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Quantifying Synthetic CannabinoidMetabolites

Single Extraction LC-MS/MS Method for Both Hydroxylated

and Carboxylated Metabolites

By Amanda Rigdon*, Paul Kennedy**, and Ty Kahler*

*Restek Corp., **Cayman Chemical

• Single SPE extraction replaces separate low and high

pH liquid/liquid extractions.

• The Ultra Biphenyl LC column separates positional

isomers that cannot be distinguished by MS/MS.

• Quantify both hydroxylated and carboxylated

JWH-018 and JWH-073 metabolites in urine.

Recent increases in the use of herbal incense containing synthetic

cannabinoids, such as JWH-018 and JWH-073, have resulted in

greater demand for testing. In response, many laboratories are now

developing methods to analyze human urine for these compounds.

Research has shown that the parent molecules are extensively

metabolized prior to excretion [1]; therefore, the more abundant

metabolites are better targets for screening assays.

Major metabolites of JWH-018 and JWH-073 include mono- and di-

hydroxylated, as well as carboxylated, compounds [1,2]. These groups

are generally extracted separately due to differences in their pKa val-

ues. Both present chromatographic challenges: the hydroxylated ana-

lytes exist as multiple positional isomers that are indistinguishable by

MS/MS detectors, and the carboxylated compounds are hydrophilic,

making them difficult to retain using RP-HPLC. Here we show the

analysis of authentic urine samples using a simplified extraction pro-

cedure and a chromatographic method that allows quantification of

clinically relevant metabolites.

Simplified Extraction Speeds up Sample Prep

Previously published methods describe the use of a high pH liquid/

liquid extraction for the analysis of synthetic cannabinoid metabo-

lites [1]. While this is suitable for hydroxylated metabolites, carboxyl-

ated metabolites require a second liquid/liquid extraction at low

pH for adequate recovery. In contrast, the SPE procedure used here

recovers both mono-hydroxylated and carboxylated metabolites. This

SPE extraction procedure allowed authentic samples to be prepared

for analysis quickly using just a single procedure.

Analysis of Positional Isomers and Unknown

Metabolites in Authentic Samples

Many JWH-018 and JWH-073 metabolites are positional isomers,

meaning they have the same molecular weight, share several com-

mon fragments, and must be chromatographically resolved because

they are indistinguishable by MS/MS detectors. The analytical method

used here provides chromatographic separation of all major isomeric

analytes (Figure 1) and was used to determine the clinically signifi-

cant positional isomer metabolites in authentic samples (Figure 2).

Quantitative results for authentic samples are presented in Table I.

All reported values met ion ratio criteria for the first qualifier MRM

transition; however, most results for JWH-018 5-hydroxypentyl did

not meet ion ratio criteria for the second qualifier. To determine if

an interfering compound was coeluting, samples were re-analyzed

using an isocratic method. Results revealed a coeluting peak with

Table I:

Quantitative LC-MS/MS results for JWH metabolites in

authentic urine samples.

*Results did not meet ion ratio criteria (±20%) for the second qualifier MRM transition.

ND = no peak detected

Compounds

Sample 1

(ng/mL)

Sample 2

(ng/mL)

Sample 3

(ng/mL)

Sample 4

(ng/mL)

Sample 5

(ng/mL)

Sample 6

(ng/mL)

JWH-018 N-pentanoic acid

9.9

11.5

22.7

1.5

< 1

44.3

JWH-018 5-hydroxypentyl

+ unknown metabolite

29.5*

14.7*

84.2*

5.4*

1.4*

48.9

JWH-073 4-hydroxybutyl

Unknown metabolite

14.2

35.2

21.6

1.70

< 1

69.7

JWH-073 N-butanoic acid 13.7

1.2

9.3

1.3*

1.4

JWH-018 4-hydroxyindole

JWH-018 5-hydroxyindole

< 1

JWH-018 6-hydroxyindole < 1

1.1

JWH-018 7-hydroxyindole

JWH-073 4-hydroxyindole

JWH-073 5-hydroxyindole

JWH-073 6-hydroxyindole

JWH-073 7-hydroxyindole

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