Supplementary Information:

Improved determination of femtogram-level organic explosives in

multiple matrices using dual-sorbent solid phase extraction and liquid

chromatography-high resolution accurate mass spectrometry

Rachel Irlam1, Mark C. Parkin1,2, Dermot Brabazon3, Matthew S. Beardah4, Michael

O’Donnell4 and Leon P. Barron1,*

1 King’s Forensics, School of Population Health & Environmental Sciences, Faculty of Life

Sciences & Medicine, King’s College London, 150 Stamford Street, SE1 9NH, London, United

Kingdom

2 Eurofins Forensics, Teddington, Middlesex, UK

3 Advanced Processing Technology Research Centre, Dublin City University, Dublin 9, Ireland

4 Forensic Explosives Laboratory, Dstl, Fort Halstead, Sevenoaks, Kent, UK

* Corresponding author (e-mail: leon.barron @ kcl.ac.uk ; Tel.: +44 20 7848 3842)

Table of contents:

List of tables:

Table S1. SPE sorbents tested and their chemical and physical properties……………...S-3

Table S2. Optimised atmospheric pressure chemical ionisation (APCI) and mass

spectrometric conditions for the chosen explosive analytes………………………………..S-3

Table S3. Properties of chosen subset (n=14) of explosives analytes………………….…S-4

Table S4. Explosives analytes and their detected ions in HRMS…………………………..S-5

List of figures:

S-1

Figure S1. LC-APCI-HRMS matrix effects for a selection of probe explosives in six

different matrices after single- and dual-sorbent SPE……………………………………….S-6

Figure S2. Recoveries of a selection of n=14 chemically diverse explosives from a range

of sample types using single- and dual-sorbent SPE combinations……………………….S-7

Figure S3. Positive and negative mode ion plots showing all ions that are removed by the

best dual-sorbent combination for each of the six matrices....................................….......S-8

Figure S4. Positive mode ion plots showing selective extraction sorbent alone vs. the best

matrix removal-selective extraction sorbent combination for each matrix…………………S-9

Figure S5. Negative mode ion plots showing selective extraction sorbent alone (left) vs.

the best matrix removal-selective extraction sorbent combination for each matrix……..S-10

List of additional experimental information:

Particular sampling and pre-treatment procedures for each sample type………S-11 – S-12

Solid phase extraction (SPE)…………………………………………………………………S-13

Determination of recovery and MS detection matrix effect………………………………..S-14

S-2

Table S1: SPE sorbents tested and their chemical and physical properties.Sorbent trade

name Manufacturer Sorbent base material

Additional functionality

Weight (mg)

Volume (mL)

Oasis HLB Waters Divinylbenzene (DVB) N-vinylpyrrolidone 200 6

Isolute ENV+ Biotage Polystyrene divinylbenzene copolymer (PS-

DVB)

Hydroxyl 200 6HyperSep Retain

PEP Thermo Fisher Urea 200 6

HyperSep SAX Thermo Fisher Trifunctional quaternary amine 200 3

HyperSep NH2 Thermo Fisher Silica-based Aminopropyl 200 3Bond Elut CN Agilent Cyanopropyl 500 3

Strata Alumina-N Phenomenex Neutral alumina Proprietary 500 3

Table S2: Optimised atmospheric pressure chemical ionization (APCI) and mass

spectrometric conditions for the chosen explosive analytes.

Negative ion mode Positive ion modeSheath gas flow rate [N2, L min-1] 50 50

Auxiliary gas flow rate [N2, L min-1] 5 5Sweep gas flow [N2, L min-1] 0 0

Discharge current [µA] 20 10Capillary temperature [°C] 250 250

Capillary voltage [kV] -25 25Tube lens voltage [V] -55 50Skimmer voltage [V] -18 18

Vaporiser temperature [°C] 300 300

S-3

Table S3: Properties of the chosen subset (n=14) of explosives analytes.

Compound Molecular Weight(g/mol)

No. of Hydrogen Bond Donors

No. of Hydrogen

Bond Acceptors

No. of Aromatic

Rings

LogS (pH = 7.00)

LogS (pH = 2.00)

LogP LogD (pH = 7.00)

LogD (pH = 2.00)

pKa (Base)pKa Conf.

limitsAtom No.

HMX 296.16 0 16 0 -2.01 -2.01 -1.71 -1.71 -1.71 -16.32 0.2 2RDX 222.12 0 12 0 -2.23 -2.23 -1.20 -1.20 -1.20 -15.06 0.2 2NG 227.09 0 12 0 -2.42 -2.42 2.41 2.41 2.41ETN 302.11 0 16 0 -3.23 -3.23 3.21 3.21 3.21

EGDN 152.06 0 8 0 -1.66 -1.66 1.51 1.51 1.51PETN 316.14 0 16 0 -3.21 -3.21 3.64 3.64 3.64

3,4-DNT 182.13 0 6 1 -3.00 -3.00 2.15 2.15 2.151,3-DNB 168.11 0 6 1 -2.83 -2.83 1.55 1.55 1.553,5-DNA 183.12 2 7 1 -2.75 -2.74 1.63 1.63 1.62 0.24 0.1 13DMDNB 176.17 0 6 0 -1.31 -1.31 1.82 1.82 1.82HMTD 208.17 0 8 0 -2.47 -1.72 0.13 0.13 -0.62 2.66 0.2 2

-0.18 0.2 7TATP 222.24 0 6 0 -4.06 -4.06 2.16 2.16 2.16TNT 227.13 0 9 1 -3.21 -3.21 1.79 1.79 1.79

1,3,5-TNB 213.1 0 9 1 -3.03 -3.03 1.36 1.36 1.36

S-4

Table S4: Explosives analytes and their ion detected at the highest intensity in HRMS.

Analyte Accurate m/z

Elemental composition

Proposed ion

DMDNB 194.11378 C6H16O4N3+ [M+NH4]+

HMTD 207.09819 C7H15O5N2+ [M+MeOH2-H2O2]+

TATP 89.05963 C4H9O2+ [M-C5H9O4]+

HMX 331.01703 C4H8O8N8Cl- [M+Cl]-

TNB 213.00252 C6H3O6N3- [M]-

3,5-DNA 183.02884 C6H5O4N3- [M]-

PETN 350.98288 C5H8O12N4Cl- [M+Cl]-

EGDN 61.98839 NO3- [NO3]-

NG 61.98839 NO3- [NO3]-

ETN 61.98839 NO3- [NO3]-

TNT 227.01862 C7H5O6N3- [M]-

3,4-DNT 182.03334 C7H6O4N2- [M]-

1,3-DNB 168.01727 C6H4O4N2- [M]-

RDX 257.00494 C3H6O6N6Cl- [M+Cl]-

NB 107.03773 C6H5ON- [M-O]-

4-NT 136.0405 C7H6O2N- [M-H]-

2,6-DNT 182.03363 C7H6O4N2- [M]-

DPA 170.09650 C12H12N+ [M+H]+

DEDPU 269.16510 C17H21ON2+ [M+H]+

1,2-DNB 168.01796 C6H4O4N2- [M]-

2-NT 136.04053 C7H6O2N- [M-H]-

3-NT 121.05334 C7H7ON- [M-O]-

DMDPU 241.13382 C15H17ON2+ [M+H]+

4-Am-2,6-DNT 196.03621 C7H6O4N3- [M-H]-

2,4-DNT 181.02571 C7H5O4N2- [M-H]-

Tetryl 241.02180 C7H5O6N4- [M-NO2]-

2-Am-4,6-DNT 196.03633 C7H6O4N3- [M-H]-

R-Salt 209.01941 C3H6O3N6Cl- [M+Cl]-

1,2-DNG 216.98737 C3H6O7N2Cl- [M+Cl]-

2,6-DA-4-NT 168.07684 C7H10O2N3+ [M+H]+

2,4-DA-6-NT 168.07683 C7H10O2N3+ [M+H]+

S-5

1,3-DNG 61.98836 NO3- [NO3]-

NQ 105.04071 CH5O2N4+ [M+H]+

DEGDN 61.98846 NO3- [NO3]-

DADP 89.05962 C4H9O2+ [M-C2H3O2]+

TEGDN 258.09302 C6H16O8N3+ [M+NH4]+

PYX 621.00659 C17H7O16N11- [M]-

TATB 257.02789 C6H5O6N6- [M-H]-

TMETN 85.02960 C4H5O2- [M-CH4O7N3]-

Picramic Acid 198.01532 C6H4O5N3- [M-H]-

HND 438.99979 C12H5O12N7- [M]-

PGDN 61.98839 NO3- [NO3]-

NM 60.00920 CH2O2N- [M-H]-

Picric Acid 227.98943 C6H2O7N3- [M-H]-

S-6

-35

-15

5

25

45

65

85

DM

DN

B

HM

TD

TA

TP

HM

X

TN

B

3,5-

DN

A

PE

TN

EG

DN

NG

ET

N

TN

T

3,4-

DN

T

1,3-

DN

B

RD

X

LC-A

PC

I-HR

MS

Mat

rix E

ffec

t [%

]

Analy te

Isolute ENV+Strata Alumina-N - Isolute ENV+HyperSep NH2 - Iso lute ENV+HyperSep SAX - Iso lute ENV+

161 ± 19

(c) Topsoil

-50

-40

-30

-20

-10

0

10

20

30

40

50

DM

DN

B

HM

TD

DA

DP

TA

TP

HM

X

TN

B

3,5-

DN

A

PE

TN

EG

DN

NG

ET

N

TN

T

3,4-

DN

T

1,3-

DN

B

RD

X

LC-A

PC

I-HR

MS

Mat

rix E

ffec

t [%

]

Analy te

(d) Cooking Oil Residue

-35

-15

5

25

45

65

85

DM

DN

B

HM

TD

TA

TP

HM

X

TN

B

3,5-

DN

A

PE

TN

EG

DN

NG

ET

N

TN

T

3,4-

DN

T

1,3-

DN

B

RD

X

LC-A

PC

I-HR

MS

Mat

rix E

ffec

t %]

Analy te

177±2

(e) Dirt Residue

-35

-15

5

25

45

65

85

DM

DN

B

HM

TD

TA

TP

HM

X

TN

B

3,5-

DN

A

PE

TN

EG

DN

NG

ET

N

TN

T

3,4-

DN

T

1,3-

DN

B

RD

X

LC-A

PC

I-HR

MS

Mat

rix E

ffec

t [%

]

Analy te

111 ± 19 120 ± 27

(f) Dried Blood

-35

-25

-15

-5

5

15

25

35

45

55

65

75

85D

MD

NB

HM

X

TN

B

3,5-

DN

A

PE

TN

EG

DN

ET

N

TN

T

3,4-

DN

T

1,3-

DN

B

LC-A

PC

I-HR

MS

Mat

rix E

ffec

t [%

]

Analy te

Oasis HLBHyperSep SAX - Oasis HLBHyperSep NH2 - Oasis HLBStrata Alumina-N - Oasis HLB

52 � 31

33 � 25

(a) River water

-35

-15

5

25

45

65

85

DM

DN

B

HM

X

TN

B

3,5-

DN

A

PE

TN

EG

DN

ET

N

TN

T

3,4-

DN

T

1,3-

DN

B

TA

TP

HM

TD

DA

DP

RD

X

LC-A

PC

I-HR

MS

Mat

rix E

ffec

t [%

]

Analy te

(b) Wastewater

Figure S1: LC-APCI-HRMS matrix effects for a selection of probe explosives in six

different matrices after single- and dual-sorbent SPE. Error bars represent the standard

deviations of triplicate extraction experiments. The key for river water and wastewater is

given in (a) and for all other matrices the key is given in (c).

S-7

Figure S2: Recoveries of a selection of n=14 chemically diverse explosives from a range

of sample types using single- and dual-sorbent SPE combinations. Dirt residue represents

swabs of road traffic signs in London, UK. Error bars represent the standard deviations of

triplicate recovery experiments.

S-8

Figure S3: Differential LC-HRMS full-scan ion plots in positive (left) and negative (right) ionisation mode showing

all additional ions that were removed by the best dual-sorbent combination for each of the

S-9

Figure S4: Positive mode LC-HRMS full-scan ion plots showing single (left) versus dual-

SPE (right) for each sample type.

S-10

Figure S5: Negative mode LC-HRMS full-scan ion plots showing single (left) versus dual-

SPE (right) for each sample type.

S-11

Sampling and pre-treatment procedures for the selected matrices

Aqueous samples

River water samples were collected in 500 mL Nalgene bottles, which had been rinsed

thrice with river water, and stored in the dark at -20 °C until analysis. Before SPE, samples

were defrosted, pooled, filtered under vacuum using GF/F glass microfibre filters (4.7 cm

i.d., Whatman, Buckinghamshire, UK) and 100 mL aliquots were taken for SPE.

Once at the laboratory, all wastewater samples were acidified to pH 2 using HCl (37 %

w/v, to minimise microbial activity) and frozen until analysis. For analysis, wastewater was

defrosted and prepared in the same way as river water samples. For the assessment of

matrix effects and recoveries, equal aliquots of all six wastewater samples were pooled to

form a representative matrix across the week. Where necessary, samples were spiked

with a standard mix of explosives prior to SPE. Also, for application of the method to

explosives screening, a single wastewater sample taken on the 11th March 2016 was

extracted and analysed, as above.

Topsoil

For soil analysis, 5 g of standardised topsoil was weighed and transferred into a ball mill

extraction cartridge with a glass bead (IKA, Oxford, UK). 20 mL of ethanol:water (50:50

v/v) were added and the sample extracted for 10 min at 3200 rpm (optimised). After being

left to settle for 10 min, approximately 10 mL of supernatant were transferred into a 20 mL

volumetric flask and diluted to 20 mL with ultrapure water. For calculation of recoveries,

soil samples were spiked after weighing with a 5 mg L -1 standard mix of analytes in

acetonitrile (2.5 µg g-1 soil) and left to dry in air before the addition of extraction solvent.

Blood, dirt- and oil-based samples/residues

The matrix-containing swabs were prepared for SPE according to the FEL Standard

S-12

Operating Procedure for the Use and Extraction of Swabs, as follows. A cotton wool swab

was wetted with ethanol:water (50:50 v/v) and, using forceps, one side then the other was

lightly wiped across the surface being examined. The swab was placed in the glass vial

containing 5 mL ethanol:water, from which it had been wetted, then agitated and

compressed thoroughly within the solvent using a glass Pasteur pipette (~1 min/side). The

solvent was then drawn up through the swab with the pipette and transferred into a 20 mL

volumetric flask. Another 5 mL ethanol:water were added to the swab and the agitation

and transfer process repeated. The resulting extract (~10 mL) was diluted to 20 mL in a

volumetric flask with water and transferred to a clean, dry Nalgene bottle. This dilution step

is an addition to the FEL protocol in an attempt to further increase recoveries.

S-13

Solid Phase Extraction (SPE)

SPE of liquid samples

Cartridges were conditioned with methanol (5 mL) and ultrapure water (10 mL) before the

100 mL sample was loaded at 5 – 10 mL min-1. Cartridges were washed with 5 mL

ultrapure water, dried for 10 min under vacuum and eluted with 2.5 mL acetonitrile.

Extracts were transferred to 2 mL septum capped crimped vials and stored at -20 °C until

analysis (Agilent Technologies, Cheshire, UK).

Optimisation of SPE procedure for wastewater

Briefly, and according to Rapp Wright et al., the sorbent selected here (Oasis HLB)

followed a thorough evaluation of 34 different commercially available cartridges [1]. This

broadly applicable phase is a copolymer of divinylbenzene and n-vinylpyrrolidone, which

incorporates both polar and non-polar character to the extraction sorbent, as well as

facilitating pi-pi stacking and induced dipole interactions. Several additional conditions

were systematically optimised including elution solvent type (methanol, acetonitrile and

ethyl acetate) and volume (1-8 mL), evaporation (either drying and reconstitution in mobile

phase or not to assess losses due to analyte volatility), sample volume (10-1000 mL) and

pH (pH 2 and 7).

S-14

Determination of recovery and MS detection matrix effect

Recoveries of the selected analytes from model solutions were determined by comparing

samples of ultrapure water spiked at 250 µg L -1 (HPLC-UV) or 25 µg L-1 (HPLC-HRMS)

(n=6 for Oasis HLB, n=3 for other sorbents) to a standard solution of analytes in

acetonitrile at the theoretical final concentration for 100 % recovery (concentration factor =

40). Recoveries from river- and wastewater (HPLC-HRMS) were evaluated by comparing

samples spiked with a standard solution of the explosives (25 µg L-1) to a matrix-matched

standard prepared at the expected concentration for 100 % analyte recovery (n=3).

Recoveries from soil (HPLC-UV) were calculated by comparing samples spiked with a

standard mix of analytes in acetonitrile (2.5 µg g-1) with a standard mix at a concentration

corresponding to theoretical 100 % recovery (n=3).

References

[1] H. Rapp-Wright, G. McEneff, B. Murphy, S. Gamble, R. Morgan, M. Beardah, L.

Barron, Suspect screening and quantification of trace organic explosives in

wastewater using solid phase extraction and liquid chromatography-high resolution

accurate mass spectrometry, Journal of Hazardous Materials 329 (2017) 11-21.

S-15