Determination of ketamine and norketamine in hair by gas chromatography/mass spectrometry using...

RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2006; 20: 3159–3162

ublished online in Wiley InterScience (www.in
P
RCM

Letter to the Editor

Figure 1. Comparison of three different derivatization methods.

To the Editor-in-Chief

Sir,

Determination of ketamine and nor-

ketamine in hair by gas chromatog-

raphy/mass spectrometry using two-

step derivatization

Ketamine (KET) is a central nervous

system depressant that produces a

rapid-acting dissociative effect. It was

developed in the 1960s as a medical

anesthetic to replace phencyclidine

and is currently used in human ane-

sthesia and veterinary medicine.1

KET is also a potent analgesic at

subanesthetic doses with effects

similar to phencyclidine, and its hallu-

cinogen effects are comparable with

those of lysergic acid diethylamide.2–4

KET undergoes liver metabolism by

CYP-450 N-demethylation to norketa-

mine (NKT). The cyclohexanone ring

also undergoes oxidative metabolism

to form a second metabolite, dehydro-

norketamine.5

Although KET is not as common as

other recreational drugs such as

Ecstasy (3,4-methylenedioxymetham-

phetamine), there has been a recent

tendency for KET abuse in Korea,

resulting in some fatalities.6–8 KET is

usually acquired as an injectable liquid

diverted from veterinary clinics; how-

ever, it is also available as a powder

that can be snorted or compressed into

pills. Because of its abuse, KET was

registered on the official list of con-

trolled substances by the Korean gov-

ernment in November 2005. As a

consequence, reliable and sensitive

analytical methods to detect KET and

its metabolites in biological samples

have become a focus of attention in

forensic science and clinical toxicology.

Several chromatographic methods

have been reported for the identification

of KET and/or NKT in biological

matrices, including high-performance

liquid chromatography (HPLC),9 gas

chromatography (GC),7,10 gas chromatog-

raphy/mass spectrometry (GC/MS),11

and liquid chromatography/tandem

mass spectrometry (LC/MS/MS).12

We have focused on GC/MS methods

for the determination of KET and its

metabolite NKT in hair, but these

methods show relatively high limits

of detection (LODs) and limits of

quantification (LOQs).13,14 To increase

detection sensitivity some of the

methods require negative chemical

ionization and/or a tandem mass

spectrometric technique, which are

not readily available in many labora-

tories.11,15

Because of the low molecular

weights and high polarity of KET

and NKT, derivatization is required

when using GC or GC/MS to improve

their GC properties and to form more

characteristic mass spectral fragment

ions. The common derivatization pro-

cedures for determining KET and NKT

by GC/MS in urine include acetylation

using heptafluorobutyric anhydride

(HFBA) or N-methylbis(trifluoroaceta-

mide) (MBTFA),11,16 and acylation using

pentafluorobenzoyl chloride (PFBC).17

To produce a stable derivative with a

high and reproducible yield, the deri-

vatization reactions were based on two

steps of TFA derivatization of analytes

using both trifluoroacetic anhydride

(TFAA) and MBTFA. The procedure

includes a consecutive formation of

trifluoroacetyl (TFA) derivatives for

simultaneous determination.

In the present study we describe a

determination method involving a

two-step derivatization of KET and

its metabolite NKT in hair samples

terscience.wiley.com) DOI: 10.1002/rcm.2682

followed by GC/MS analysis using

electron ionization (EI). A preliminary

application to the analysis of hair

samples from suspected KET abusers

suggests that this method is suitable

and feasible.

The reference compounds KET

(100mg/mL), NKT (100mg/mL),

KET-d4 (10mg/mL), and NKT-d4

(10mg/mL) were purchased from Cer-

illiant (Austin, TX, USA). TFAA was

purchased from Alltech (Deerfield,

IL, USA) and MBTFA was obtained

from Acros Organics (Geel, Begium).

Extract-CleanTM empty reservoirs

(4.0 mL) and polyethylene frits were

obtained from Alltech. HPLC-grade

methanol, ethyl acetate, and acetone

were supplied by J. T. Baker (Phillips-

burg, NJ, USA). The water was purified

with a MAXIMA water purification

system (ELGA, High Wycombe, UK).

Working standard solutions (0.1, 1.0,

10.0mg/mL) of KET and NKT, and a

combined standard solution of the

internal standards (1.0mg/mL) KET-

d4 and NKT-d4, were prepared in

methanol. All solutions were found

to be stable for about 6 months when

stored at �208C.

Drug-free hair to be used as a matrix

for control and calibration samples was

obtained from a 38-year-old male

volunteer. Head hair samples were

received from the Narcotics Depart-

ment at the Seoul District Prosecutors’

Office, which were taken from possible

KET abusers tested positive for its use

during a screening test of urine

samples by GC/MS. These samples

were generally cut as close as possible

to the skin from the posterior vertex.

Copyright # 2006 John Wiley & Sons, Ltd.

Figure 2. (A) EI mass spectra of the trifluoroacetyl (TFA) derivatives of KET, KET-d4,

NKT, and NKT-d4. (B) SIM chromatograms of the quantification ions for TFA

derivatives of KET (at m/z 270), KET-d4 (at m/z 274), NKT (at m/z 284), and

NKT-d4 (at m/z 288).

3160 Letter to the Editor

Total length was measured and special

treatments such as coloring and

bleaching were noted.

Hair samples (30 mg) were washed

with water (10 mL) and acetone

(10 mL), air-dried and cut with scissors

into small fragments (<1 mm) before

transfer to a test tube (12� 100 mm)

containing 150mL of the combined

internal standard solution. The hair

samples were extracted with 2 mL of

0.25 M methanolic HCl at 508C for 1 h

under ultrasonication. The hair was

then filtered with a fritted reservoir

(Extract-CleanTM) and the filtrate was

dried under a nitrogen stream at 458Cat 30 kPa. TFA derivatives of KET

and NKT were formed by reaction of

the sample with TFAA (50mL) and

ethyl acetate (50mL) in a dry heating

block at 408C for 60 min, followed

by drying under a nitrogen stream.

For the second derivatization, MBTFA

(40mL) was added to the sample,

which was incubated at 1208C for

30 min. Single derivatizations with

either TFAA or MBTFA were also

carried out in the same manner for

comparison. Finally, an aliquot (1mL)

of the reaction mixture was injected

into the GC/MS instrument.

GC/MS analyses were performed with

an Agilent Technologies 5975i mass

spectrometer equipped with a 6890N

gas chromatograph and a 7683B auto-

sampler (Agilent, Santa Clara, CA, USA).

Data acquisition and analysis were

performed using standard software

supplied by the manufacturer (MSD

Chemstation D.02.00). Separation was

achieved with a capillary column (DB-

5MS, 30 m� 0.25 mm i.d., 0.25mm,

J&W Scientific, Folsom, CA, USA) with

helium as the carrier gas at a flow rate

of 0.9 mL/min. The GC temperature

program was as follows: initial

temperature was 908C for 0.5 min,

increased to 2208C at a rate of 158C/

min, then increased to 3008C at a rate of

358C/min, and held for 0.55 min. The

split ratio was 1:9. The injector and the

transfer line temperatures were 260

and 2808C, respectively. The mass

spectrometer was operated under

positive ion EI conditions (70 eV) with

selected ion monitoring (SIM) for

quantification. The ion groups of the

derivatized analytes were monitored,

for quantification and qualification, in

the elution order as follows (quantifi-


cation ions are given in parentheses):

NKT-d4, m/z (288); NKT, m/z (284), 256;

KET-d4, m/z (274); KET, m/z (270), 236.

The dwell time for each ion was

100 ms.

Since external contaminants on the

hair, including KET and NKT, must be

considered as a possible source of

false-positives, a KET positive hair

sample was washed with water and

Rapi

acetone. Neither KET nor NKT was

detected in the acetone wash, sugges-

ting that the hair-washing procedure

was suitable for decontamination.

The derivatization yield was

obtained from comparison of the peak

area of each analyte from three differ-

ent derivatization methods. A two-step

derivatization utilizing both TFAA and

MBTFA gave much better selectivity

d Commun. Mass Spectrom. 2006; 20: 3159–3162

DOI: 10.1002/rcm

Figure 2. (Continued)

Letter to the Editor 3161

and recovery for KET and NKT than

single-step derivatizations. The effects

of temperature (30–608C) and time (30–

60 min) for trifluoroacetylation with

TFAA/ethyl acetate were studied for

each analyte. The optimum conditions

for the derivatization of the drugs were

408C and 60 min. The second TFA

derivarization with MBTFA was car-

ried out according to the procedure

described by Huang et al.16 The deri-

vatization yield of the analytes was

considerably increased by using a two-

step derivatization method, giving a

yield 2–2.5 times higher than other

single-step derivatization methods

(Fig. 1).


Chemical structures and EI mass

spectra, recorded with the quadrupole

mass spectrometer, of the derivatized

analytes are depicted in Fig. 2(A). The

main features of derivatization are

improved overall chromatographic

selectivity and non-tailing peak shapes,

from the new compounds with altered

polarity and volatility properties. The

TFA derivatives of KET and NKT were

well separated with good peak shapes

and no interference from the matrix

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

Six-point (KET) and eight-point

(NKT) calibration curves were estab-

lished with three replicates at each

concentration. The calibration curves

Rapi

were linear in the concentration ranges

of 0.15–25.0 ng/mg (r2¼ 0.9997) for

KET and 0.1–40.0 ng/mg (r2¼ 0.9998)

for NKT, indicating good linear

regression. The LODs were 0.03 ng/mg

for KET and 0.01 ng/mg for NKT,

based on the concentration corre-

sponding to a signal plus 3 standard

deviations (SD) from the mean of five

replicates of drug-free hair. The LODs

obtained for KET and NKT were nearly

10–25 times lower than those pre-

viously reported for other GC/MS

methods.13,14 The LOQ, defined as

the lowest concentration determined

with a precision (coefficient of vari-

ation, CV) less than 20%, was 0.11 ng/mg

for KET and 0.05 ng/mg for NKT.

Analytical recovery, accuracy, and

precision experiments were carried

out at three concentrations (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 control (QC) samples

with the analytes, KET and NKT,

at three different concentrations (0.5,

4.0, and 20.0 ng/mg). The intra-day

accuracy and precision of detecting

KET or NKT ranged from �13.3 to

1.4% and 0.5 to 5.6%, respectively, and

the inter-day accuracy and precision

values ranged from �13.4 to 0.9% and

2.2 to 11.5%, respectively. Considering

the complexity of the hair matrix, we

regard these results as satisfactory.

Analytical recoveries of analytes were

also examined at three concentration

levels (0.5, 4.0, and 20.0 ng/mg) in five

replicates each. Excellent analytical

recoveries were obtained: 96.6–

107.3% for KET and 97.3–102.2% for

NKT.

This method was validated by

analysis of a hair sample from a

suspected KET abuser. The SIM chro-

matogram for the sample (Fig. 2)

shows no interfering peaks from

endogenous substances or co-extracted

compounds. The concentrations mea-

sured in this sample (19.7 mg) were

4.50 ng/mg for KET and 0.35 ng/mg

for NKT.

In conclusion, this reliable and

highly sensitive GC/MS method for

the determination of KET and NKT in

human hair includes two steps of TFA

derivatization of analytes after acidic

hydrolysis/extraction procedure. Deri-


DOI: 10.1002/rcm

Table 1. Validation data for the analysis of KET and NKT in hair

KET NKT

Concentration range (ng/mg) 0.15–25.0 0.10–40.0Linearitya (r2) 0.9997 0.9998LODb (ng/mg) 0.03 0.01LOQc (ng/mg) 0.11 0.05Recovery (% mean� SD, n¼ 5)

0.5 ng/mg 105.8� 8.6 102.2� 3.14.0 ng/mg 107.3� 2.8 101.7� 3.020.0 ng/mg 96.6� 8.1 97.3� 1.1

Intra-day precisiond (% CV, n¼ 3)0.5 ng/mg 5.6 2.04.0 ng/mg 1.2 0.920.0 ng/mg 0.5 0.9

Intra-day accuracye (% bias, n¼ 3)0.5 ng/mg �13.3 �5.84.0 ng/mg 1.4 �1.320.0 ng/mg 0.6 0.0

Inter-day precision (% CV, n¼ 5)0.5 ng/mg 7.4 11.54.0 ng/mg 6.4 3.320.0 ng/mg 2.2 2.7

Inter-day accuracy (% bias, n¼ 5)0.5 ng/mg �13.4 0.94.0 ng/mg �1.9 �1.620.0 ng/mg �0.9 �1.0

a Linearity is described by the correlation coefficient for the calibration curve.b Limit of detection (LOD) was based on the concentration corresponding to a signal plus 3standard deviations (SD) from the mean of five replicates of drug-free hair.c Limit of quantification (LOQ) was defined as the lowest concentration with precision within20% (% CV).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 con-centration]� 100.

3162 Letter to the Editor

vatization using both TFAA and

MBTFA was the key to improving

the chromatographic selectivity and

sensitivity over single-step procedures

with either TFAA or MBTFA. This

method has substantial benefits over

previous methods for the determi-

nation of KET and NKT, such as more

effective removal of matrix interfer-

ences, higher sensitivity and better

repeatability. This method was vali-

dated by the successful determination

of KET and NKT in a hair sample from

a KET abuser.


AcknowledgementsThis work was supported by the KoreaMinistry of Science and Technology. Pre-sented in part at the 11th Annual Meeting ofthe Society of Hair Testing (Vadstena,Sweden, May 2006).

Jin Young Kim1*, Moon Kyo In1, andJeong-Han Kim2

1

Drug Analysis Laboratory, ForensicScience Division, Supreme Prosecu-

tors’ Office, Seoul 137-730, Korea2School of Agricultural Biotechnology,

Seoul National University,Seoul 151-742, Korea

Rapi

*Correspondence to: J. Y. Kin Drug Analy-sis Laboratory, Forensic Science Division,Supreme Prosecutors’ Office, 1730-1Seocho-dong, Seocho-gu, Seoul 137-730,Korea.E-mail: [email protected]

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Received 27 April 2006Revised 21 July 2006

Accepted 24 July 2006


DOI: 10.1002/rcm

Determination of ketamine and norketamine in hair by gas chromatography/mass spectrometry using...

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