RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2007; 21: 1339–1342

ublished online in Wiley InterScience (www.in

P

RCM

Letter to the Editor

To the Editor-in-Chief

Sir,

Determination of 11-nor-D9-tetra-

hydrocannabinol-9-carboxylic acid

in hair using gas chromatography/

tandem mass spectrometry in nega-

tive ion chemical ionization mode

Cannabis sativa, also known as hemp, is

one of the most widely abused drugs

worldwide. A dry, pulverized green or

brown mix of flowers and leaves of the

hemp plant is usually smoked in

cigarettes. Cannabis contains more

than 420 chemical substances includ-

ing at least 61 cannabinoids.1 D9-

Tetrahydrocannabinol (D9-THC) is

the most prominent psychoactive

cannabinoid component and is exten-

sively metabolized in humans into

11-hydroxy- and 8-hydroxy-D9-tetra-

hydrocannabinol, and finally to

11-nor-D9-tetrahydrocannabinol-9-car-

boxylic acid (THCCOOH).2–4

Generally, the identification of

D9-THC, cannabidiol, and cannabinol

in decontaminated hair indicates

exposure to cannabis, while the deter-

mination of the major metabolite

THCCOOH is recommended to dis-

tinguish passive exposure from active,

intentional ingestion.5–8 The concen-

tration level of THCCOOH in hair is

lower than that of the parent drug

D9-THC because of the weak incorp-

oration of the acidic metabolite into the

hair matrix.9–11 Due to the aforemen-

tioned reason and matrix-induced

interferences, the analysis of the com-

pound at low concentration levels in

hair has posed a significant technical

challenge.12 The proposed cut-off con-

centration for the detection in hair of

THCCOOH is 0.2 pg/mg from the

Society of Hair Testing and 0.05 pg/

mg under the federal regulations of the

United States.13,14 To satisfy these

analytical requirements, a method

providing high sensitivity, specificity

and data reproducibility is required.

Several mass spectrometric methods

have been reported for the detection of

THCCOOH in hair samples, including

gas chromatography/mass spectrom-

etry with electron ionization (GC/

MS-EI),15 GC/MS with negative ion

chemical ionization (NCI),6,16,17 two-

dimensional GC/MS with electron

capture chemical ionization (ECCI),18

and gas chromatography/tandem

mass spectrometry with negative ion

chemical ionization (GC/MS/MS-

NCI).8,19–21 We have focused on GC/

MS/MSmethods for the determination

of THCCOOH in hair. Analysis by

GC/MS/MS provides reliable data for

use in forensic toxicology and results in

a substantial increase in detection

sensitivity by the combination of NCI

and MS/MS.20,22

In this study our objective is to

establish and validate a GC/MS/MS

method for the determination of

THCCOOH in human hair using the

NCI-MS of its pentafluoropropyl

derivative. The method was success-

fully applied to the analysis of

THCCOOH in hair samples from

cannabis abusers.

The reference compoundsTHCCOOH

(100mg/mL) andTHCCOOH-d9 (100mg/

mL) were purchased from Cerilliant

(Austin, TX, USA). The derivatizing

agents, pentafluoropropionic anhydride

(PFPA) and pentafluoro-1-propanol

(PFPOH), were obtained from Acros

Organics (Geel, Belgium). Acetic acid

was purchased from Wako (Osaka,

Japan). HPLC-grade methanol, n-hexane,

ethyl acetate, and isopropyl alcohol were

supplied by J. T. Baker (Phillipsburg, NJ,

USA). The water was purified with a

MAXIMA water purification system

(ELGA, High Wycombe, UK).

Working standard solutions of

THCCOOH (0.1, 1.0, 10.0 ng/mL)

and of the internal standard

THCCOOH-d9 (1.0 ng/mL) were pre-

pared in methanol. All solutions were

stored at �208C in the absence of light

until use.

Drug-free hair to be used as a matrix

for control and calibration sampleswas

obtained from a 39-year-old male

volunteer. Head hair samples were

received from the Narcotics Depart-

ment at the Seoul District Prosecutors’

terscience.wiley.com) DOI: 10.1002/rcm.2956

Office. The samples had been taken

from possible cannabis abusers who

had tested positive for its use during a

confirmatory test of a urine sample by

GC/MS.23 These hair samples were

generally cut as close as possible to the

skin from the posterior vertex. The

total length was measured and special

treatments such as coloring and

bleaching were noted.

Hair samples (25mg) were washed

with isopropyl alcohol (10mL) three

times, air-dried and cut with scissors

into small fragments (<1mm) before

transfer to a silanized test tube

(12� 100mm) containing 75pg of a

deuterated internal standard. The hair

samples were hydrolyzed by incu-

bation in 1mL of 1.0M sodiumhydrox-

ide at 958C for 30min. The digested

solutionwas then acidifiedwith 200mL

of concentrated acetic acid and 1.5mL

of 0.1M acetate buffer solution (pH

4.5), followed by liquid-liquid extrac-

tion with n-hexane/ethyl acetate (9:1,

v/v) for 20min. The organic layer

was evaporated under a stream of

nitrogen at 458C and 30 kPa. The

residuewas dried in a vacuumdesicca-

tor over P2O5-KOH for at least 15min,

derivatized with 25mL of PFPOH and

50mL of PFPA in a dry heating block

at 708C for 30min, followed by evap-

oration under a stream of nitrogen.

Sample extracts were reconstituted

with 100mL of ethyl acetate, and then

filtered through a 0.2mm PVDF filter

(13mm Millex filter, Millipore, Bill-

erica, MA, USA) using a Teflon syr-

inge. An aliquot (1mL) of the filtered

extract was injected into the GC/MS/

MS instrument.

GC/MS/MS analyses were per-

formed with a Waters Quattro micro

GC tandem quadrupole mass spec-

trometer (Waters/Micromass, Man-

chester, UK) equipped with an Agilent

Technologies (Foster City, CA, USA)

6890N gas chromatograph and 7683B

autosampler. Data acquisition and

analysis were performed using stan-

dard software supplied by the manu-

facturer (Waters, MassLynx V4.0).

Separation was achieved with a capil-

lary column (DB-5MS, 30m� 0.25mm

i.d., 0.25mm, J&W Scientific, Folsom,

Copyright # 2007 John Wiley & Sons, Ltd.

1340 Letter to the Editor

CA, USA) with helium as the carrier

gas at a flow rate of 1.0mL/min. The

GC temperature program was as

follows: initial temperature was

1008C for 1.0min, increased to 2758Cat a rate of 358C/min, held for 3.0min,

then increased to 3008C at a rate of

25 8C/min, and held for 3.5min. The

splitless injection mode was used with

a purge-on time of 1.0min. The injector

and the GC interface temperatures

were 260 and 2758C, respectively.

The mass spectrometer was operated

under NCI conditions using the

multiple reaction monitoring (MRM)

mode for quantification. The NCI

mode used methane as a reagent

gas in all MS measurement. MS/MS

experiments were based on collision-

induced dissociation (CID) occurring

in the collision cell of the tandem

quadrupole. The argon collision gas

pressure was maintained at 2.5mTorr.

To determine the retention time and

characteristic ions for each compound,

the primary NCI mass spectra of the

derivatized analyte and internal stan-

dard were recorded in full-scan mode

(m/z 50–650).

The chemical structures, full-scan

mass spectra and product ion mass

spectra of the derivatized analyte and

400300200100

%

0

100

%

0

100

NCI-MS spectrum of THCCOOH-2PFP

147.06

128.03474.

432.19148.13 275.93320.59

NCI-MS/MS spectrum of the selected ion (m/z60473.

448.99310.95149.03

193.22

O

C

C5H11

O

O

O

CF2CF3

CHO 2CF2CF3

C27H28F10O5MW: 622

(a)

Figure 1. Representative full-scan MS an

THCCOOH and (b) THCCOOH-d9.

Copyright # 2007 John Wiley & Sons, Ltd.

internal standard are depicted in

Figs. 1 (a) and 1(b), respectively. For

the derivatized THCCOOH, the pro-

minent [M–HF]� ion (m/z 602) was

selected as the precursor ion. In MS/

MS m/z 602 lost HF to m/z 582. Loss

of CF3 from m/z 582 produced m/z 513,

which underwent further loss of HF

and F to form the product ion at m/z 474

(Fig. 1(a)).24,25 For THCCOOH-d9, the

corresponding [M–HF] ion (m/z 611)

was selected at the precursor ion and

m/z 483 as the product ion.

The collision energies were 15 eV

for THCCOOH and 13 eV for

THCCOOH-d9, adjusted to optimize

the signal for the selected product

ions. The electron multiplier was set

at 650V. Each transition was alter-

nately monitored with a dwell time

of 80ms.

Derivatization allows improved

overall chromatographic selectivity

and non-tailing peak shapes, leading

to new compounds with altered

polarity and volatility properties. The

pentafluoropropyl derivatives of

THCCOOH and its internal standard

were readily ionized in the NCI mode,

and the observed noise was low,

allowing sensitive detection of the

analyte. The derivatized analytes were

m/z600500

Scan CI-1.90e8602.32

19513.25

603.25

622.15

623.29

2)1.04e883

513.02

582.09583.50

200100

%

0

100

%

0

100

NCI-MS spectrum of TH

146.95

127.93

109.87 148.08

NCI-MS/MS spectrum o

253128.30

(b)

d MS/MS spectra under NCI conditions of th

Rapi

well separated and no interference

originating from chemical background

was observed (see Fig. 2(b)).

A six-point calibration curve was

established with three replicates at

each concentration. The calibration

curve was linear in the concentration

range of 0.1–10.0 pg/mg (r2¼ 0.997) for

THCCOOH, indicating good linear

regression. The sensitivity of the met-

hod was evaluated by determining the

limit of detection (LOD) and the limit

of quantification (LOQ) for the analyte.

The LOD and LOQ of analytical

method were determined to be 0.02

and 0.05 pg/mg, defined as the con-

centration giving a signal plus 3 and 10

standard deviations from the mean of

eight replicates of drug-free hair,

respectively. Analytical recovery,

accuracy, and precision experiments

were carried out at three concen-

trations (low, middle, high), covering

the calibration range (Table 1). The

intra-day (n¼ 3) and inter-day (n¼ 5)

accuracy (% bias) and precision (% CV)

were assessed by spiking quality con-

trol (QC) samples with the analyte at

three different concentrations (0.3, 1.0,

and 7.0 pg/mg). The intra- and inter-

day accuracy ranged from �10.0 to

1.4% and the intra- and inter-day

m/z600500400300

CCOOH-d9-2PFP Scan CI-1.59e6611.09

483.08 609.90

484.15

612.16

631.11

632.18

f the selected ion (m/z611)3.90e5483.11

350.11

306.11.98 478.72522.14

611.00

O

C

D3C

D3C

O OCH2CF2CF3

CD3

O

O

CF2CF3

C27H19D9F10O5MW: 631

e pentafluoropropyl derivatives of (a)

d Commun. Mass Spectrom. 2007; 21: 1339–1342

DOI: 10.1002/rcm

(a) Blank hair processed without internal standard

Time9.008.508.007.507.006.506.00

%

0

100

9.008.508.007.507.006.506.00

%

0

100

9.008.508.007.507.006.506.00

%

0

100

MRM of 3 Channels CI-061226_BLK_11602.2 > 474.1

6.82e3Area

602.2 > 512.97.69e3

Area

611.2 > 483.11.43e4

Area

(b) Blank hair

Time9.008.508.007.507.006.506.00

%

0

100

9.008.508.007.507.006.506.00

%

0

100

9.008.508.007.507.006.506.00

%

0

100

MRM of 3 Channels CI-061226_IS_11602.2 > 474.1

1.29e4Area

602.2 > 512.91.15e4

Area

611.2 > 483.15.53e5

Area

7.45

Time9.008.508.007.507.006.506.00

%

0

100

9.008.508.007.507.006.506.00

%

0

100

9.008.508.007.507.006.506.00

%

0

100

MRM of 3 Channels CI-061226_cal2_11602.2 > 474.1

3.84e4Area

7.48

602.2 > 512.99.25e3

Area

7.48

611.2 > 483.12.62e5

Area

7.45

(c) Hair sample spiked at 0.5 pg/mg of THCCOOH

Time9.008.508.007.507.006.506.00

%

0

100

9.008.508.007.507.006.506.00

%

0

100

9.008.508.007.507.006.506.00

%

0

100

061226_sample_09602.2 > 474.1

1.34e4Area

7.48

602.2 > 512.95.00e3

Area

7.48

611.2 > 483.11.49e5

Area

7.45

(d) Positive hair sample of THCCOOH at 0.19 pg/mg

MRM of 3 Channels CI-

Figure 2. GC/MS/MS chromatograms of (a) blank hair processed without internal standard, (b) blank hair, (c) drug-fortified

hair, and (d) drug-user hair samples for the MRM transitions (m/z 602! 474 and 602! 513 for THCCOOH;m/z 611! 483 for

THCCOOH-d9).

Letter to the Editor 1341

precisions were, respectively, in the

range 2.1–13.4% and 3.0–12.2% for the

analyte. Considering the complexity of

hairmatrix and theweak incorporation

of acidic compound into the hair

matrix we regard these results as

satisfactory. Analytical recoveries of

analyte were also examined at three

different concentrations (0.3, 1.0, and

7.0 pg/mg) in five replicates each.

Excellent analytical recoveries of

83.1–85.1%. were obtained.

The developed method was vali-

dated by analysis of hair samples from

possible cannabis abusers. Figure 2

shows the representative chromato-

Copyright # 2007 John Wiley & Sons, Ltd.

grams of blank hair, drug-fortified

hair, and drug-user hair samples with

the MRM transitions (m/z 602! 474

and 602! 513 for THCCOOH; m/z

611! 483 for THCCOOH-d9). The

chromatograms show no interfering

peaks from endogenous substances or

co-extracted compounds. The concen-

trations of THCCOOH measured in

twelve hair samples ranged from

0.14–0.85 pg/mg with an average of

0.35 pg/mg.

To summarize, this reliable and

highly sensitive GC/MS/MS method

for the determination of THCCOOH in

human hair employs the pentafluoro-

Rapi

propyl derivatization of analytes after

acidic hydrolysis and liquid-liquid

extraction procedures. Optimization of

sample preparation by the dilution and

filtration of sample extracts before GC/

MS/MS analysis was the key to improv-

ing the chromatographic sensitivity and

measurement repeatability of the target

analyte. The pentafluoropropyl deriva-

tives were readily ionized in the NCI

mode because of the electronegativity of

the pentafluoropropyl moiety,25,26 and

therefore the use of derivatization for

NCI and MS/MS analysis resulted in

greatly improved sensitivity and

more informative fragmentation of

d Commun. Mass Spectrom. 2007; 21: 1339–1342

DOI: 10.1002/rcm

Table 1. Validation data for the analysis of THCCOOH in hair

THCCOOH

Concentration range (pg/mg) 0.10–10.0Linearitya (r2) 0.997LODb (pg/mg) 0.02LOQc (pg/mg) 0.05Recovery (% mean� SD, n¼ 5)0.3 pg/mg 83.1� 5.71.0 pg/mg 83.2� 7.17.0 pg/mg 85.1� 6.8

Intra-day precisiond (% CV, n¼ 3)0.3 pg/mg 13.41.0 pg/mg 2.17.0 pg/mg 3.8

Intra-day accuracye (% bias, n¼ 3)0.3 pg/mg �10.01.0 pg/mg 0.37.0 pg/mg �5.0

Inter-day precision (% CV, n¼ 5)0.3 pg/mg 12.21.0 pg/mg 3.07.0 pg/mg 6.7

Inter-day accuracy (% bias, n¼ 5)0.3 pg/mg �3.31.0 pg/mg 1.47.0 pg/mg �4.2

a Linearity is described by the correlation coefficient for the calibration curve.b Limit of detection (LOD) and climit of quantification (LOQ) was based on the concentrationcorresponding to a signal plus 3 and 10 standard deviations from the mean of eight replicatesof drug-free hair, respectively.d Expressed as the coefficient of variation of the peak area ratios of analyte/internal standard.e Calculated as [(mean calculated concentration – nominal concentration)/nominal concen-tration] �100.

1342 Letter to the Editor

the analyte. The described met-

hod provides high sensitivity, improved

repeatability, and effective removal of

matrix-induced interferences. Further-

more, themethodwas validated by

the successful determination of

THCCOOH in hair samples from can-

nabis abusers.

AcknowledgementsThis work was supported in part bygrant M10640010000-06N4001-00100 fromNational R&D Program of Ministry ofScience and Technology (MOST) andKorea Science and Engineering Foundation(KOSEF). The authors are grateful to Dr TimJenkins and Dr Peter Hancock of WatersCorporation for their kind proofreading ofthis manuscript and Ji Yeon Kim of WatersKorea for her professional advice.

Jin Young Kim*and Moon Kyo In

Drug Analysis Laboratory,Forensic Science Division,

Supreme Prosecutors’ Office, Seoul137-730, Korea

Copyright # 2007 John Wiley & Sons, Ltd.

*Correspondence to: J. Y. Kim, DrugAnalysis Laboratory, Forensic ScienceDivision,Supreme Prosecutors’ Office, 706, Banporo,Seocho-gu,Seoul 137-730, Korea.E-mail: paxus@spo.go.krContract/grant sponsors: National R&DProgram of Ministry of Science and Tech-nology (MOST) and Korea Scienceand Engineering Foundation (KOSEF).Contract/grant number: M10640010000-06N4001-00100.

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Received 10 January 2007Revised 29 January 2007

Accepted 30 January 2007

d Commun. Mass Spectrom. 2007; 21: 1339–1342

DOI: 10.1002/rcm