An Evaluation of 25B-, 25C-, 25D-, 25H-, 25I- and 25T2-NBOMe via LC–MS-MS: MethodValidation and Analyte Stability

Robert D. Johnson1*, Sabra R. Botch-Jones2, Tiffany Flowers1 and Connie A. Lewis1

1Tarrant County Medical Examiner’s Office, 200 Feliks Gwozdz Pl, Fort Worth, TX 76104, USA, and 2FTox Consulting,

12 Green Street #1, Woburn, MA 01801, USA

*Author to whom correspondence should be addressed. Email: rdjohnson@tarrantcounty.com

As potent serotonin (5-HT2A) receptor agonists, the NBOMe class ofdrugs including 25B-, 25C-, 25D-, 25H-, 25I- and 25T2-NBOMe is fre-quently abused due to the intense hallucinations that they induce.From the limited literature available, the concentration of theseNBOMe compounds reported in postmortem cases is exceedinglylow. In most instances, published concentrations are <0.50 ng/mL.Therefore, the need for a sensitive, rapid and comprehensive analyt-ical method for the quantification of these compounds was evident. Inaddition to the more publicized analog 25I-NBOMe, evaluation of 25B-,25C-, 25D-, 25H and 25T2- in whole blood, plasma and urine was con-ducted. This publication presents the data obtained from the valida-tion of a liquid chromatography–tandem mass spectrometry methodfor the simultaneous quantification of these six NBOMe analogs. Themethod utilizes ultra-performance liquid chromatography technologyfor the separation followed by positive electrospray ionization of eachanalog. Limits of quantification for these analogs ranged from 0.01 to0.02 ng/mL (10–20 pg/mL) with typical linear dynamic ranges of0.01–20 ng/mL. Data for recovery, intraday control accuracy and pre-cision, matrix effects, ion suppression/enhancement and analytestability are included. Validation was completed in whole blood, plas-ma and urine. Short run times and high sensitivity afforded by thisnewly validated analytical method that allows for the detection ofthese six analogs in the most common toxicological matrices andcan be applied to both ante- and postmortem specimens.

Introduction

As occurrence of synthetic compounds such as N-benzylpipera-

zine, 4-methyl-N-methyl-cathinone (mephedrone) and 3,4-

methylenedioxypyrovalerone (MDPV) decline in our laboratory,

others like ‘N-Bomb’s’ (NBOMe) have increased in prevalence.

The successful validation of a liquid chromatography–tandem

mass spectrometry (LC–MS-MS) method for the detection of

these compounds is the focus of this manuscript as we are

now encountering these drugs in both human performance

and postmortem toxicology cases. 2-(4-Iodo-2,5-dimethoxy-

phenyl)-N-[(2-methoxyphenyl)methyl]ethanamine (25I-NBOMe)

is just one of many of the 2C dimethoxyphenyl-N-[(2-methoxy-

phenyl)methyl]ethanamine derivatives that have been encoun-

tered in the Tarrant County Medical Examiner’s Office.

Obtained through the now typical channels, that is, the internet

or drug paraphernalia shops and the more traditional route, a

local dealer, they can be found as liquid solutions, powders, on

blotter paper or laced on other substances intended for

ingestion. In November 2013, the US Drug Enforcement

Administration made three of these compounds (25I-, 25C- and

25B-NBOMe) Schedule I, illegal drugs under the Controlled

Substances Act for the next 2 years due to the lack of approved

medical use or for human consumption (1). The ingestion of

these compounds has either caused or been a contributing factor

in the deaths of numerous individuals with at least 19 deaths in

the US between March 2012 and August 2013 (1, 2).

Sharing a core phenethylamine structure, the 2C-substances

(2C-I, 2C-C and 2C-B) differ from the NBOMe compounds

by the addition of a 2-methoxybenzyl group on the nitrogen.

As potent serotonin (5-HT2A) receptor agonists, 25I-NBOMe,

25C-NBOMe, 25B-NBOME, 2C-I, 2C-C and 2C-B can impair work-

ing memory and cognitive processes (2). These compounds are

abused, in part, because they cause intense hallucinations due to

the stimulation of the 5-HT2A discussed previously. (1) The hal-

lucinogenic effects experienced following use are due in part to

the effect that they have on the 5-HT2A receptors. Per the

American College of Medical Toxicology’s Case Registry, in

2012, several of the participating sites across the USA reported

cases of NBOMe exposure (3). It is expected that the number

of reported cases will increase as the drugs become more

prevalent.

From the limited literature available, the concentration of

these NBOMe compounds seen in postmortem cases is exceed-

ingly low (1–3). In most instances, published concentrations are

,0.50 ng/mL (4). Therefore, the need for a sensitive, rapid and

comprehensive analytical method for the quantification of these

compounds was evident. Using recent work published by several

groups as a guide, we have expanded the number of NBOMe

compounds and present a validated LC–MS-MS method applica-

ble to numerous matrices (2, 4–6). We have also taken what we

previously learned in the evaluation of other new psychoactive

substances to examine these compounds more fully during the

validation process (2, 7). In addition to the more publicized ana-

log 25I-NBOMe, evaluation of 25B-, 25C-, 25D-, 25H and 25T2- in

whole blood, plasma and urine was conducted. We report the

evaluation of the analytical method and an examination into

the postextraction stability of these compounds.

Materials and methods

Chemicals and solutions

All aqueous solutions were prepared using deionized water (DI)

that was obtained by a US Filter water purification system (US

Filter Corporation, Snellville, GA, USA). All chemicals were pur-

chased in the highest possible purity and used without any

further purification. 25B-NBOMe, 25C-NBOMe, 25D-NBOMe,

25H-NBOMe, 25I-NBOMe and 25T2-NBOMe were obtained by

Lipomed Reference Standards (Lipomed, Inc., Cambridge, MA,

USA). The internal standard MDPV-d8 was purchased from

Cerilliant (Cerilliant Corporation, Round Rock, TX, USA).

Methanol, acetonitrile containing 0.1% formic acid, ammonium

hydroxide, acetic acid, potassium phosphate monobasic and

# The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Journal of Analytical Toxicology 2014;38:479–484

doi:10.1093/jat/bku085 Special Issue

hydrochloric acid were purchased from Fisher Scientific

(Pittsburgh, PA, USA). Formic acid (97%) was purchased from

MP Biomedicals (MP Biomedicals, Inc., Santa Ana, CA, USA).

When necessary, the pH of a solution was measured using a

Corning model 430 pH meter (Corning Life Sciences, Acton,

MA, USA) connected to a Corning 3-in-1 model pH electrode.

Two separate 10 mL stock solutions of the six NBOMe analogs

were prepared independently at 100 ng/mL in methanol. Each of

these stock solutions was derived from a unique lot of metha-

nolic standard obtained from the manufacturer. These two

stock solutions were subsequently identified as calibrators and

controls. MDPV-d8 was employed as the internal standard for

these experiments and was prepared at a concentration of

1,000 ng/mL in 10 mL of methanol.

The aqueous portion of the ultra-performance liquid chroma-

tography (UPLC) mobile phase was 2.0 mM ammonium formate

containing 0.2% formic acid. The primary organic component of

the mobile phase was HPLC grade acetonitrile containing 0.1%

formic acid. The elution gradient employed for these experi-

ments utilized a mixture of these components at an initial ratio

of 60 : 40. This ratio was adjusted to 5 : 95 (aqueous buffer : ace-

tonitrile containing 0.1% formic acid) at 4.00 min and returned to

60 : 40 (aqueous buffer : acetonitrile containing 0.1% formic

acid) at 4.01 min. An equilibration time of 0.50 min was added

to the end of this gradient elution profile for a total HPLC run

time of 4.51 min.

Instrumentation

Analyte separation was achieved using a Shimadzu Nexera UPLC

system (Shimadzu, Kyoto, Japan) equipped with an Allurew PFP

guard column (10 � 2.1 mm i.d.) from Restekw (Restek

Corporation, Bellefonte, PA, USA), followed immediately by a

Phenomenex Kinetexw PFP (50 � 2.1 mm i.d., 1.7 mm particles)

analytical column (Phenomenex, Torrance, CA, USA).

Identification and quantitation were accomplished using an AB

Sciex 4000 QTrapw hybrid triple quadrupole LC–MS-MS system

(AB Sciex, Framingham, MA, USA) that utilized nitrogen as the

sheath and curtain gas. Control of the UPLC system, integration

of the chromatographic peaks and communication with the

mass spectrometer were accomplished using Analystw software

version 1.6 (AB Sciex).

LC–MS-MS and LC–MS-MS-MS methods

For all determinations, the UPLC was operated in the gradient

mode (discussed previously) with a flow rate of 0.70 mL/min.

The sample injection volume was 5 mL. The UPLC column was

equilibrated for 30 min prior to use under the initial mobile

phase conditions of the analytical method. Following use, the

column was washed and stored in a 50 : 50 mixture of acetoni-

trile containing 0.1% formic acid : H2O. Initial ionization evalua-

tion of these compounds by direct injection into the 4000

QTrap indicated that positive chemical ionization creating the

[MþH]þ ions was much more effective in signal production

than negative chemical ionization, which formed the [M2H]2

ions. Positive electrospray ionization (ESI)–MS-MS conditions

were optimized separately for each of the seven compounds by

infusing the desired compound at a concentration of �100 ng/mL,

prepared by dilution from the stock solutions using methanol,

into the 4000 QTrap at a constant rate of 5 mL/min. Tuning the

MS for the desired ions and transitions was then accomplished

using the autotune feature of the Analystw software. The instru-

mental operating conditions were as follows: scan type, multiple

reaction monitoring (MRM); ion spray voltage, 5,000 V; entrance

potential, 10 V; collisionally activated dissociation gas, medium;

curtain gas, 10; ion source gas 1, 70; ion source gas 2, 30; source

temperature, 5008C and declustering potential, collision energy

and collision cell exit potential were all compound specific and

set by the instrument during method development.

Calibrator and control preparation

Calibrators were prepared in certified-negative human whole

blood from one set of original stock standard solutions of six

NBOMe compounds. Controls were prepared in a similar manner

to calibrators, using certified-negative human whole blood as

diluent but employing the second set of original stock solutions.

Calibration curves were routinely prepared at concentrations

ranging from 0.01 to 20 ng/mL. A minimum of seven calibrators

were used to construct each calibration curve employed for

quantitation. Controls used for the determination of accuracy, pre-

cision and stability were prepared at 0.04, 0.40 and 1.0 ng/mL.

Controls used to determine the extraction efficiency, or percent

recovery, of this method were prepared at concentrations of 0.10

and 10 ng/mL.

Quantitation of the six NBOMe compounds in samples was

achieved via an internal standard calibration procedure. Peak

area ratios for each compound were determined for every sample

analyzed. The peak area ratio was calculated by dividing the area

of the analyte peak by the area of the internal standard peak.

Calibration curves were derived by plotting a linear regression

of the analyte/internal standard peak area ratio versus the analyte

concentration for each respective calibrator. A weighting factor

of 1/x was utilized for each calibration curve. These calibration

curves were then used to determine the concentrations of the

six NBOMe compounds in controls and specimens.

Sample preparation and extraction procedure

Samples were prepared and extracted in the following manner,

with slight modification to recent publications by Poklis et al.

(1, 2, 4). Five hundred microliter aliquots of calibrators, controls

and specimen fluids were transferred to individual 16 � 100 mm

disposable culture tubes. To each sample, 10 ng of internal stan-

dard was added as 10 mL of the 1,000 ng/mL stock internal stan-

dard solution. Two milliliters of 0.10 M potassium phosphate

buffer (pH 6.0) were added to each tube. The samples were

then vortexed briefly. Centrifugation at 820 � g for 4 min provid-

ed removal of cellular debris. Following centrifugation, the ex-

tracts were transferred to UCT Clean Screenw DAU mixed

mode solid-phase extraction (SPE) columns (UCT, Inc., Bristol,

PA, USA), which had been preconditioned with 1.0 mL methanol,

followed by 1.0 mL 0.10 M phosphate buffer (pH 6.0). Care was

taken not to dry the column prior to extract addition. Column

flow rates of 1–2 mL/min were maintained in each SPE step

using a UCT positive pressure manifold SPE processor with a pos-

itive pressure of 3 psi. Once each sample had passed through its

respective column, the columns were washed with 2.0 mL of DI

water, 1.0 mL of 0.10 M acetic acid and 2.0 mL of methanol. The

SPE columns were then dried completely with 25 psi of positive

pressure for 5 min. The analytes were eluted using 3.0 mL of 2%

ammonium hydroxide in a 80 : 20 mixture of methylene chlo-

ride : isopropanol, which was prepared fresh daily, into 6 mL

480 Johnson et al.

disposable culture tubes containing 200 mL of DI water and

100 mL of 1% HCl in methanol. Eluents were evaporated

under a warm air blower until only �100 mL of water remained.

This aqueous solution was transferred to LC sample vials for

LC–MS-MS analysis.

Recovery

The recovery of each analyte was determined using the following

procedure. Briefly, two groups, X and Y, of controls prepared

using certified-negative human whole blood as the diluent was

extracted in the same manner as described immediately previ-

ously. Group X was spiked with a precisely known amount of

the six NBOMe compounds prior to extraction. Group Y was

spiked with the same precisely known amount of the six

NBOMe compounds following the SPE elution step. Upon analy-

sis, the average response factor obtained fromGroup Xwas divid-

ed by the average response factor obtained from Group Y to yield

the percent recovery value (100 � X/Y ¼ % recovery) for each

of these compounds.

Results and discussion

The procedure described provides a rapid, reproducible and ac-

curate method for the determination of six NBOMe compounds.

Chemical structures of these six compounds can be seen in

Figure 1. This procedure incorporates SPE and LC–MS-MS utiliz-

ing positive ESI ionization in the MRM mode. SPE provided a

cleaner sample than alternative liquid–liquid extraction proce-

dures that may be utilized. This was necessary to achieve the

extremely low detection limits that may be required with this

class of compounds.

Chromatographic peaks for these NBOMe compounds experi-

enced no interference from endogenous sample matrix compo-

nents. This was demonstrated by extracting a sample of

drug-negative matrix using the above-described SPE procedure

and injecting that extracted sample while simultaneously infus-

ing a neat standard of the six NBOMe compounds into the

4000 QTrap. During this process, no significant deviation in the

baseline occurred at the retention time of any of the six NBOMe

compounds. Therefore, neither ion suppression nor ion en-

hancement was determined to be an issue with this procedure.

All analytes were eluted from the column and detected in

,4.5 min utilizing this UPLC method. Figure 2 shows a represen-

tative LC–MS-MS chromatogram. Mass spectral parameters in-

cluding precursor and product ions, declustering potential

(DP), collision energy (CE), and cell exit potential (CXP) for

each of the six NBOMe compounds can be seen in Table I

below. Typical retention times were 2.38, 2.32, 2.34, 1.81, 2.51

Figure 1. Chemical structures of 25B-NBOMe, 25C-NBOMe, 25D-NBOMe, 25H-NBOMe, 25I-NBOMe and 25T2-NBOMe.

25B-, 25C-, 25D-, 25H-, 25I- and 25T2-NBOMe via LC–MS-MS 481

and 2.49 min for 25B-NBOMe, 25C-NBOMe, 25D-NBOMe,

25H-NBOMe, 25I-NBOMe and 25T2-NBOMe, respectively.

The linear dynamic range (LDR), limit of detection (LOD) and

limit of quantitation (LOQ) were initially determined by analysis

of extracted certified-negative human whole blood, plasma

and urine calibrators ranging in concentrations from 0.005 to

40 ng/mL. The LDR for each compound was determined follow-

ing this analysis. The experimentally determined LODs, LOQs

and LDRs for 25B-NBOMe, 25C-NBOMe, 25D-NBOMe, 25H-

NBOMe, 25I-NBOMe and 25T2-NBOMe can be seen in Table II.

The correlation coefficients for each of these calibration curves

exceeded 0.995 when a weighting factor of 1/X was employed.

For this method, the LOD was defined as the lowest concen-

tration of analyte having a minimum signal-to-noise ratio of

five, ion ratios within 20% of the average of the calibrators and

a retention time within 5% of the average of the calibrators.

The LOQ was defined as meeting all LOD criteria, plus having

an experimentally determined value within +20% of its pre-

pared concentration. Table III shows the average recovery for

each compound from whole blood when extracted using this

newly validated procedure at two different concentrations.

Carryover contamination was initially investigated and subse-

quently monitored by the use of solvent blank injections. An ace-

tonitrile blank injected following the highest extracted calibrator

showed no carryover contamination. Subsequently, blanks were

Figure 2. Representative chromatogram of MDPV-d8, 25B-NBOMe, 25C-NBOMe, 25D-NBOMe, 25H-NBOMe, 25I-NBOMe and 25T2-NBOMe in an extracted 1 ng/mL whole bloodquality control sample. Chromatographic peaks represent ions monitored in MRM mode for each compound. Peaks were obtained from a 5 mL injection.

Table ILC–MS-MS Instrument Parameters for Each of the NBOMe Compounds Analyzed

Analyte Precursor ion (m/z) Product ions (m/z) DP CE CXP

25b-NBOMe 1 381.9 121.0 31 33 625b-NBOMe 2 90.9 31 63 1425c-NBOMe 1 336.0 121.0 41 25 825c-NBOMe 1 91.0 41 69 225d-NBOMe 1 316.1 121.0 21 25 625d-NBOMe 2 91.1 21 63 1425h-NBOMe 1 301.9 121.1 21 27 1025h-NBOMe 2 90.9 21 51 625i-NBOMe 1 428.0 121.0 56 31 1425i-NBOMe 2 91.0 56 81 025T2-NBOMe 1 362.2 121.0 96 25 825T2-NBOMe 2 91.0 96 81 0

Table IILOD, LOQ and LDR Data for Six NBOMe Compounds

Analyte LOD (ng/mL) LOQ (ng/mL) LDR (ng/mL)

25B-NBOMe 0.005 0.01 0.01–2025C-NBOMe 0.01 0.02 0.02–2025D-NBOMe 0.005 0.01 0.01–2025H-NBOMe 0.01 0.02 0.02–2025I-NBOMe 0.005 0.01 0.01–2025T2-NBOMe 0.005 0.01 0.01–20

482 Johnson et al.

used throughout the sample sequence to verify that no sample-

to-sample contamination occurred.

Intraday (within day) accuracy and precision were examined

for this extraction. Accuracy was measured as the relative error

between the experimentally determined and target concentra-

tions of a sample. Precision was measured as the relative standard

deviation (RSD) for the experimentally determined concentra-

tions. Whole blood, plasma and urine controls at 1 ng/mL as

well as additional whole blood controls at 0.04 and 0.40 ng/mL

were prepared, extracted and analyzed on the same day. For in-

traday analyses, a calibration curve was extracted along with five

replicates of each control concentration. The intraday relative

error and RSD data can be seen in Tables IV and V. In general, rel-

ative error values ranged from 0.4 to 13% while RSD values

ranged from 1 to 9% for the 1 ng/mL controls extracted from

whole blood, plasma and urine. For the 0.04 and 0.40 ng/mL

whole blood controls, relative error values ranged from 2 to

18% while RSD values ranged from 1 to 6%.

Stability

Sample extracts were stored in a refrigerator at 48C for 7 days and

then reanalyzed to determine the stability of each compound in

the sample vial following extraction and analysis. A new calibra-

tion curvewas extracted and validated with newly extracted con-

trols; this new curve was used to quantify the stored samples. As

can be seen in Table VI, both the relative error and RSD increased

dramatically following storage. This increase was more prevalent

in the whole blood samples. The compound that demonstrated

the most deterioration over the 7-day storage period was

25I-NBOMe. This compound had an RSD of 39% in the whole

blood extract following storage. The mechanism of this deterio-

ration as well as the discrepancy between sample matrices is be-

yond the scope of the current work. However, it is clear that in

an effort to maintain a high degree of accuracy, sample extracts

should be analyzed following extraction and that storing them

for numerous days prior to analysis is not advised.

Conclusion

An LC–MS-MS method that is rapid, reliable and sensitive has

been developed for the identification and subsequent quantita-

tion of six NBOMe compounds in biological specimens. With

LOQs as low as 0.01 ng/mL (10 pg/mL), the method offers suffi-

cient sensitivity to detect any of these compounds following use.

This validation included the most commonly analyzed matrices;

whole blood, plasma and urine, while utilizing a deuterated inter-

nal standard of similar structure to ensure extraction and chro-

matographic characteristics consistent with the analytes of

interest. This method can be readily applied to postmortem or

human performance toxicology cases suspected to involve this

class of drugs.

References

1. Poklis, J.L., Devers, K.G., Arbefeville, E.F., Pearson, J.M., Houston, E.,

Poklis, A. (2014) Postmortem detection of 25I-NBOMe [2-(4-iodo-

2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine] in

fluids and tissues determined by high performance liquid chromatog-

raphy with tandem mass spectrometry from a traumatic death.

Forensic Science International, 234, e14–e20.

Table VIntraday Accuracy and Precision for a 0.04 ng/mL and a 0.40 Extracted Controla

NBOMe 25B 25C 25D 25H 25I 25T2

0.04 ng/mL whole blood controlAverage 0.038 0.036 0.039 0.037 0.038 0.036SD 0.002 0.002 0.002 0.005 0.001 0.001Relative error 5.0 2.5 5.0 5.0 2.5 2.5RSD 6.4 5.3 4.1 1.3 3.7 5.0

0.40 ng/mL whole blood controlAverage 0.358 0.387 0.377 0.393 0.344 0.355SD 0.02 0.03 0.02 0.007 0.02 0.02Relative error 11.5 3.2 3.0 4.0 18 13RSD 6.7 7.5 4.7 1.7 6.5 5.5

an ¼ 5 at each concentration for each compound.

Table VIStability of 1 ng/mL Extracts Following Storage at 48C for 7 Daysa

NBOMe 25B 25C 25D 25H 25I 25T2

Whole bloodAverage 0.648 0.924 0.958 1.076 0.492 0.622SD 0.20 0.19 0.15 0.15 0.19 0.21Relative error 35.2 7.6 4.2 7.6 50.8 37.8CV 31.2 20.2 15.2 13.7 39.0 34.5

PlasmaAverage 0.884 1.002 1.06 1.096 0.786 0.908SD 0.17 0.14 0.14 0.04 0.16 0.19Relative error 11.6 0.2 6.0 9.6 21.4 9.2RSD 19.4 14.2 13.1 4.1 20.2 21.4

UrineAverage 0.977 1.100 1.123 1.020 0.937 0.943SD 0.04 0.02 0.01 0.10 0.06 0.05Relative error 2.3 10.0 12.3 2.0 6.3 5.6RSD 4.2 1.9 0.84 10.0 6.3 5.0

an ¼ 5 at in each specimen type for each compound.

Table IVIntraday Accuracy and Precision for a 1 ng/mL Extracted Controla

NBOMe 25B 25C 25D 25H 25I 25T2

Whole bloodAverage 0.994 0.996 0.906 0.91 0.996 0.958SD 0.09 0.05 0.03 0.02 0.08 0.06Relative error 0.06 0.04 0.94 0.90 0.04 0.42RSD 9.0 5.3 3.0 2.2 8.0 6.0

PlasmaAverage 1.008 1.062 1.02 0.996 0.99 1.012SD 0.04 0.03 0.03 0.01 0.05 0.04Relative error 0.80 6.2 2.0 0.40 1.0 1.2RSD 3.6 3.0 2.7 1.3 5.2 3.7

UrineAverage 1.108 1.136 1.036 1.006 1.124 1.106SD 0.01 0.03 0.03 0.04 0.02 0.02Relative error 10.8 13.6 3.6 0.60 12.4 10.6RSD 1.0 2.9 2.7 3.5 1.9 1.8

an ¼ 5 at in each specimen type for each compound.

Table IIIRecovery Data for Six NBOMe Compounds at 0.10 and 10 ng/mL from Whole Blood

Analyte 0.10 ng/mLa (%) 10 ng/mLa (%)

25B-NBOMe 62 8925C-NBOMe 73 9125D-NBOMe 82 9425H-NBOMe 85 9325I-NBOMe 54 8725T2-NBOMe 74 88

aData presented as the average percent recovery for each compound. n ¼ 5 for each analyte at each

concentration.

25B-, 25C-, 25D-, 25H-, 25I- and 25T2-NBOMe via LC–MS-MS 483

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484 Johnson et al.