PRODUCTION OF SEIZED DRUG 

ANALYSIS STANDARDS BY INKJET 

PRINTING 

Jeanita S. Pritchett1, Jeffrey A. Lawrence2, Karen W. Phinney3 , and Jennifer R. 

Verkouteren2

National Institute of Standards and Technology

1.Chemical Science Division 2. Materials Measurement Science 

Division 3. Biomolecular Measurement Division

100 Bureau Dr. Gaithersburg MD, 20899
Introduction

 The availability of standards of illicit/designer drugs to help confirm the identity of drugs of 

abuse poses a challenge for forensic laboratories1.

 Limited number are available commercially, and even fewer can be obtained as certified 

reference materials (CRMs).

 Due to this, laboratories must often prepare in-house reference standards (by chemical 

synthesis or through isolation of the desired compound from seized materials)2.

 Each laboratory must confirm the identity of its in-house standard and demonstrate their suitability for 

the intended purpose

 Lack of readily available reference standards has lead to identification efforts based 

primarily on mass spectrometry; however, these approaches have limited applicability 

because of the potential for different compounds to yield nearly identical mass spectra3.

 Currently, NIST does not offer any pure standards for seized drugs but does have a number 

of matrix-based Standard Reference Materials (SRMs) for drugs of abuse in hair, blood, and 

urine.
Objectives

 The goal of the proposed research is to produce cost 

effective, easy to use seized drug standards by inkjet 

printing technology for analysis by LC-MS(/MS) or GCMS.

 If successful, this method could be expanded to the 

development of quantitative standards for drug 

analysis and to the production of materials for quality 

assurance programs.

 This work would also serve as a foundation for possible 

NIST reference material (RM or SRM) development in 

the future.
Inkjet Printing Technology

 Piezoelectric drop-on-demand (DoD) inkjet printing 

is a versatile method for the quantitative delivery of 

micro volumes of solution4.

 Inkjet printing technology will be used to deposit 

known amounts (nanoliter-picoliter volumes) of pure 

substances (illicit drugs), or mixtures of substances 

onto inert substrates.

 At the time of use, the compounds will be desorbed 

by a small amount of solvent, then used in analysis.
Inkjet Printing Schematic

Pressure Control 

System

Movable stage

Dried blood 

spot card

DoD dispenser (50 μm 

orifice)

Piezoelectric waveform 

generator

Computer

Sample 

reservoir 

Optimized parameters:

Standard singular wave

•Voltage pulses (dwell voltage=32.0 V; rise 

time=4 μs; dwell time=30 μs; fall time = 4 μs)

•Frequency: 500 Hz

•Average mass : ~60 ng

•Average volume per drop = ~75 pL

•Average ejection velocity ~2.0 m/s

•Uncertainties of drop dispensed: 1 % RSD (by 

mass) which corresponds to1% RSD by volume 

and 0.3% RSD by diameter
Inkjet printing apparatus 4 single use samples printed on a dried 

blood spot card 

(Dimensions per spot: 2.4 mm x 2.6 mm)

Sample 

reservoir 

DOD dispenser
Benefits of Inkjet Printed Standards

 Noncontact, high-throughput deposition of precise quantities (nanoto picoliter deposits)5

.

 Reproducibility of optimized inkjet printers have been reported 

better than 1% relative standard deviation for day-to-day 

measurements6.

 Dynamic ranges in deposited analyte concentrations (105) can be 

achieved by varying the number of drops printed5. 

 Very small amount of compound needed for each printed standard 

(cost effective).

 Single use materials reduce potential contamination from multiple 

uses of the same batch of material.
Application of Inkjet Printing 

Technology

 Has been applied to diverse range of applications

 Printing of DNA microarrays 7

 Dispensing metallic solder for interconnects in the 

electronic industry8

 Production of RDX (1,3,5-trinitro-13,5 triazcyclohexane) 

test materials for trace level explosive analysis to 

evaluate ion mobility spectrometers 5.
Factors to consider 

1. Identification of suitable substrates and solvents 

for printing

2. Determination of quantities of drug compounds 

that need to be deposited for extraction and use 

with LC-MS and/or GC-MS

3. Efficiency of release of the printed substances 

from the substrate

4. Stability of printed compounds on different 

substrates and under varying storage conditions

Preliminary work will focus on Methamphetamine (MAMP), a common drug 

of abuse, to access feasibility before investigating other substances
Substrates, solvents, and extraction 

methods

 Substrates

 Teflon 

 Whatman cards

 DMPK-A, B, C

 DMPK-A, B, C-IND

 Deposition solvents

 Isobutanol

 Isobutanol: Methanol (9:1) 

(solubility issues)

 Stock solution (100 ng/μL)

 Nominal masses of 10 ng, 50ng, 

and 500 ng were first tested.

 Extraction solvents

 MeOH

 IPA

 0.2 N H2SO4

Transparent teflon film with Al foil 

backing
Desorption Methods

•Vortex-mix (1hr)9

•Remove (cotton swab, teflon, or card)

•Evaporate solvent to dryness under N2 at 40 oC (add 

50 μL MeOH: conc HCl (9:1))

•Transfer to clean centrifuge tube with ethyl acetate 

(precipitate excess cellulose fibers)

•Centrifuge; filter supernatant; evaporate to dryness.

•LC-MS: reconstitute in 100 μL Solvent AAnalyze in 

SIM mode (150 and 155 m/z monitored)

•GC-MS-derivatize with HFAA (30 min at 65 oC

Analyze in SIM mode (254 and 258 m/z monitored).

Cotton swab (spike in IS)

Peel and submerge (spike in IS)

Cut and submerge (solvent contains IS)
LC-MS parameters for MAMP analysis

 LC/MSD with multimode electrospray

ionization in the positive mode.

 Column: Phenomenex Luna C18 15 cm x 2.0 

mm; 5μm); 20 oC; 0.25 mL/min flow rate

 Gradient : Initially 0.1% acetic acid in H2O: 

MeOH (92:8) for 3 min, ramped to 90:10 at 

10 min, then ramped back to 92:8 at 15 

min, and equilibrated for 5 min.

 MSD Parameters:

 Fragmentor: 70 V

 Drying gas: 12 L/min; 350 oC

 Nebulizer Pressure: 25 psig

 Vaporizer temperature: 150 oC

 Capillary Voltage: 3500 V

MAMP (150 m/z)

MAMP-d5(155 m/z)

Reconstructed selected ion chromatograms for 

MAMP calibrant (1:1)
GC-MS parameters for MAMP analysis

 HP 5973 Mass Selective Detector

 Column: DB-5MS capillary column 

(0.25 mm x 30 m x 0.25 μm); splitless

injection

 Temperature program: 120 oC (initial), 

1 min (hold time), 5o C/min (heating 

rate) to 150 oC, 8 min (hold time) for a 

total run time of 15 min.

 Monitoring (m/z 254 and 258) began 

at 7 min.

7 . 1 0 7 . 2 0 7 . 3 0 7 . 4 0 7 . 5 0 7 . 6 0 7 . 7 0 7 . 8 0 7 . 9 0

0

5 0 0 0 0

1 0 0 0 0 0

1 5 0 0 0 0

2 0 0 0 0 0

2 5 0 0 0 0

3 0 0 0 0 0

T i m e - - >

A b u n d a n c e

I o n 2 5 4 . 0 0 ( 2 5 3 . 7 0 t o 2 5 4 . 7 0 ) : 3 . D

 7 . 3 3

7 . 1 0 7 . 2 0 7 . 3 0 7 . 4 0 7 . 5 0 7 . 6 0 7 . 7 0 7 . 8 0 7 . 9 0

0

5 0 0 0 0

1 0 0 0 0 0

1 5 0 0 0 0

2 0 0 0 0 0

2 5 0 0 0 0

3 0 0 0 0 0

T i m e - - >

A b u n d a n c e

I o n 2 5 8 . 0 0 ( 2 5 7 . 7 0 t o 2 5 8 . 7 0 ) : 3 . D

 7 . 2 7

MAMP (254 m/z)

MAMP-d5(258 

m/z)
Summary of recovery from Teflon

~Amount 

Deposited

MeOH Cotton 

Swab 

(ng/sample) Recovery %

~Amount 

Deposited

IPA Cotton Swab 

(ng/sample) Recovery %

~Amount 

Deposited Submerged in 

MeOH 

(ng/sample)

% 

Recovery

10 ng 0.83 8.29 10 ng 0.13 1.26 10 ng ND

50 ng 6.64 13.29 50 ng 7.83 15.66 50 ng ND

500 ng 210.61 42.12 500 ng 179.09 35.82 500 ng 308.60 61.72

Important notes:

1. Recoveries from the 10 ng and 50 ng samples are very low; decided not to proceed with low concentration 

samples (All subsequent printing was done to yield 500 ng deposits).

2. While submersion of teflon into solvent provided higher recoveries compared to the cotton swab method, the 

adhesive backing lead to undesirable, inconsistent chromatograms.

3. MeOH, has slightly higher recovery than IPA (similar to literature).
Recovery from Whatman cards

Card Stocks

3 mL H2SO4 desorption 

(ng/sample) Recovery % Card Stocks

3 mL MeOH Desorption 

(ng/sample) Recovery %

DMPK-A 275.6694502 55.13389005 DMPK-A 277.643265 55.52865299

DMPK-B PPT NA DMPK-B 378.4267106 75.68534211

DMPK-C 362.8086883 72.56173766 DMPK-C 353.7828365 70.75656729

DMPK-A-IND PPT NA DMPK-A-IND 257.6502374 51.53004748

DMPK-B-IND PPT NA DMPK-B-IND 406.4521899 81.29043797

DMPK-C-IND PPT NA DMPK-C-IND 366.6126089 73.32252178

•500 ng DMPK-B-IND deposits was chosen because it yielded the highest recoveries and permitted visualization 

of the spots.

•Intraday and Interday precisions are both better than 4% RSD. PPT from 

H2SO4

extraction
Storage Conditions

 Immediately after printing, 

cards were placed in either 

tin storage containers or N2-

filled storage bags

 Stored at -20 oC (freezer), 4 

oC (refrigerator), 25oC (room 

temperature), and 37 oC

(incubator)

 Samples were analyzed at 

various time points 

 (ie < 1week after printing, 1 

month, 2 months etc...)

Tin 

Storage 

Container N2

-filled PET lined 

foil bag
MAMP stability results by LC-MS

0

100

200

300

400

500

600

t= < 1 week t=1 month t= 2 months

Tin Storage Containers (n=3)

25 o C

4 oC

37 oC

-20 oC

Nanograms Recovered

Sampling Time

0

100

200

300

400

500

600

t= < 1 week t=1 month t= 2 months

N2Storage Containers; (n=3)

25 oC

4 oC

37 oC

-20 oC

Nanograms Recovered

Sampling Time
MAMP stability results by GC-MS

0

100

200

300

400

500

600

t= < 1 week t=1 month t= 2 months

Tin Storage Containers (n=3)

25 oC

4 oC

37 oC

-20 oC

Nanograms Recovered

Sampling Time

0

100

200

300

400

500

600

t= < 1 week t=1 month t= 2 months

N2Storage Container (n=3)

25 oC

4 oC

37 oC

-20 oC

Nanograms Recovered

Sampling Time
Stability of MAMP at various storage 

conditions

Tin (LC-MS) 1 month 2 months

25 oC 61.7 45.6

4 oC 80.2 63.7

37 oC 50.8 34.1

-20 oC 76.3 68.6

Stability was assessed in triplicate at all storage conditions; the results are expressed as % 

of initial concentration

N2 (LC-MS) 1 month 2 months

25 oC 64.9 59.5

4 oC 132.5 68.1

37 oC 53.6 74.1

-20 oC 74.9 111.1

Tin (GC-MS) 1 month 2 months

25 oC 69.2 62.6

4 oC 78.0 83.3

37 oC 60.6 56.2

-20 oC 84.8 109.2

N2(GC-MS) 1 month 2 months

25 oC 72.9 74.7

4 oC 122.4 86.9

37 oC 55.3 65.9

-20 oC 83.5 126.6
MAMP stability discussion

 Appears that storage at 4oC and -20oC mitigates 

the degree of degradation of MAMP on the 

DMPK-B-IND cards.

 Reduction trend similar to Saussereau et. al. report 

that observed~50-59% (4 oC) and ~85-87% (-20oC) 

of MAMP remained in dried blood spots on filter 

paper after 6 months compared to the initial 

measurement10.

 The data is too preliminary to determine if there is 

benefit to sealing in the N2-filled storage bag.

 Sample analysis will continue to determine if a more 

definitive trend exists.
MAMP stability discussion

 Appears that storage at 4oC and -20oC mitigates 

the degree of degradation of MAMP on the 

DMPK-B-IND cards.

 Reduction trend similar to Saussereau et. al. report 

that observed~50-59% (4 oC) and ~85-87% (-20oC) 

of MAMP remained in dried blood spots on filter 

paper after 6 months compared to the initial 

measurement10.

 The data is too preliminary to determine if there is 

benefit to sealing in the N2-filled storage bag.

 Sample analysis will continue to determine if a more 

definitive trend exists.
Amphetamine on DMPK cards

 Proceeded to determine if amphetamine would be a suitable analyte for this methodology.

 Deposited, desorbed, and analyze using the same procedure as MAMP.

 LC-MS and GC-MS parameters were identical as those used for MAMP except for the SIM ions.

Reconstructed selected ion 

chromatograms for AMP calibrant

(1:1) by LC-MS

AMP-d3

(139 m/z)

AMP

(136 m/z)

5 . 0 0 5 . 1 0 5 . 2 0 5 . 3 0 5 . 4 0 5 . 5 0 5 . 6 0 5 . 7 0 5 . 8 0 5 . 9 0

0

20000

40000

60000

80000

100000

120000

140000

T i m e - - >

A b u n d a n c e

I o n 2 4 0 . 0 0 ( 2 3 9 . 7 0 t o 2 4 0 . 7 0 ) : 1 1 2 0 1 2 2 0 . D

 5 . 4 5

5 . 0 0 5 . 1 0 5 . 2 0 5 . 3 0 5 . 4 0 5 . 5 0 5 . 6 0 5 . 7 0 5 . 8 0 5 . 9 0

0

20000

40000

60000

80000

100000

120000

140000

T i m e - - >

A b u n d a n c e

I o n 2 4 4 . 0 0 ( 2 4 3 . 7 0 t o 2 4 4 . 7 0 ) : 1 1 2 0 1 2 2 0 . D

 5 . 4 0

Reconstructed selected ion 

chromatograms for AMP calibrant

(1:1) by GC-MS

AMP (240 m/z)

AMP-d5

(244 m/z)
AMP stability results by LC-MS

0

10

20

30

40

50

60

70

80

90

100

t= < 1 week t=1 month t= 2 months

Nanograms recovered

Sampling Time

Tin storage containers (n=3)

25 oC

4 oC

37 oC

-20 oC

0

10

20

30

40

50

60

70

80

90

100

t= < 1 week t=1 month t= 2 months

Nanograms Recovered

Sampling Time

N2storage containers (n=3)

25 oC

4 oC

37 oC

-20 oC

Recoveries from the initial measurement are much lower than MAMP (~15% vs ~80%)
AMP stability results by GC-MS

0

20

40

60

80

100

120

140

t= < 1 week t=1 month t= 2 months

Nanograms Recovered

Smapling Time

Tin Storage Container (n=3)

25 oC

4 oC

37 oC

-20 oC

0

10

20

30

40

50

60

70

80

90

100

t= < 1 week t=1 month t= 2 months

Nanograms Recovered

Sampling Time

N2Storage Containers (n=3)

25 oC

4 oC

37 oC

-20 oC
Stability of AMP at various storage 

conditions

Tin (LC-MS) 1 month 2 months

25 oC 95.8 33.8

4 oC 105.8 50.8

37 oC 47.8 11.8

-20 oC 92.4 57.4

Stability was assessed in triplicate at all storage conditions; the results are expressed as % 

of initial concentration

N2 (LC-MS) 1 month 2 months

25 oC 88.7 31

4 oC 96.5 54.7

37 oC 33.7 9.9

-20 oC 97.9 62.3

Tin (GC-MS) 1 month 2 months

25 oC 80.7 59.27

4 oC 129.9 87.7

37 oC 54.6 30.6

-20 oC 120.5 100.3

N2(GC-MS) 1 month 2 months

25 oC 100.1 71.9

4 oC 100.0 94.4

37 oC 38.4 34.1

-20 oC 110.9 99.9
AMP stability discussion summary

 Recovery of AMP from the cards appeared to be extremely low.

 Attributed this to crystallization in the print solution, which lead to a lower 

amount actually being deposited

 Despite this, when looking at relative amounts recovered over time, AMP 

appears to be more stable at 4oC and -20oC 

 Reduction trend similar to Saussereau et. al. report that observed~56-60% (4 

oC) and ~86-96% (-20oC) of AMP remained in dried blood spots on filter 

paper after 6 months compared to the initial measurement10.

 A new stock solution of AMP was prepared and used for printing 

immediately after preparation.

 Recovery was improved; however, it was still less than desired (>80%).

 Other extraction solvents will be explored to improve release efficacy of AMP 

from the DMPK-B-IND cards. 
Summary/ Future Work

 This work has demonstrated the feasibility of utilizing 

inkjet printing technologies to produce single use, illicit 

drug testing material.

 Further characterization of the test materials will 

continue to better understand the stability of these 

compounds over time as well as efficacy of release 

from the printing substrates.

 In the future, this study will be expanded to include 

other illicit drugs (ie cocaine) and mixtures of drugs to 

serve as a foundation for potential NIST reference 

materials for seized drug analysis.
References

1. Laks, S., Pelander, A., Vuori, E., Ali-Tolppa, E., Sippola, E., and Ojanpera, I., Anal. Chem., 76 

(2004) 7375-7379.

2. ENFSI Guidelines on the Use of Reference Materials in Forensic Drug Analysis, available 

from www. Enfsi.eu

3. Maher, H.M., Awad, T., DeRuiter, J., and Clark, C.R., J. Chromatogr. Sci., 50 (2012)1-9.

4. Lee, E. Microdrop Generation; CRC Press: Boca Raton, FL, 2003.

5. Windsor, E., Najarro, M., Bloom, A., Benner, Jr., Fletcher, R., Lareau, R., and Gillen, G., Anal. 

Chem., 82 (2010) 8519-9524

6. Verkouteren, R.M. and Verkouteren, J.R. Langmuir 2011, 27, 9644-9653.

7. Theriault, T. P.; Winder, S. C.; Gamble, R. C. DNA Microarrays-A Practical Approach; Schena, 

M., Ed.; Oxford University Press, Oxford,1999; Chapter 6.

8. Hayes, D. J.; Wallace, D. B.; Cox, W. R. Proc. IMAPS Int. Conf. High Density Packag. MCMs 

1999, 242.

9. Procedure for Whatman DMPK Cards found at 

(http://www.whatman.com/References/Initial%20Protocol%20for%20Whatman%20DMPK

%20Cards%2019May09.pdf)

10. Saussereau, E.; Lacroix, C.; Gaulier, J.M.; Goulle, J.P. Journal of Chromatography B, 885-

886(2012)1-7

Funding:

National Research Council Postdoctoral Fellowship

NIST Forensics Measurement Challenge 2012