
bition of programmed cell death [7]. After diffusion into the muscle
cells, NO activates the enzyme soluble guanylate cyclase and induces
vasodilation. The pharmacological mechanism of chemical substances
such as isosorbide mononitrate (ISMN), isosorbide dinitrate, glycerol
trinitrate and PETN is to act as NO donors. With prolonged non-
intermittent administration, most organic nitrates induce nitrate toler­
ance [8,9]. In contrast, even with long-term use of PETN, no nitrate
tolerance has been described [10]. In addition, PETN and/or its me­
tabolites have unique antioxidant properties. In a study with cultured
endothelial cells, the active PETN metabolite PETriN was shown to in­
crease mRNA and protein levels of heme oxygenase (HO)-1. Pretreat­
ment with PETriN protected endothelial cells from peroxide-mediated
toxicity [11]. In 2022, our group confirmed these effects by demon­
strating that PETN pretreatment protects endothelial cells from oxida­
tive stress in response to thrombin-treatment [12].
Due to the above-described vasodilative and vasoprotective effects
the potential for PETN to have a positive impact in pregnant women
with impaired placental blood flow has been explored. Impaired
placentation is associated with risk of developing fetal growth restric­
tion (FGR) and preeclampsia. These placenta-associated pregnancy
complications occuring in 3 to 10 % of all pregnancies cause iatrogenic
prematurity as a major risk factor for perinatal mortality and morbidity.
To date, there are no therapeutic approaches for these diseases that have
had a successful effect on pregnancy outcomes [13–15]. At the Univer­
sity Hospital Jena, a monocentric randomized controlled pilot study in
111 patients with impaired placental blood flow showed that the
administration of Pentalong® reduced the risk of developing FGR or
fetal death by 39 % and of preterm birth by 73 % [13,14]. A consecu­
tively performed multicenter randomized controlled trial aimed to
confirm the risk-minimizing effect of PETN on the development of FGR
and preterm birth in a larger number of patients [15,16]. To gather
safety data on placental drug transfer, maternal plasma samples were
collected from study participants at the study center in Jena during
regular study-visits and umbilical cord plasma was collected at birth to
Fig. 1. Chemical structures and relative molecular masses (Mr) of PETN and its
provide a perspective on whether PETN and/or its metabolites (PEXN) metabolites.

2
M. Städtler et al.
concentration of 79.3 ng/mL was determined 7.1 h after administration
[2,18]. In a study including 23 male volunteers who orally ingested 80
mg Pentalong®, plasma samples were taken before and within 36 h
following administration. These samples were analyzed for PEDN and
PEMN as described by Stalleicken et al. [19]. PEMN could be quantified
in all samples, PEDN could only be detected until 24 h after application.
The mean maximum concentration detected in this study was 9.7 ng/mL
for PEDN with a median maximum time of 3 h and 41.6 ng/mL for PEMN
with a median maximum time of 5 h [20].
PEXN concentrations reported in the different studies have been
determined using thin layer chromatography, gas chromatography
(GC)-mass spectrometry (MS) methods or two separate GC–MS methods
using negative chemical ionization (NICI) after derivatization with N,O-
bis[trimethylsilyl]trifluoro-acetamide (BSTFA). In regard to quantifica­
tion of PETN and its metabolites in rat plasma, a method using thin layer
chromatography was described in 1974. 14C PETN was administered to
rats by gavage. The plasma samples were extracted three times with
ethyl acetate. The extracts were concentrated under a stream of nitro­
gen. Aliquots were used for 14C assay by liquid scintillation spectrometry
[21]. Weber et al. described a GC–MS method for quantification PEMN
and PEDN in male volunteers in 1995. Plasma concentrations of PEDN
and PEMN were assayed by GC/MS after adding isosorbide-5-nitrate as
internal standard, liquid–liquid extraction of the samples, derivatization
with BSTFA and detection by chemical ionization MS in the negative ion
mode and ammonia as reagent gas. The lower limit of quantification
(LLOQ) for PEDN was 0.5 ng/mL and 1.0 ng/mL for PEMN. Accuracy
and precision as determined by inter-day variation of back-calculated
calibrator concentrations were between 101.2 ± 8.6 % at the lower
and 101.0 ± 3.1 % at the upper LOQ for PEDN and between 96.4 ± 8.8
% and 100.2 ± 2.3 % for PEMN, respectively [18]. In 1999 Hames et al.
[22] and Schutz et al. [23] described a simplified and specific quantifi­
cation to detect PETN metabolites. Hames et al. described a sensitive and
selective routine method for simultaneous determination of PEDN and
PEMN in human plasma. Isosorbide-5-mononitrate served as the inter­
nal standard. Analytes were isolated from plasma by extraction with
hexane/acetic ester (1:4) and subsequently derivatized to its trime­
thylsilyl ether with BSTFA. Quantification was performed by MS after
GC separation and NICI using methane as reagent gas. All analytes were
quantified via the fragment ion at m/z 46. The method was validated
according to GLP guidelines. The LLOQ was 1 ng/mL for PEMN and 0.2
ng/mL for PEDN. A maximum value of 10 % (PEDN) and 9 % (PEMN)
was calculated for method accuracy, and 22 % (PEDN) and 16 % (PEMN)
for precision. In the method of Schutz et al., human plasma samples were
exhaustively extracted with acetic acid ethlyester/hexane (4:1), the
combined phases were concentrated to dryness under nitrogen flow, and
then incubated with BSTFA. ISMN served as the internal standard.
Plasma samples were analyzed by gas chromatography (NICI mode).
Specific ions for each metabolite were used for identification (PEMN: m/
z 351, PEDN: m/z 324, PETriN: m/z 62, ISMN: m/z 217). The LLOQ was
1.0 ng/mL for PEMN and 0.2 ng/mL for PEDN and PETriN. The recovery
was 72 % for PEMN and 94 % for PEDN and PETriN. They concluded
that the recoveries of their method were not as high as in the published
method of Weber et al., but did not require ammonia as reagent gas and
still offered a very good detection and quantification limit. Furthermore,
there was no differentiation between PETriN and PEDN or PEMN in the
sample preparation [23]. So far, no applications of these two last-
mentioned methods in either human or animal samples have been
described in the literature.
Since the aforementioned GC–MS methods required very time-
consuming sample preparations as well as high sample volumes (about
1 mL of plasma) and were probably not sufficiently sensitive for the
analysis of samples from the upcoming clinical and animal studies, the
aim of the presented study was to develop and validate a new method for
the simultaneous and sensitive determination of PEXN in limited serum
and plasma samples using liquid chromatography (LC)-MS. So far,
various LC-MS methods for the determination of PETN in the context of
3
Journal of Chromatography B 1234 (2024) 124028
explosives analyses [24–30], methods for the determination of PETN
residues on plastics, nylon, metallic surfaces and other debris residues
[24,31,32], in soil samples [33–36], in groundwater and seawater
samples [34,37,38], plant tissue [39] und hand swabs [40] has been
described. A LC-MS based method described by Brust et al. also covered
the PETN metabolites PE, PEMN, PEDN, and PETriN [29,30]. Following
validation, we aimed to assess levels of PETN and its metabolites in
plasma samples from pregnant woman and umbilical cord samples, as
well as in maternal and fetal ovine plasma samples. With this new
method an assessment regarding the placental transfer of PEXN from
mother to fetal can be evaluated.
2. Material and methods
2.1. Chemicals and reagents
PEMN was synthesized by Exclusive Synthesis AG (Groningen,
Netherlands). PETN and PETriN were provided by Dottikon Exclusive
Synthesis AG (Dottikon, Switzerland). The metabolite PE for synthesis
was obtained from Merck (Darmstadt, Germany) and PEDN from
Selectlab Chemicals (Munster, Germany).
1,3-Dinitroglycerine (1,3-DNG) and 2-Mononitroglycerine (2-MNG)
were purchased from Cerilliant Corporation (Texas, USA). Ammonium
formate and formic acid were obtained from Sigma-Aldrich (Steinheim,
Germany); water (LC grade), acetonitrile, and methanol from Fisher
Scientific (Schwerte, Germany); DMSO, ethyl acetate, dichlormethane,
2-propanol and all other chemicals from Merck (Darmstadt, Germany).
2.2. Biosamples
Pooled ovine serum was ordered from Fiebig Nährstofftechnik
(Idstein, Germany). Study and blank maternal and fetal ovine serum
samples were provided by the Ritchie Centre, Hudson Institute of
Medical Research (Clayton Vic., Australia). Experiments were approved
by the Monash Medical Centre Animal Ethics Committee A
(MMCA2016/01) under guidelines established by the National Health
and Medical Research Council of Australia code of practice for the care
and use of animals for scientific purposes (8th Edition, 2013).
Human blank plasma samples used for method development were
obtained from a local blood bank. Authentic maternal plasma and um­
bilical cord plasma samples from participants of the multicenter trial
were submitted to the authors’ laboratory by the clinic of obstetrics.
These participants have consented to the collection of blood for research
purposes by means of an informed consent form approved by the rele­
vant ethics committee (Reg.-Nr.: 2022–2602-Material from
21.04.2022). All plasma samples were handled according to the insti­
tutional protocol and regulations and were stored at − 80 ◦ C.
2.3. Sample preparation
To 200 µL of the ovine sample, 100 μL methanol/water (50:50, v/v)
and 50 µL of the internal standard (IS) mixture (0.001 mg/mL 1,3-DNG
and 0.001 mg/mL 2-MNG) and 1,000 μL of an ethyl acetate/i-propanol/
dichloromethane mixture (60:20:20, v/v/v) were added in a reaction
tube. To 100 µL of the human samples, 50 µL methanol/water (50:50, v/
v) and 25 µL of the IS and 500 µL of an ethyl acetate/i-propanol/
dichlormethane mixture (60:20:20, v/v/v) were added in a reaction
tube. The mixtures were shaken for 15 min at 1,650 rpm and centrifuged
at 2,500 x g. Eight hundred µL (ovine), respectively 400 µL (human
samples) of the supernatant were transferred into an autosampler vial
and evaporated to dryness under a gentle stream of nitrogen. The res­
idue was dissolved in 50 μL water/acetonitrile mixture (95:5, v/v). A 10
μL aliquot was injected into the LC-MS system.
M. Städtler et al.
2.4. LC-MS method
The method was developed and validated on a Q Exactive Focus mass
spectrometer coupled to a Dionex ultra-high performance LC system
(Thermo Fisher Scientific, Dreieich, Germany). Chromatographic sepa­
ration was performed by gradient elution with 10 mM ammonium
formate buffer with 0.1 % formic acid (mobile phase A) and acetonitrile
with 0.1 % (v/v) formic acid (mobile phase B) on a Nucleoshell RP 18
plus; column (100 × 3 mm, 2.7 μm). The column oven temperature was
set to 35 ◦ C. The chromatographic gradient was programmed as follows:
1.0 min hold at 99 % A, 1.0 to 4.0 min increase to 99 % B, both steps with
a flow rate of 0.5 mL; 4.0 to 4.5 min hold at 0 % A with a flow gradient to
0.7 mL/min; steep decrease to starting conditions and flow rate of 0.5
mL/min and hold for 3 min. The autosampler temperature was set to
8 ◦ C. Draw speed, dispense speed, and dispense delay were 5 µL/s, each.
An injection wash was performed with 20 µL of a mixture of water/
methanol (90:10 v/v) after draw with a wash speed of 5 µL/s. High
resolution MS was performed in negative full scan mode after heated
electrospray ionization. MS conditions were as follows: sheath gas flow
rate 50 arbitrary units; aux gas flow rate 20 arbitrary units; spray voltage
− 3.50 kV; capillary temperature 320 ◦ C; s-lens RF level of 55; aux gas
heater temperature 350 ◦ C. Full scan evaluation was performed over a
scan range from m/z 55 to m/z 400 with a resolution of 17,500. Quan­
tification was performed using the following exact mass traces of the
analytes; PE as formate adduct at m/z 181.0711, PEMN as formate
adduct at m/z 226.0566, PEDN as formate adduct at m/z 271.0425,
PETriN as formate adduct at m/z 316.0270, 2-MNG at m/z 182.0297,
1,3-DNG at m/z 227.0153. For PETN the decay of nitrate was monitored
at m/z 61.9868. All ions were extracted with 20 ppm mass accuracy. For
data evaluation, Xcalibur 2.1.0 and LC quan 2.6.0 software were used to
obtain peak areas.
2.5. Preparation of stock solutions, calibration standards and control
samples
Duplicate stock solutions of each analyte with a concentration of
10.0 mg/mL, each, were prepared in DMSO. Stock solutions of PEMN,
PEDN and PETriN were prepared by separate weighing of the same
batches of the respective substances. Stock solutions of PE and PETN
could be prepared from different analyte batches. Working solutions of
PE and PETN (1,000, 100 and 10 µg/mL) and of PEMN, PEDN and
PETriN (1,000, 100, 10, 1, and 0.1 µg/mL) were prepared in methanol/
water (50:50 v/v) by dilution of each stock solution. The internal
standard stock solution of 1,3-DNG and 2-MNG (1 µg/mL, each) was
prepared in methanol/water (50:50 v/v).
Spiking solutions for calibration standards (Cal) and quality control
(QC) samples were prepared by adding the appropriate amount of the
corresponding working solution to obtain respective concentrations for
eight calibrators and five quality control samples (Table 1). Spiking
solutions for calibrators were prepared two times higher than the
serum/plasma concentration, whereas the spiking solutions for the QC
samples were 100 times higher. All solutions were stored at − 20 ◦ C.
Calibration standards were prepared freshly every day using 200 μL
of ovine blank serum or 100 µL human blank plasma and 100 μL or 50 μL
Table 1
Analytes, concentrations of calibrators (Cal) and concentrations of quality control (QC) samples.
Analyte Cal in plasma/serum [ng/mL]
Cal 1 Cal 2 Cal 3 Cal 4 Cal 5 Cal 6
PEMN 0.1 0.5 1.0 5.0 10.0 20.0
PEDN 0.1 0.5 1.0 5.0 10.0 20.0
PETriN 0.1 0.5 1.0 5.0 10.0 20.0
PETN 10.0 15.0 20.0 25.0 30.0 40.0
*
For human QC high 35 ng/mL and for human QC very high 50 ng/mL were used based on the experience from the validation of the human samples, the QCs for the
validation of ovine samples were set high for PETN.
Journal of Chromatography B 1234 (2024) 124028
spiking solution, respectively. Pools of ovine QC samples were prepared,
aliquoted and thawed for each measurement. Human QC samples had to
be prepared freshly every day by spiking the respective spiking solution.
All samples were extracted as described above. QC samples were stored
at − 80 ◦ C.
2.6. Method validation
Method validation was performed according to international guide­
lines [41–43]. The following parameters were evaluated for ovine serum
and human plasma: selectivity, matrix effects (ME), recovery (RE),
linearity, accuracy, precision, stability and limit of quantification (LOQ).
To verify selectivity, maternal (n = 2) and fetal (n = 4) ovine blank
serum samples and human blank plasma samples (n = 6, from different
sources; non-pregnant), were analyzed for peaks interfering with the
detection of the analytes or the ISs. Two zero samples (human/ovine
blank sample + IS) were analyzed to check for absence of the respective
analyte signals caused by the IS or their potential impurities or degra­
dation products. Two ovine/human blank samples spiked with the QC
very high solutions were analyzed to check for absence of signals
interfering with those of the ISs.
RE and ME were determined according to the simplified approach
described by Matuszewski et al. [44].
For the validation of ovine matrix, three sets of samples with six
different sources of ovine serum were only prepared at low concentra­
tion (Cal. 4). Furthermore, an additional 100 ng/mL sample for PETN
was tested. For the validation in the human matrix, three sets of samples
with 6 different sources of human plasma were prepared at low con­
centration (Cal. 4) and high concentration (Cal. 7). Sample set 1 rep­
resenting the neat standard, sample set 2 representing blank matrix
spiked after extraction, and sample set 3 consisting of blank matrix
spiked before extraction. RE results were obtained by comparison of the
absolute peak areas of sample set 3 with those of the corresponding
peaks in sample set 2. MEs were estimated by comparing the peak areas
of sample set 2 with those in set 1. The acceptance criteria were defined
according to forensic guidelines [41] and international validation
standards [43], with a minimum RE of 50 %, a RSD below 25 % and ME
between 75 and 125 % and a RSD of 20 %.
The calibration model was tested by analyzing six replicates at each
concentration level for each matrix analyzed on the same day. Non-
weighted and weighted (1/x and 1/x2) linear and quadratic regression
models were evaluated using analyte/IS ratios as response.
Accuracy and precision were determined by duplicate analysis of QC
samples (ovine/human) on eight days using daily calibrations. Accuracy
was calculated in terms of bias, while within-day and time-different
precision were calculated as relative standard deviations (RSD). Accu­
racy (bias) within ± 20 % of the accepted reference value and precision
within 20 % RSD were considered acceptable [43]. The LLOQ was
defined as the lowest concentration that deviated less than 20 % from
the specified concentration.
Bench-top stability experiments were also performed. For this pur­
pose, 9 QC low and 9 QC very high samples, both ovine and human, were
extracted and pooled into one extract for each concentration and matrix.
Eight aliquots of the pooled extract were transferred into vials and
QC in plasma/serum [ng/mL]
Cal 7 Cal 8 QC very low QC low QC med QC high QC very high
40.0 60.0 0.3 3.0 7.5 35.0 50.0
40.0 60.0 0.3 3.0 7.5 35.0 50.0
40.0 60.0 0.3 3.0 7.5 35.0 50.0
50.0 60.0 – 12.5 75.0 225.0* 320.0*
4
M. Städtler et al.
injected under the conditions of a regular analytical run at time intervals
of 45 min over a total run time of 5 h. Bench top stability was tested by
regression analysis in which the absolute peak areas of each analyte at
each concentration were plotted against the injection time. Instability of
the processed samples is indicated by a negative slope that is signifi­
cantly different from zero (p < 0.05).
For evaluation of freeze/thaw stability in ovine serum and human
plasma samples, QC samples (low, med and high) were analyzed before
(control samples, n = 6 each) and after four freeze/thaw cycles (stability
samples, n = 6 each). For each freeze/thaw cycle, the samples were
frozen at − 20 ◦ C for at least 21 h, thawed, and kept at room temperature
for at least 3 h. The concentrations of the QC samples were calculated
based on the daily calibration curves. For stability, there are two criteria
which have to be fulfilled; the ratio of means (stability/control) has to be
within 90–110 %, and the 90 % confidence interval has to be within
80–120 % from the control sample [41]. Additionally, the long-term
stability of human samples was tested. For this, two sets of QC high
and low, were frozen at − 20 ◦ C and − 80 ◦ C. One set was frozen unin­
terrupted for 4 weeks and the other set was thawed once per week. Each
set contained three samples, which comprised two different pooled se­
rums samples and one pooled plasma samples.
2.7. Applicability
Ovine serum samples and human plasma samples were analyzed
using the newly developed method. External QC material could neither
be purchased for ovine serum nor were human specimens available for
external quality control.
2.8. Animal experiments
At the Ritchie Centre Hudson Institute of Medical Research, Australia
an animal model for fetal growth restriction is well established using
single umbilical artery ligation (SUAL) to induce placental insufficiency.
This model-system served as an animal model to quantify the placental
transfer of PETN and its metabolites from the mother to the fetus in
pregnancies growth restriction and normal grown fetuses [45–47].
Ewes were sourced from Monash Animal Research Platform (n = 8).
Each ewe underwent sterile surgery on day 105 of pregnancy (term
148–150 days gestation) to induce FGR via SUAL as previously
described [14–16]. Briefly, ewes were sedated via sodium thiopental
and were then intubated and anaesthesia was maintained via gaseous
isoflurane. Ewes were randomly allocated for surgery or sham surgery
where the umbilical cord of control fetuses was exposed and handled,
but not ligated (control, normal grown). SUAL is undertaken via ligation
of one of the fetal umbilical arteries resulting in placental atrophy and
Fig. 2. Timeline of Pentalong® Administration in ewes and maternal and fetal blood sampling.
5
Journal of Chromatography B 1234 (2024) 124028
subsequent disruption of placental function with fetal chronic hypo­
xaemia. All fetuses were instrumented with a right femoral artery
catheter, to enable blood sampling. The fetus was returned to the uterus
and catheters were exteriorized through the right flank of the ewe.
Catheters were inserted into the maternal carotid artery for blood
sampling.
The pregnant sheep, weighing approximately 60 kg, were fed with 2
tablets of Pentalong® 50 mg daily. A baseline serum sample, was
collected before first application of Pentalong® on day 1. Fetal arterial
blood samples (~200 μL) were collected prior to PETN administration.
Whole blood was collected and centrifuged to collect serum. Samples
were taken 2 h, 4 h and 24 h on three consecutive days after feeding. The
24 h sample represents the sample before the next feeding with PETN
(Fig. 2). Sampling was performed in ewes (maternal) as well as in NL and
L fetuses.
2.9. Clinical trial in humans
In a prospective, randomized, double-blind, placebo controlled,
parallel grouped, multi-center trial pregnant patients presenting with
impaired uterine perfusion at mid gestation, who consented to partici­
pate in the PETN-trial, were randomized to oral intake of Pentalong® 50
mg or placebo [15]. Patients should take the investigational medical
product two times daily, one tablet in the morning and one tablet in the
evening until 37 weeks of pregnancy. Plasma samples were taken during
the regular monthly control visits (6 visits in the case of a 40-week
pregnancy) and umbilical cord plasma was collected at birth at the
study center in Jena. Umbilical cord samples of five participants could
be collected. Theses samples and the corresponding maternal samples (n
= 18) were analyzed. All samples above the upper calibration range
were analyzed again after four-fold dilution. The analyzes was per­
formed blinded for study group assignment.
3. Results
3.1. Method validation
The selectivity experiments showed no interfering signals for PEMN,
PETN, and the ISs for both matrices when analyzing the blanks. For
PETriN, no interfering peaks were observed in 5 of 6 ovine blank sam­
ples. In one ovine sample a signal of corresponding to 4 % of the
abundance in Cal 1 was observed. In the human samples, minor inter­
fering signals were observed the intensity of which, however, was only
about 1 % of the respective signal at Cal. 1. For PEDN, minor signals
could be detected for both matrices, resulting in approximately 0.1 %
signal intensity of PEDN signal in Cal. 1 for ovine samples and
M. Städtler et al. Journal of Chromatography B 1234 (2024) 124028

approximately 1 % signal intensity of Cal. 1 for human samples. In

Ratio
particular, PE showed signals exceeding the intensity of a sample with a

13.5
10.9
9.6
concentration of 0.5 ng/mL in two human sources. No PE interference

–
RSD [%]
was detected in maternal ovine samples, but in all fetal samples. The

Area

13.3
17.0
15.5
observed signal had a slightly different retention time, but showed high

–
intensity and therefore overlapped with the PE signal. No interfering
peaks for ISs signals could be detected when analyzing the blank samples

93.3
84.3
98.2
[%]
ME
spiked with the QC_high solution.

–
The calibration experiments showed, that the response depended on

Analytes, concentration of tested calibrator, recovery (RE), matrix effects (ME) and relative standard deviation (RSD) in %, calculated by analyte area (area) or area ratio analyte/IS (ratio).

Ratio
analyte concentrations for all analytes. The best correlation between the

16.2
8.8

4.1
area ratios of PETN to IS was obtained using weighted linear calibration

–
RSD [%]
models with a weighting factor of 1/x in ovine serum and human

Area

15.5
18.5
17.2
plasma. For the other analytes, a weighted quadratic calibration model

–
with a weighting factor of 1/x2 was chosen for both specimens.
RE and ME results of both matrices are shown in Table 2. PE is not

58.9
66.7
70.8
[%]
RE
shown, because of the failed selectivity experiments. Average RE were

–
well above the pre-defined minimum value of 50 % for ovine and human
samples. Average ME were within the generally applied acceptance

Cal 7 human

Conc. [ng/
limits for this parameter (75–125 %) with exception of PEMN (54.8 %)
and PEDN (57.7 %) in ovine samples. The generally applied acceptance

40.0
40.0
40.0
mL]
limit for the variability of ME of 20 % RSD was (slightly) exceeded for
PETriN and PETN in ovine samples and all three PEXN at low concen­

Ratio

11.7
18.3
16.3
trations in human samples. This variability was reduced to RSD values

–
below 20 % for almost all analytes and concentration levels when

RSD [%]
looking at relative ME, i.e., ME results calculated using ratios of analytes

Area

22.1
22.2
23.1
vs IS. The only exception was PEDN in ovine samples where the vari­

–
ability of relative ME was actually larger than that of absolute ME.

ME [%]

103.5

105.3
The back-calculated concentrations of all calibration samples met the

93.3
acceptance limits suggested by Shah et al. [42]. Accuracy, repeatability,

–
and time-differentiated intermediate precision results are given in

Ratio

36.8
Table 3 for ovine and Table 4 for human samples.

8.5

7.1
–
PE is not shown in Table 4, because of the failed selectivity experi­

RSD [%]
ments. PEMN, PEDN and PETriN in ovine samples fulfilled the strict

Area

16.9
51.3
8.2
forensic validation parameters at all tested concentrations levels. While

–
all parameters were acceptable for PETN at QC_low, stronger deviations
were observed for the other concentrations for all parameters. In human

62.1
81.1
76.6
[%]
RE

samples the acceptance criteria were met for all parameters for PEMN

–
and PEDN in QC_very_low and QC_low, but slightly do moderately
Cal 4 human

exceeded the generally applied acceptance limits for accuracy or inter­

Conc. [ng/

mediate precision at the other concentrations levels. PETriN did not

fulfill the validation criteria. The described method is not fully valid for
mL]

5.0
5.0
5.0
–

PE and PETN in ovine and human samples.

The results of the stability check, show that PEMN, PEDN and PETriN
Ratio

11.0
29.1
17.9
15.1

were stable in extracts for a period of 5 h at autosampler conditions of +

RSD [%]

8 ◦ C in ovine and human samples. During the three freeze/ thaw cycles,
Area

15.1
12.0
34.7
22.2

PETN showed a degradation of about 70 % in human samples, whereas

PETriN showed a high increase of around 100 % and PEMN an increase
of around 60 %, PEDN seemed stable. For the ovine samples, slight
ME [%]

100.0

changes of around 10 % increase/decrease were observed for PEMN,

54.8
57.7

77.1

PEDN, PETriN and PETN. In the long-term stability test (four weeks) was
observed that the concentration in the PETN extracts decreased by
Ratio

12.0
23.2
22.0
12.7

approximately 50 %, PEMN, PEDN, and PETriN increased up to 30 %.

RSD [%]

A larger effect was observed in the lower concentration. For all

Area

71.1
56.4
70.0
29.8

analytes, larger effects were observed for the samples stored at − 20 ◦ C in

comparison to samples stored at − 80 ◦ C (data not shown). Few varia­
tions were detected whether the samples were thawed several times or
RE [%]

107.42
171.2

97.0
80.6

constantly frozen.
The LLOQs in ovine samples are 1.0 ng/mL for PETriN and 0.1 ng/
mL for PEMN and PEDN. The LLOQs in human samples are 5.0 ng/mL
Cal 4 ovine

for PETriN and 0.3 ng/mL for PEMN und PEDN. For PE and PETN no
Conc. [ng/

LLOQ could be determined in both matrices.

mL]

100
5.0
5.0
5.0

3.2. Application
Analytes

PETriN
Table 2

PEMN
PEDN

PETN

In total, 8 ewes were included in the animal study. Two sheep

(19,211, 19,215) were pregnant with one L fetus, one sheep (19,013)

6
M. Städtler et al. Journal of Chromatography B 1234 (2024) 124028

was pregnant with one NL fetus, five sheep (19,018, 19,037, 19,049,
19,107, 19,115) with two fetuses, one NL and one L, each. 184 samples

precision RSD
Intermediate
were collected from 21 animals at nine timepoints (Fig. 2). Five samples
(19,013 maternal sample at baseline, 19,049 maternal sample on day 1

12.7

60.5
6.0
7.9
6.3
[%]
2 h, 19,115 maternal sample day 3 4 h, 19,018 L fetus day 2 2 h, 19,107
L fetus day 2 4 h) were missing. The timeline of feeding and sample

Repeatability
collection is depicted in Fig. 2. The results of the ovine samples are

RSD [%]
shown in Table 5 and 6.
PETN could not be detected in any of the samples. PETriN was never

10.8

60.5
5.7
7.1
5.0
detected in a concentration above the LLOQ. In 15 samples (8 maternal
Analytes, nominal concentration, accuracy expressed as bias in %, repeatability and inter-mediate precision as RSD in % for QC low, QC med, QC high and QC very high in ovine serum.

and 7 fetal samples) distinct signals representing the exact mass of

Accuracy
bias [%]

− 7.7
− 5.9
6.9
− 5.9
− 50.6
PETriN and the correct relative retention time compared to a calibrator,
analyzed in the same run, indicated PETriN presence in about 10 % of
QC very high

the fetal and maternal samples. PEDN and PEMN were detected in 91 %
[ng/mL]
Nominal

of the samples taken at 2 h and 4 h after application, and were not

320.0
50.0
50.0
50.0
50.0
conc.

detected in baseline and 24 h samples. The sample chromatogram of an

authentic ovine sample and the corresponding calibrator 4 is shown in
precision RSD
Intermediate

Fig. 3 A/B.
Two hours after application the mean concentration of PEDN was
5.3 ng/mL in maternal ovine samples and 7.3 ng/mL for PEMN, after
16.7

34.6
5.9
9.1
5.8
[%]

four hours the mean concentrations increased slightly for PEDN to 6.9
ng/ml and for PEMN to 9.3 ng/ml. In the fetal ovine samples, the mean
Repeatability

concentrations at two and four hours after application, were about half
RSD [%]

of the maternal mean concentration (Table 7 and 8).

5.5
2.8
9.1
3.8
34.6

During the PETN study, plasma and corresponding cord blood sam­
ples from 5 of 50 Jena participants could be collected. For the five cord
Accuracy
bias [%]

blood samples in four cases maternal samples at time of delivery were

− 11.9

− 49.7
− 4.1
8.7
− 3.5

available. Additionally, 9 maternal samples retrieved during study visits

of these mothers have been analysed. Results are displayed in supple­
[ng/mL]
Nominal
QC high

mentary table 1. All placebo samples and samples which were taken at
225.0
35.0
35.0
35.0
35.0
conc.

baseline (before Pentalong® administration) and after 37 weeks of

pregnancy (end of Pentalong® administration) were negative for PEXN.
precision RSD

PEDN was detected in three maternal verum samples with concentra­

Intermediate

tions of 27, 21 and 2 ng/mL during pregnancy. PEMN was detected in

four maternal verum samples during pregnancy. The corresponding
19.6

26.9
6.0
9.2
8.1
[%]

PEMN concentrations were 149, 152, 70 and 0.8 ng/mL. The sample
chromatogram of an authentic human sample and the corresponding
Repeatability

calibrator 3 is shown in Fig. 3 C/D. Just one mother gave birth before 37
RSD [%]

weeks pregnancy and under Pentalong® administration. PEDN and

14.3

26.9
3.5
4.2
5.2

PEMN was detected in the respective maternal sample with a PEDN

concentration of 3.3 ng/mL and a PEMN concentration of 57 ng/mL. The
Accuracy

accompanying umbilical cord plasma concentrations were 2.3 ng/mL

bias [%]

− 9.0
− 1.2
− 2.4
− 11.3

− 16.0

for PEDN and 73 ng/mL for PEMN. PETN and PETriN were never
detected. To summarize, in one of the five cord blood samples, as well as
in the corresponding maternal sample PEDN and PEMN could be
[ng/mL]
Nominal
QC med

detected.
conc.

25.0

75.0
7.5
7.5
7.5

4. Discussion
precision RSD
Intermediate

4.1. Method development

38.0

16.3
6.0
9.7
9.8
[%]

To achieve high sensitivity as well as high selectivity high resolution

Repeatability

mass spectrometry was used for identification and quantification. Using

negative ionization full scan MS, all analytes could be detected as
RSD [%]

deprotonated analytes except PE. Furthermore, in-source cleavage of the

5.9
4.0
6.3
8.8
16.3

NO–3 moiety was observed for PETN and its metabolites PETriN, PEDN
and PEMN. No other fragments/cleavages could be detected, when the
Accuracy
bias [%]

analytes were exposed to different levels of fragmentation energy.

− 12.0
0.1
− 3.5
− 0.6

− 9.1

However, as ammonium formate was used as mobile phase component,

abundant formate adducts could also be detected for all analytes. These
[ng/mL]
Nominal
QC low

mass spectrometric properties are in accordance with those described

conc.

12.5

12.5
3.0
3.0
3.0

after atmospheric pressure chemical ionization of PETN and PEXN in

explosives described by Brust et al. [29,30] To achieve highest sensi­
tivity several ionization parameters such as spray voltage, capillary
Analytes

PETriN
Table 3

PEMN
PEDN

PETN

temperature, s-lens RF level, aux gas heater temperature as well as gas

PE

settings were optimized with respect to deprotonated analyte molecules

7
M. Städtler et al. Journal of Chromatography B 1234 (2024) 124028

Table 4
Analytes, nominal concentration, accuracy expressed as bias in %, repeatability and inter-mediate precision as RSD in % for QC very low, QC low, QC med, QC high and
QC very high in human plasma.
Analytes QC very low QC low QC med

Nominal Accuracy Repeatability Intermediate Nominal Accuracy Repeatability Intermediate Nominal Accuracy
conc. [ng/ bias [%] RSD [%] precision RSD [%] conc. [ng/ bias [%] RSD [%] precision RSD [%] conc. [ng/ bias [%]
mL] mL] mL]

PEMN 0.3 − 16.6 9.1 13.1 3.0 − 12.1 3.9 10.8 7.5 − 18.5
PEDN 0.3 10.9 6.1 18.2 3.0 15.8 5.1 19.9 7.5 12.0
PETriN – – – – 3.0 76.9 8.3 31.8 7.5 164.9

as well as the formate adducts (data not shown). Additionally, the in­
fluence of post column infusion of formate buffer as well as ammonia
solution was tested, but no significant signal enhancement could be
observed (data not shown). Since the formate adducts showed signifi­
cantly higher signals than the deprotonated analytes, the adducts were
used for identification/quantification of PE, PEMN, PEDN, and PETriN,
whereas the NO–3 cleavage was used for PETN. With the final method it
was possible to detect all 5 analytes.
For the optimization of the chromatographic system, different col­
umns, mobile phases and their compositions and flow rates, as well as
different column temperatures were tested (data not shown). A suffi­
cient baseline separation of the analytes of interest could be achieved on
the selected RP 18 column using a mobile phase composition of
ammonium formate buffer and acetonitrile in the above-mentioned
gradient. To the authors knowledge, this is the first LC-MS-based
method for the simultaneous analysis of PETriN, PEDN, and PEMN in
biological samples resulting in a sufficient chromatographic separation
and mass-spectrometric detection.
So far, only few methods have been described for the extraction of
PETN and its metabolites from human plasma. In 1983, Yu et al.
described a combination of liquid–liquid extraction (LLE) and solid
phase extraction. They extracted PETN with ethyl acetate, followed by a
SPE using a C18 cartridge [48]. In 1997, Stalleicken et al. developed a
method for the extraction of PEXN based on two different separations.
For PETriN and PETN, a neutral LLE with dichloromethane was per­
formed, whereas PEMN and PEDN were extracted at basic conditions
with a tert-butyl methyl ether/n-pentane mixture [19]. Two years later,
a method for the extraction of PEMN, PEDN, and PETriN was developed
by Schutz et al. [23] but no application has been published for this
method so far.
To achieve a manageable method of extraction, various organic
solvents (e.g., dichloromethane, ethyl acetate, n-hexane, ether) and
solvent combinations were tested for the LLE of PEXN in the present
study (data not shown). The best results, with respect to recovery and
reproducibility were obtained with a combination of ethyl acetate/i-
propanol/dichloromethane (60:20:20, v/v/v). Also, the extraction
with dichloromethane showed good results, especially for PETriN and
PETN. As the literature indicated that PEMN and PEDN are the primary
target analytes in human samples, this solvent combination was chosen.
Other extraction methods such as the more laborious and cost-intensive
SPE were also considered, but did not show better results than LLE (data
not shown). As Schutz et al. described an exhaustive extraction in their
method, a single, double and triple extraction was also tested, but no
significant differences in RE were found. In order to perform a simple
and time efficient LLE, a single-step extraction of the samples was cho­
sen for the final method.
With respect to an ideal IS, the most suitable would have been the
stable isotope labeled analytes. However, these would have had to be
synthesized in a particularly cost-intensive manner. In the search for
alternatives, ISMN, isosorbide dinitrate, trinitroglycerol, 2-mononitro­
glycerol (2-MNG), 1,2-dinitroglycerol, and 1,3-dinitroglycerol (1,3-
DNG) were examined (data not shown), while 2-MNG and 1,3-DNG
seemed to be the best candidates. During validation, 2-MNG could
better compensate extraction and ionization effects leading to better
repeatability and accuracy. 2-MNG was therefore used as internal
standard. A chromatogram of the internal standard (2-MNG) and PEXN

Table 5
Individual PEMN concentrations in ng/mL (3 days) in maternal, fetal non-ligated (NL), and fetal ligated (L) ovine serum samples at baseline, 2 h/4h/24 h after
application of 100 mg PETN to the mother just after baseline and 24 h blood collections.
number type Day 1 Day 2 Day 3

Baseline 2h 4h 24 h 2h 4h 24 h 2h 4h

19,013 maternal * 7.0 11 0.2 7.7 8.9 0.2 10 13

NL n.d. 3.7 9.4 0.4 5.0 6.7 0.5 3.6 12
19,018 maternal n.d. 7.6 9.1 n.d. 5.7 6.1 n.d. 5.7 7.3
L n.d. 2.4 6.5 0.5 * 4.2 0.3 1.8 6.5
NL n.d. 2.8 7.0 0.6 2.7 5.0 0.2 2.2 5.7
19,037 maternal n.d. 4.6 7.2 n.d. 3.4 3.6 n.d. 4.6 6.1
L n.d. 1.3 4.0 0.3 1.8 1.9 0.3 1.9 4.2
NL n.d. 1.2 4.2 0.2 2.1 2.9 0.2 2.5 4.1
19,049 maternal n.d. * 8.1 n.d. 7.3 6.9 n.d. 7.3 7.4
L n.d. 1.0 3.9 0.3 4.2 5.4 0.5 4.2 5.8
NL n.d. 1.2 4.6 0.2 4.9 7.0 0.3 5.4 8.1
19,107 maternal n.d. 3.2 5.7 0.3 0.2 1.3 0.5 2.5 8.3
L n.d. 1.0 3.6 0.9 0.6 * 1.4 1.4 4.5
NL n.d. 1.2 4.5 0.8 0.4 0.9 1.1 1.5 5.8
19,115 maternal n.d. 2.9 4.3 0.3 4.7 4.9 0.1 * 4.3
L n.d. 0.9 2.7 0.5 1.7 3.6 0.3 2.1 3.9
NL n.d. 1.2 3.6 0.3 2.0 3.9 0.3 3.1 4.9
19,211 maternal n.d. n.d. n.d. n.d. 0.3 0.5 n.d. 25 27
L n.d. n.d. n.d. n.d. n.d. 0.3 n.d. 6.7 17
19,215 maternal n.d. 29 33 0.3 9.7 19 0.5 12 20
L n.d. 12 26 1.2 3.8 11 0.8 4.9 13
*
missing sample, n.d. not detectable.

8
M. Städtler et al. Journal of Chromatography B 1234 (2024) 124028

QC med QC high QC very high

Repeatability Intermediate Nominal Accuracy Repeatability Intermediate Nominal Accuracy Repeatability Intermediate
RSD [%] precision RSD [%] conc. [ng/ bias [%] RSD [%] precision RSD [%] conc. [ng/ bias [%] RSD [%] precision RSD [%]
mL] mL]

3.9 18.9 35.0 − 20.8 3.0 18.7 50.0 − 25.8 3.5 16.6
8.2 24.7 35.0 0.1 3.7 19.6 50.0 − 3.0 2.9 19.7
3.3 34.1 35.0 56.9 2.7 24.9 50.0 29.1 4.2 28.4

in plasma is shown in Fig. 3. detectable in higher concentration, therefore an additional sample of

The calibrator concentrations were chosen with respect to the cur­
rent study of Pabst et al. [20], where a maximum mean concentration for
PEDN of 9.7 ng/mL and a maximum mean concentration for PEMN of
41.6 ng/mL was determined. There are no guidelines for recommended
concentrations in human so far. Since no reference values were available
in sheep, a broad concentration range was defined for validation (see
Table 1) in order to cover the concentrations in both matrices as well as
possible.
4.2. Method validation
The developed method is selective for analysis of PETN, PETriN,
PEDN and PEMN, because either no signals were detected or any
observed signals were negligibly small and therefore not interfering with
the analysis. In contrast, relevant interfering signals for PE were detec­
ted in two human sources and also in two ovine samples. For the human
samples the interfering peaks were at the corresponding retention time
of PE. As the human blank samples had been obtained from the local
biobank, a PETN intake could not be excluded, although this seems
unlikely. In the ovine sample, interfering signals were only observed in
fetal samples but not in the maternal samples. Although they had a
slightly different retention time, PE signals could not be sufficiently
distinguished from the overlapping signal in the dead volume. A selec­
tive analysis of nitrate-free PE in matrix is therefore not possible with the
present method. The calibration model experiments showed a concen­
tration dependence for all analyte signals. Unfortunately, PETN is only
PETN (100 ng/mL) in ovine matrix for RE and ME was added and QC
concentrations were modified. RE and ME for ovine serum samples were
obtained with only one concentration (Cal. 4), due to a limited sample
volume of different sheep samples. For human samples, two concen­
trations, Cal. 7 and Cal. 4, respectively were used for the verification of
RE and ME. In the ovine and human matrix, the RE were always over 50
% and similar to or only slightly lower than the recoveries of 72 % for
PEMN and up to 94 % for PEDN/PETriN reported by Schutz et al. [23].
The better results reported by the latter group might be explained by the
fact, that they used another extraction solution and, more importantly,
extracted each sample three times. In the present study, simplicity of
workup was preferred over potential minor gains in RE that might have
been achieved by repeated extraction steps. No severe ME could be
observed and average ME values were within the generally applied
acceptance range with exception the one for PEMN and PEDN in ovine
samples. However, even for the latter two analytes satisfactory LLOQs
were obtained. The RSD of ME for PETriN and PETN in ovine and all
analytes in human samples at low concentrations (slightly) exceeded the
generally applied acceptance limit of 20 %. However, the variability of
absolute ME was (partly) compensated by the IS leading to less vari­
ability in relative ME. For this reason, ME were considered acceptable
for the purpose of the present study. The observed differences in matrix
effects between human and ovine is most likely due to a combination of
inter-species variability and the large variability in ovine samples. The
latter may in turn be due to differences in composition of maternal and
fetal ovine samples as also indicated by the results of the selectivity

Table 6
Individual PEDN concentrations in ng/mL (3 days) in maternal, fetal non-ligated (NL) and fetal ligated (L) ovine serum samples at baseline, 2 h/4h/24 h after
application of 100 mg PETN to the mother just after baseline and 24 h blood collections.
number type Day 1 Day 2 Day 3

Baseline 2h 4h 24 h 2h 4h 24 h 2h 4h

19,013 maternal * 2.4 2.4 0.1 3.2 1.4 0.1 3.5 2.2
NL 0.2 4.9 4.3 0.2 3.4 1.9 0.2 3.6 4.9
19,018 maternal n.d. 0.5 0.7 n.d. 0.4 0.7 n.d. 0.5 0.7
L 0.1 0.7 1.5 0.1 * 0.9 0.1 0.8 1.8
NL 0.1 0.5 1.5 0.1 0.9 0.8 n.d. 0.8 1.8
19,037 maternal n.d. 0.8 1.3 n.d. 0.5 0.6 n.d. 0.7 1.1
L 0.1 0.7 1.3 0.1 0.6 0.7 n.d. 0.7 1.4
NL 0.1 0.6 1.3 n.d. 0.7 0.8 n.d. 1.0 0.9
19,049 maternal n.d. * 1.0 n.d 1.3 0.9 n.d. 1.2 1.0
L 0.1 0.6 1.8 n.d. 2.1 1.5 n.d. 1.5 2.0
NL n.d. 0.5 1.5 n.d 1.6 1.4 n.d. 1.6 1.4
19,107 maternal 0.1 1.4 3.1 0.1 n.d. 1.0 0.1 1.7 3.1
L 0.1 1.7 3.3 0.8 0.1 * 0.2 1.3 4.9
NL 0.1 1.7 3.3 2.7 0.2 1.3 0.3 1.5 5.2
19,115 maternal 0.0 0.6 0.7 0.1 0.7 0.6 0.1 * 0.5
L n.d. 0.4 0.6 0.1 0.5 0.6 0.1 0.5 0.6
NL n.d. 0.4 0.7 0.1 1.7 0.6 0.1 0.6 0.5
19,211 maternal 0.4 0.1 0.1 0.2 0.1 0.1 0.1 3.6 3.6
L 0.2 0.2 0.2 0.2 0.2 0.2 0.2 4.9 6.0
19,215 maternal 0.2 1.6 2.1 0.2 1.0 1.6 0.2 2.3 3.4
L 0.2 1.7 2.3 0.2 0.9 1.7 0.2 1.7 2.9
*
missing sample.

9
M. Städtler et al. Journal of Chromatography B 1234 (2024) 124028

Fig. 3. Chromatograms of the extracted masses of the internal standard 2-MNG, pentaerythritol mononitrate (PEMN), pentaerythritol dinitrate (PEDN), pentaer­
ythritol trinitrate (PETriN) in ovine calibrator 4 with a concentration of 5.0 ng/ml, each (A); serum from authentic non-ligated fetus 19107, 24 h after application of
100 mg PETN to the mother (Day 2 baseline) (B); human calibrator 3 with a concentration of 1.0 ng/ml, each (C); and diluted (1:4) authentic human maternal plasma
sample taken at visit 3 after twice daily intake of 50 mg PETN (D).

experiments. A further elucidation was not possible in this study due to
the limited availability of individual blank matrix samples.
Accuracy, repeatability, and time-different intermediate precision
were calculated. PEMN, PEDN and PETriN in ovine samples fulfilled the
requirements for all three validation parameters. No validation data
exist so far for animal matrices in the literature. The described method is
not valid for PETN and PE, which is already evident from the previous
validation results. In particular, the precision of PEMN and PEDN in
human samples slightly or moderately exceeded the generally applied
acceptance criteria, especially for intermediate precision for PEDN and
the accuracy for PEMN in higher concentration. The bias could possibly
be explained by the different stock solution for PEMN and/or stability
issues (see below). The deviation in human samples could also result
from the fatty human samples compared to the ovine samples. These are

Table 7
Highest observed (CH) and average (Cmean) concentrations (3 days) of PEMN in maternal, fetal non-ligated (NL) and fetal ligated (L) ovine serum samples at Baseline
(BL), 2 h/4h/24 h after application of 100 mg PETN to the mother.
BL 2h 4h 24 h

Maternal n fetal NL fetal L Maternal n fetal NL fetal L n Maternal n fetal NL fetal L n Maternal n fetal NL fetal L n
=8 n=6 n=7 = 22 n = 18 = 20 = 24 n = 18 = 20 = 16 n = 12 = 14

CH [ng/ n.d. n.d. n.d. 29.0 5.4 12.0 33.2 12.1 26.0 0.5 0.8 1.4.
mL]
Cmean n.d. n.d. n.d. 7.3 2.6 2.7 9.3 5.6 6.4 0.2 0.4 0.5
[ng/
mL]
RSD [%] n.d. n.d. n.d. 97.4 56.8 103.9 86.4 45.9 96.4 112.3 63.4 77.2

Table 8
Highest observed (CH) and average (Cmean) concentrations (3 days) of PEDN in maternal, fetal non-ligated (NL) and fetal ligated (L) ovine serum samples at Baseline
(BL), 2 h/4h/24 h after application of 100 mg PETN to the mother.
BL 2h 4h 24 h

Maternal n fetal NL fetal L Maternal n fetal NL fetal L n Maternal n fetal NL fetal L n Maternal n fetal NL fetal L n
=8 n=6 n=7 = 22 n = 18 = 14 = 24 n = 18 = 14 = 16 n = 12 = 14

CH [ng/ 0.2 0.2 0.2 3.6 4.9 4.9 3.6 5.2 6.0 0.2 2.7 0.8.
mL]
Cmean 0.1 0.1 0.1 1.3 1.5 1.1 1.4 1.9 1.8 0.1 0.3 0.1
[ng/
mL]
RSD [%] 122.0 77.9 49.6 84.8 87.6 97.5 74.2 78.6 83.0 87.8 247.8 114.3

10
M. Städtler et al.
difficult to extract due to the high fat content. Further processing steps
(filtration, additional centrifugation steps, dilution) did not reduce the
variability in the results. For PEMN, the other parameters are within
(repeatability) or slightly above (intermediate precision) the acceptance
criteria. For PEDN the accuracy and repeatability are within the
acceptance criteria. Therefore, the developed method was considered
valid for analyzing PEMN and PEDN in context of the purpose of studies.
PETriN showed high discrepancies in accuracy in human samples. As the
same working solutions were used for these samples as for ovine sam­
ples, which met the validation criteria, a systematic error for preparing
the solution could be excluded. The stability issue could also be
excluded, as QC samples were prepared freshly every day and stability of
the working solutions was given at the time of validation. Therefore, the
strong discrepancies in accuracy for PETriN can only be explained by
extraction of human samples. A further modification of the extraction
procedure was waived considering that PETriN seems an unimportant
metabolite in humans. In any case, sensitive qualitative analysis for
PETriN is still possible in human samples. In comparison with the
combined method of Schutz et al. [23] the newly developed method had
higher derivations for intermediate precision, whereas the other results
are comparable.
The results of the stability check show that PEMN, PEDN and PETriN
were stable in extracts for a period of 5 h at autosampler conditions of +
8 ◦ C in ovine and human samples. During the three freeze/ thaw cycles
PETN showed a degradation of about 70 % in human samples, which
resulted in higher peak areas of PETriN after the freeze–thaw cycle. This
seems in contrast to the observations by Schutz et al. [23] who
mentioned no stability problems during storage at − 20 ◦ C and two
freeze/thaw cycles. However, from their publication it is not clear if
their stability experiments were done in solution or plasma samples.
During the long-term stability experiment, four weeks of storage, the
concentration in the PETN extracts decreased by approximately 50 %,
regardless of whether the samples were thawed or frozen without
interruption. PETN degradation was slightly smaller when stored at
− 80 ◦ C than when stored at − 20 ◦ C and in the higher concentration
(data not shown). PEMN, PEDN, and PETriN showed little variation
from the freshly prepared solutions when the samples were thawed
several times than when they were frozen constantly. This suggests that
when thawed several times, the nitrates possibly degrade to the next
metabolite and thus the concentrations of the metabolites either in­
crease or remain constant. The stability of the last metabolite PE was not
monitored. Since all PETN-metabolites were in the same solution, it
cannot be distinguished to which extent each metabolite was degraded
to the following one.
As mentioned above, the LLOQ for PEMN, PEDN and PETriN ranged
from 0.1 to 5 ng/mL. It is noticeable that the LLOQs in human samples
were higher than in ovine samples. This resulted from the fatty human
samples. So far, no LLOQ for animal matrices has been reported in the
literature. In comparison with the LLOQ acquired by Schutz et al. [23]
with an LLOQ of 1.0 ng/mL for PEMN and 0.2 ng/mL for PEDN and
PETriN in human plasma, the LLOQ of the new method for PETriN was
higher, while the LLOQ of PEDN was similar and of PEMN was better.
The LLOQ of PEMN and PEDN seems to be sufficient for human samples.
PETriN could not be detected in human samples. This might be due to
the comparatively high LLOQ. Nevertheless, a presence of PETriN could
be observed in the ovine samples, where even lower concentrations of
PEMN/PEDN were detected. Therefore, the authors conclude that in
most of the analyzed samples PETriN was no longer present at the time
of sample collection. This is in line with the conclusion by Weber et al.
[18] and Pabst et al. [20], who both stated that neither PETN nor PETriN
were quantifiable in plasma at an analytical LLOQ of 50 pg/mL.
Compared to previously published methods, the new method
described here eliminates the need for exhaustive extraction and
derivatization. Furthermore, a considerably lower sample volume is
used following the trend to lower sample volumes in modern bio­
analytical methods.
11
Journal of Chromatography B 1234 (2024) 124028
4.3. Application
The newly developed approach was used to analyze 184 ovine
samples of the animal study and 13 maternal plasma samples and five
umbilical cord plasma samples taken from the participants of the PETN-
trial at the study center in Jena. Unfortunately, the identification criteria
(at least two analyte specific ions/fragments, acceptable relative ion
ratio and acceptable relative retention time [49]), for a reliable identi­
fication of PETriN were not met as the signal intensity was low.
Nevertheless, since method validation showed no interfering signals and
PETriN signals were not detected in any of the blank samples, it can be
assumed that the observed signals belong to PETriN (Fig. 3 B).
PEDN and PEMN were detected in nearly all maternal, NL and L
samples taken 2 h and 4 h following intake of Pentalong, while it was not
detected in samples taken at baseline. In some samples collected 24 h
after Pentalong® application, just before the next fed, a very low con­
centration of PEMN (in 32 of 42 maternal and fetal samples) and PEDN
(in 3 maternal samples of overall 42 samples) was still detectable.
Despite the similar weight of the sheep (about 60 kg), there were strong
inter-individual differences in the concentrations for PEMN and PEDN 2
h and 4 h after Pentalong® administration (Table 7 and 8). PEDN con­
centrations did not follow a comparable pattern, with in part increasing
and in part decreasing concentrations found in the samples taken 2 h and
4 h following feeding. In contrast for PEMN, all maternal as well as all
fetal concentrations were higher at 4 h than at 2 h indicating that CH
(highest observed concentration) of this metabolite occurs later than 2 h
after drug administration. As PEMN is the last active metabolite in the
metabolic pathway of PETN, it is not surprising that the time of maximal
concentration is observed at later time points than for PEDN. With
respect to interindividual differences, fetal PEDN concentrations were
comparable with maternal ones. In contrast, PEMN was detected at
higher levels in maternal samples.
In conclusion, a transfer of PEDN and PEMN from the mother to the
fetus was observed in this animal model of FGR. Regarding the question
whether differences in drug transfer to non-ligated and ligated fetuses
are observable, statistical analysis were performed (Fig. 4).
For further statistical calculations two mother–child couples were
excluded. For these two sheep with ligated fetuses, implausible PEDN
and PEMN concentrations were found. In one case no or only traces of
PEDN and PEMN could be detected on day one and two, while in the
second case the determined PEMN concentrations on day one and two
were almost three times higher than in all other sheep.
PEMN and PEDN concentrations at 2 h and 4 h were higher in
samples taken from NL fetus compared to L fetus serum samples. To
determine whether there is a statistical difference between the L and NL
fetuses’ individual concentration ratios were used. NL and L fetal ana­
lyte concentrations were divided by the corresponding maternal analyte
concentration. The retrieved ratios for all 2 h samples of day 1, day 2 and
day 3 were averaged to obtain a mean concentration ratio (see Fig. 4 A/
B). The same calculation was performed for the results of the 4 h samples
(see Fig. 4 A/B). No significant differences between the two groups could
be observed. Additionally, to adjust for possible time-dependent differ­
ences the area under the curve (AUC) was calculated for both analytes
over the period from 0 h to 4 h. As described for the concentration ratios,
ratios of the AUC were calculated and averaged over the 3 days. The
results are shown in Fig. 4 C. A significant difference in passage (p =
0.026) and a trend towards significance were found for PEM and PEDN,
respectively. Thus, the performed ligation of the umbilical artery with a
consecutive reduction in blood flow to the fetus might also reduce the
transfer of PEXN from mother to child. Additionally, a reduced meta­
bolic activity in the impaired fetuses might reduce the fetal metabolism
reducing PEDN to PEMN resulting in lower concentrations of PEMN.
In the human samples collected from study participants of the pla­
cebo group, as well as in the maternal samples collected later than 37
weeks of gestation, when study medication was stopped, as well as in the
corresponding fetal samples no PEXN could be detected (supplementary
M. Städtler et al.
Fig. 4. Ratio of PEMN concentration fetus/mother at 2 h and 4 h [A], ratio of PEDN concentration fetus/mother at 2 h and 4 h [B] and AUC ratio fetus/mother at 0–4
h of PEDN and PEMN [C] of ovine samples (non ligated “NL”, ligated “L”). A Man-Wittney-U test was performed, a p-value smaller than 0.05 represents a signif­
icant difference.
table 1). In the samples taken from study participants assigned to the
verum group and delivery occurred before or at 37 weeks of gestation
PEMN and PEDN was found in maternal and umbilical cord samples.
Interestingly, the here observed concentrations were above those
described by Pabst et al. [20], where a mean maximum concentration for
PEDN of 9.7 ng/mL and for PEMN of 41.6 ng/mL were reported. In our
samples a maximum concentration of 28 ng/mL for PEDN and 152 ng/
mL for PEMN could be determined. This could be explained by the
higher concentration of PETN intake (2 x 50 mg vs 1 x 80 mg Penta­
long®) and the administration of the drug for several weeks leading to a
steady state condition. Furthermore, gender specific difference cannot
be excluded. In the one umbilical cord plasma sample taken at delivery
in week 32 PEMN and PEDN could be detected (supplementary table 1).
To summarize, the detected consistently higher concentrations of
PEMN compared to PEDN correspond to data of Pabst et al. [20] and
Weber et al. [18]. Furthermore, these results proof that PETN metabolite
12
Journal of Chromatography B 1234 (2024) 124028
(s) are transferred from the mother to the fetus. Again, the higher con­
centration of PEMN in the umbilical cord plasma in comparison to the
corresponding maternal sample indicates that besides the transfer of
PEXN to the fetus, fetal metabolism converts PEXN to PEMN, leading to
an increased concentration of PEMN. Since we were only able to collect
a single umbilical cord samples at delivery during the period of study
medication application, the description of our result has to remain
descriptive. However, this is the first time that PETN metabolites were
detected in umbilical cord plasma and plasma of pregnant women at
delivery, demonstrating that PETN metabolites pass the placental
barrier.
Surprisingly, both PEDN and PEMN concentrations in human sam­
ples were significantly higher than in ovine samples although the same
doses of Pentalong® were administered. Besides differences in body
weight possible interspecies differences in PETN metabolism in human
and ovine organism might serve as an explanation for this observation.
M. Städtler et al.
In conclusion, the presented LC-MS/MS method for the simultaneous
analysis of PEMN, PEDN and also PETriN, allows a rapid and simple
extraction and was successfully applied to detect the analytes in human
and ovine samples. In principle, the present method is suitable for the
quantitative determination of PEDN and PEMN as well as a qualitative
detection of PETriN in maternal and fetal ovine serum as well as in
human plasma and cord blood. As a result, it could be demonstrated for
the first time that PEMN and PEDN can be detected in fetal samples upon
maternal intake of PETN during pregnancy. Concentrations determined
for PEMN and PEDN differ between the two species, with low biological
variability within the ovine matrix reflecting the applicability of the
developed method. Due to the observed instability of PEXN in serum and
plasma samples, should be refrigerated at − 80 ◦ C after sampling and
analyzed as soon as possible in future studies.
CRediT authorship contribution statement
Mariann Städtler: Conceptualization, Investigation, Methodology,
Validation, Visualization, Writing – original draft. Daniela Wissen­
bach: Conceptualization, Methodology, Validation, Visualization,
Writing – original draft. Dirk K. Wissenbach: Conceptualization,
Methodology, Validation. Laura Franke: Investigation. Jana Pastu­
schek: Resources. Ekkehard Schleussner: Supervision. Beth Allison:
Investigation, Resources. Frank T. Peters: Conceptualization, Supervi­
sion, Writing – review & editing. Tanja Groten: Conceptualization,
Supervision, Writing – review & editing.
Declaration of competing interest
The authors declare the following financial interests/personal re­
lationships which may be considered as potential competing interests:
Mariann Staedtler reports financial support was provided by Mittel­
deutsche Gesellschaft für Frauenheilkunde und Geburtshilfe e. V.
(MGFG). Tanja Groten reports financial support was provided by
German Research Foundation.
Data availability
Data will be made available on request.
Acknowledgements
The presented study was financially supported by the German
Research Foundation (T.G., GR1955/4-1) and the Mid-German Society
for Gynecology and Obstetrics. (M.S.). The authors thank Yvonne Hei­
mann for supporting us with human samples and Armin Siering for their
assistance and helpful discussions.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jchromb.2024.124028.
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