JPM Vol. 31, No.

3
June 1994:141-148

Derivatization With Acetic Anhydride:

Applications to the Analysis of Biogenic Amines and Psychiatric
Drugs by Gas Chromatography and Mass Spectrometry
Glen B. Baker, Ronald T. Coutts, and Andrew Holt

Neurochemical Research Unit, Department of Psychiatry and Faculty of Pharmacy and Pharmaceutical Sciences,
University of Alberta, Edmonton, Alberta Canada

Acetylation with acetic anhydride, under both aqueous and anhydrous conditions, has been
utilized to derivatize various biogenic amines and psychotropic drugs for subsequent analysis
by gas chromatography (GC) or gas chromatography-mass spectrometry (GC-MS). Under
basic aqueous conditions, acetic anhydride derivatizes phenols and amines but not alcohols;
under anhydrous conditions, all three functions are acetylated. Primary amines, once deriva-
tized with acetic anhydride, can be further derivatized with other reagents; these diderivatives
have proven useful for subsequent analysis by GC or GC-MS. Examples of applications of
derivatization with acetic anhydride to analysis of biogenic amines, antidepressants, antipsy-
chotics, and some of their metabolites are presented.

Keywords: Acetic anhydride, Gas chromatography, Mass Spectrometry, Biogenic amines,

Antidepressants, Antipsychotics

Introduction
Many of the gas chromatography (GC) and combined
gas chromatography-mass spectrometry (GC-MS) tech-
niques that have been applied to neurochemical studies in
biological psychiatry have involved derivatization of the
neurochemicals or drugs of interest. Derivatization may
serve a number of purposes: to facilitate extraction of the
compounds of interest from biological media; to improve
detection sensitivity; to alter chromatographic properties
such as polarity and peak shape; and/or to alter retention
times on chromatographic columns to facilitate the separa-
tion of the compounds of interest from other substances in
the extract. Many derivatizing reagents are available, but
this review will concentrate on acetic anhydride, a simple
molecule that has had relatively wide applications to bio-
Address reprint requests to Dr. Glen B. Baker, Neurochemical
Research Unit, Department of Psychiatry, University of Alberta,
Edmonton, Alberta, Canada, T6G 2R7.
Received August 23, 1993; revised and accepted November 22,
1993.
Journal of Pharmacologicaland Toxicological Methods 31, 141-148 (1994)
© 1994 Elsevier Science Inc.
655 Avenue of the Americas, New York, NY 10010
chemical analyses in biological psychiatry. Several aspects
of acetic anhydride are very relevant to such analyses and
should be emphasized at this point. Amines and phenols
(but not alcohols) can be derivatized with acetic anhydride
under basic aqueous conditions (Chattaway, 1931; Welsh,
1955; Goldstein etal., 1959; Hagopian etal., 1961; Brooks
and Homing, 1964; Sharman, 1969), and the resultant ace-
tylated derivatives are more lipophilic than the parent
amines or phenols, thus facilitating extraction of the neuro-
chemicals or drugs of interest into organic solvents. Under
anhydrous conditions, amines, phenols, and alcohols can
be derivatized with acetic anhydride. It is also important
that primary amines, once derivatized with acetic anhy-
dride, can often be further derivatized with another acylat-
ing agent; such compounds have proven to have high sen-
sitivity in GC and GC-MS analytical systems. Several
examples demonstrating the wide application of acetyla-
tion with acetic anhydride to analysis of biogenic amines
and antidepressant and antipsychotic drugs and their meta-
bolites are now provided in this review.
1056-8719/94/$7.00
142
Analysis of Endogenous Amines in Tissues
and Body Fluids
2-Phenylethylamine (PEA)
This arylalkylamine has been implicated in the etio-
logy and/or pharmacotherapy of a number of neurologi-
cal and psychiatric disorders (Paterson et al., 1990;
Baker et al., 1993). It is present in very low concentra-
tions in brains of drug-free laboratory animals and
humans, a factor that has created considerable difficul-
ties in quantitative studies of this amine. However,
extractive acetylation with acetic anhydride followed by
derivatization with a second reagent under anhydrous
conditions has proven to be very useful in overcoming
this problem. Martin and Baker (1977) derivatized PEA
in basified aqueous brain extracts with acetic anhydride,
extracted the resultant N-acetylPEA into an organic sol-
vent, then derivatized further with pentafluoropropionic
anhydride (PFPA) under anhydrous conditions. The
final derivative, which was shown to be N-acetyl-N-
pentafluoropropionylPEA, proved to be very sensitive to
analysis by GC with electron-capture detection (GC-
ECD). This analytical procedure revealed levels of PEA
in brain similar to those previously demonstrated with
mass spectrometric and radioenzymatic procedures.
Hampson et al. (1984a) utilized aqueous acetylation
followed by derivatization with pentafluorobenzoyl
chloride (PFBC) under anhydrous conditions to measure
PEA in brain and urine samples; final analysis was by
GC-ECD. The combination of acetylation with acetic
anhydride and subsequent reaction with PFBC has also
been used to measure PEA in brain tissue and plasma
employing GC-MS with negative chemical ionization
(NCI) (Durden, 1991; Durden et al., 1991b). The struc-
tures of N-acetyl-N-pentafluoropropionylPEA and N-
acetyl-N-pentafluorobenzoylPEA are shown in Figure 1.
5-Hydroxytryptamine (5-HT) and Tryptamine (T)
These two indolealkylamines are both formed meta-
bolically from the amino acid tryptophan, and both have
been implicated in the etiology of depression and the
actions of antidepressant drugs (Grahame-Smith, 1992;
Mousseau, 1993). In a study of levels of T in rat brain,
Warsh et al. (1977) employed extractive acetylation of
basified brain extracts by adding a solution of acetic
anhydride in an organic solvent and shaking the two
phases; after taking the solution containing N-acetylT to
dryness, the residue was reacted with trifluoroacetic
anhydride (TFAA) under anhydrous conditions to pro-
duce a derivative of T that was analyzed by GC-MS. We
developed a similar assay procedure that permits simul-
taneous assay of 5-HT and T using GC-ECD (Baker et
JPM Vol.31, No. 3
June 1994:141-148
al., 1980). The method employs homogenization of
brain tissue in perchloric acid, extraction with the liquid
ion-exchanger di-(2-ethylhexyl)phosphate (DEHPA) in
chloroform, back-extraction with 0.5 N HC1, basification
of the aqueous phase with NaHCO3, acetylation under
aqueous conditions and extraction with ethyl acetate.
The organic phase is then taken to dryness, and the
residue is reacted with pentafluoropropionic anhydride
(PFPA) in the presence of ethyl acetate. The resultant
spirocyclic derivatives (Blau et al., 1977) have excellent
properties for GC-ECD analysis (see Figure 2 for struc-
tures), and a similar procedure has also been utilized by
Martin et al. (1988) and Durden and Boulton (1988) to
analyze T in rat brain regions, using GC-MS-NC1 for
analysis.
Phenolic Phenylethylamines
Because of their amphoteric nature, phenolic amines
are often difficult to extract efficiently from aqueous
solutions, but they can be extracted in high yield by first
acetylating them with acetic anhydride in basified
aqueous medium and extracting the acetylated products
into organic solvents. Under these conditions, primary
and secondary amino groups are converted to amides
and phenolic hydroxyl groups are converted to O-ace-
tates (Welsh, 1955; Goldstein et al., 1959; Hagopian et
al., 1961; Laverty and Sharman, 1965; Sharman, 1969;
LeGatt et al., 1981a,b; Wood and Rao, 1990). The
resultant lipophilic neutral products are readily extracted
by organic solvents.
0
II
/C-CH s
O - - CH2CH2N
\C=O
I
C2F5
0
II
~C--CHs
O- CH2CH2N~c=O
F
Figure 1. Structures of N-acetyl-N-pentafluoropropionyl-2-phen-
ylethylamine (I) and N-acetyl-N-pentafluorobenzoyl-2-phenyleth-
ylamine (II).
G.B. BAKERET AL.
DERIVATIZATIONWITHACETICANHYDRIDE
~NCOCF 2CFs
I anhydrous conditions to prepare derivatives of m- and
CHsCO0~ "NCOCF2CFs
TI
Figure 2. Structures of the derivatives formed from tryptamine
(I) and 5-hydroxytryptamine (II) by reaction with acetic anhy-
dride under aqueous conditions followed by reaction with penta-
fluoropropionic anhydride under anhydrous conditions.
A procedure developed in our laboratories (Coutts et
al., 1981; LeGatt et al., 1981a,b) allows for simultane-
ous assay of PEA, T and the phenolic amines m- and
p-tyramine (m- and p-TA), 3-methoxytyramine (3-MT),
normetanephrine (NME) and 5-HT in brain tissue and
urine. Its application to brain tissue is summarized in the
flow diagram in Figure 3. In this procedure, the organic
extract of acetylated amines is treated with a small
quantity of 10 N NH4OH to hydrolyze the acetylated
phenolic groups of m- and p-TA, 3-MT, and NME
(LeGatt et al., 1981b; Coutts et al., 1980, 1981). The
NH4OH is then neutralized with HC1, the samples are
vortexed again, and the ethyl acetate phase is retained.
The selective hydrolysis step can be omitted and the
N-acetylated, phenolic O-acetylated derivatives reacted
directly with the perfluoroacylating reagent to produce
N-acetyl, N-TFA, and O-acetyl derivatives (in the case
of NME, the alcohol group is also derivatized with
TFAA). However, it was found in our GC procedure
that an interfering substance co-chromatographed with
p-TA under these conditions. In addition, the hydrolysis
step frees the phenolic group for subsequent reaction
with a perfluoroacylating reagent (TFAA in this case),
and results in increased sensitivity to GC-ECD com-
pared with the derivative formed when hydrolysis is
omitted (see Figure 4 for comparison of the two deriva-
tives). Detailed mass spectral analysis of the structures
of the derivatives of the phenolic amines are presented
in the paper by Coutts et al. (1984). PEA forms the
same derivative when carried through either procedure,
but we have found that by incorporating the hydrolysis
step, a cleaner blank for PEA is provided. Unfortu-
nately, T and 5-HT cannot be analyzed concomitantly
using the hydrolysis technique since they appear to
deteriorate to some extent under these conditions; hence
143
a separate aliquot must be taken for analysis of T and
5-HT prior to hydrolysis.
Durden et aL (1991a) used aqueous acetylation with
acetic anhydride followed by reaction with PFBC under
p - T A for subsequent analysis by GC-MS-NC1; the tech-
nique was applied to analysis of these amines in rat
brain regions.
Acetylation of phenolic substituents with acetic anhy-
dride under basic aqueous conditions followed by deri-
vatization of the alcohol groups under anhydrous condi-
tions has also been employed for analysis of
(3-methoxy-4-hydroxyphenyl)ethylene glycol (MHPG)
and (3,4-dihydroxyphenyl)ethylene glycol (DHPG), me-
tabolites of noradrenaline, in urine and tissue samples
(Sharman, 1969; Kahane et al., 1976; Takahashi et al.,
1977; Warsh et al., 1981; Li et al., 1985; Baker et al.,
1986; Wood and Rao, 1990). Acetylation of the pheno-
lic hydroxyl groups of MHPG and DHPG produces
derivatives that are readily soluble in an organic solvent.
Subsequent reaction with perfluoracylating or trimethyl-
silylating reagents under anhydrous conditions deriva-
tizes the alcohol hydroxyl groups and results in the
formation of molecules suitable for analysis by GC or
GC-MS (see Figure 5).
Analysis of Antidepressant and Antipsychotic
Drugs and Some of Their Metabolites in
Tissues and Body Fluids
Antidepressants
Gas chromatography with nitrogen-phosphorus detec-
tion (GC-NPD) has proven to be a popular technique for
analysis of tricyclic and other antidepressants and their
metabolites extracted from plasma or serum samples
(Cooper, 1988 for review). The procedure commonly
utilized involves basification of the samples, extraction of
the drugs into an organic solvent, back-extraction into
acid, basification of the acid phase and extraction with an
organic solvent, and injection of a portion of the extract
onto the GC. However, some workers have found it
useful to include an acetylation step with acetic anhydride
in the assay to provide improved separation of the parent
drugs and their metabolites (Jorgenson, 1975) and avoid
adsorption of primary and secondary amines onto GC
columns (Gupta et al., 1977). The acetylation assay pro-
cedure permits simultaneous analysis of tertiary amine
antidepressants (e.g., imipramine), which are not deriva-
tized, and their corresponding secondary amine metabo-
lites (e.g., desipramine) [Gupta et al., 1983]. Further-
more, secondary amines, such as maprotiline (Gupta et
al., 1977; Drebit et al., 1988) and fluoxetine (Goodnough
144 JPM Vol. 31, No. 3
June 1994:141-148

Brain homoqenate in perchloric acid.

Centrifuge and retain supernatant
Hake sliqhtly alkaline.
Shake wTth'di-(2-ethylhexyl)
p_hosphoric acid in chloroform.
Centrifuge.
Retain bottom layer.
J, Shake with O.5N HCI.
Centrifuge.
Retain top layer.
Hake slightly alkaline (pH 7.8).
Acetylate.
Extract with ethyl acetate. Figure 3. An assay procedure for the si-
Portion for analysis of < Retain organic layer. multaneous analysis of 2-phenylethyla-
tryptamine and 5-HT. mine, tryptamine, m-tyramine, p-tyramine,
Shake with I/IO the volume 3-methoxytyramine, normetanephrine, and
of ION NH4OH 5-hydroxytryptamine (modified from
Neutralize with conc. HCI. Baker et al., 1981).
Take to dryness Centrifuge.
under N2.
Retain organic layer.
Take to dryness under N2.

Residue. Residue.
React with PFPA. React with TFAA.
Partition between Partition between
cyclohexane and sodium cyclohexane and sodium
borate buffer. bbrate buffer.
Retain cyclohexane layer. Retain cyclohexane layer.
4, 4,
l ul on GC l ul on GC

et al., 1993), can be analyzed simultaneously with their
N-desmethylated metabolites. The procedure has also
been applied to the analysis of the secondary amine
viloxazine (Fazio et al., 1984). Reaction with acetic an-
hydride followed by GC-MS analysis has also been em-
ployed for simultaneous analysis of doxepin and N-des-
methyldoxepin (Frigerio et al., 1977) and for separation
of the tricyclic antidepressant trimipramine from a num-
ber of its desmethylated and hydroxylated metabolites
(Maurer, 1989). Most assays (e.g., Weder and Bickel,
1968; Belvedere et al., 1975; Jorgenson, 1975; Gupta et
al., 1976, 1977; Sioufi and Richard, 1980; Maurer, 1989)
employ reaction with acetic anhydride under anhydrous
conditions after previous extraction of the drugs, but we
have found that the acetylation step can be conducted
under aqueous conditions early in the procedure (Drebit
et al., 1988; Goodnough and Baker, 1994; Goodnough et
al., 1993). In an assay for maprotiline and N-desmethyl-
maprotiline (Drebit et al., 1988), we basified the plasma
and urine samples using NaHCO3 and acetylated directly.
In subsequent studies with other antidepressants, we have
found that initial basification of the samples with Na2CO 3
followed by extraction with an organic solvent such as
ethyl acetate may produce cleaner samples; the organic
extract can be taken to dryness and the residue taken up
in water and basified for acetylation as described by
Drebit et al. (1988). This modified procedure with an
initial extraction is useful if only the parent drug and its
demethylated metabolite are of interest, but the recovery
of phenolic hydroxylated metabolites will be reduced.
Although the procedure of acetylation under aqueous
conditions has not yet been employed extensively for
the analysis of hydroxylated metabolites of the tricyclic
antidepressants, we have found that direct aqueous ace-
tylation under basic conditions (produced by the addi-
tion of solid NaHCO3) of plasma and urine samples
from patients treated with imipramine provides for si-
multaneous analysis of imipramine (underivatized), des-
ipramine (N-acetylated), 2-hydroxyimipramine (O-ace-
tylated), and 2-hydroxydesipramine (N,O-diacetylated)
G. B. BAKERET AL. 145
DERIVATIZATIONWITHACETICANHYDRIDE

HO-O - CH2CH2NH2

I AA
NH40H
CHsCOO-O-- CH2 C H 2 N H C O C H s (a) Ho-O-cH2CH2NHCOCHs

(b~[TFAA I TFAA
.COCH .COCH
CHsCOO__O_CH2CHzN/ s CFsCoo_O_cH2CH2N/ s
\COCFs \COCFs
Figure 4. Derivatives formed from p-tyramine by reacting with acetic anhydride (AA) under aqueous conditions followed by
trifluoroacetic anhydride (TFAA) under anhydrousconditions. In one scheme (a), hydrolysisof the acetylatedphenol is included, while
in the other (b) it is omitted (modifiedfrom Baker et al., 1981).

(R Drebit et al., unpublished). As described later in this Antipsychotics

review, this procedure has also been utilized in analysis
of these drugs and metabolites in in vitro experiments
with CYP2D6 isozyme expressed in a human cell line
(Coutts et al., 1993; Su et al., 1993). The structures of
the derivatives are shown in Figure 6.
Tranylcypromine (TCP) is a monoamine oxidase
(MAO)-inhibiting antidepressant that is similar structur-
ally to amphetamine. This drug is a primary amine, and
aqueous acetylation with acetic anhydride followed by
reaction with PFPA (Calverley et al., 1981) or PFBC
(Hampson et al., 1984b) under anhydrous conditions
have been employed for its analysis in extracts of brain
tissues; GC-ECD was used for analysis, but the methods
should also be applicable to GC-MS analysis.
CHsO tCOC2Fs
CHsCOOL
~ CH- - CH2OCOC2F
5 plasma has also been used for derivatization of pimo-
CHsCOO OCOC2Fs
I dure has not been reported, to our knowledge, for ex-
CHsCOO-~ CH- - CH2OCOC2F
s traction and analysis of these metabolites.
Acetylation of organic extracts of body fluids with
acetic anhydride under anhydrous conditions to separate
N-desmethylated and hydroxylated metabolites of phe-
nothiazines from the parent drugs prior to analysis using
GC has been employed by several researchers (e.g.,
Driscoll et al., 1964; Johnson et al., 1965; Rose et al.,
1964). Hansen and Larsen (1974) utilized reaction with
acetic anhydride under anhydrous conditions to deriva-
tize perphenazine (which contains an alcohol hydroxy
group) after extraction from human whole blood and
prior to GC analysis. Javaid and coworkers have re-
ported that reaction with acetic anhydride under anhy-
drous conditions is a useful workup procedure for
subsequent GC-NPD analysis of a number of alcohol-
containing antipsychotics (Javaid et al., 1980a,b, 1981;
Chang et al., 1985). Reaction with acetic anhydride
under anhydrous conditions after drug extraction from
zide; analysis was subsequently conducted using GC-
MS in the chemical ionization mode (Reed, 1988). Pre-
sumably N-desmethylated and phenolic hydroxy
metabolites of antipsychotic drugs could be readily ace-
tylated under aqueous conditions, although this proce-

TI
Figure 5. Structures of derivatives of MHPG (I) and DHPG (II)
formed after reaction with acetic anhydrideunder aqueous condi-
tions followed by reaction with pentafluoropropionic anhydride
under anhydrousconditions.
Use of Acetic Anhydride to Investigate
Detailed Metabolism of Psychotropic Drugs
As mentioned previously in this review, acetylation
under aqueous conditions provides for simultaneous
146
I body fluid samples from rats and humans (Coutts et al.,
C H 2 C H 2 C H 2N(CH s)2
I /CH s
CH2 CH2CH2 N \ C - CH s
II
II o Young, 1991; Caccia and Garattini, 1992). The acetyla-
O tion strategy should prove to be a very useful one for
II
I agent, has had important implications for neuroche-
CH2CH2CHz N(CHs)2
0
Ill
II
CHsCO,~~
I /CH s
CH2CH2CH2N\ C - CHs
II
IV o agents, providing derivatives that often have excellent
Figure 6, Structures of derivatives of imipramine (I), desipra-
mine (II), 2-hydroxyimipramine (III) and 2-hydroxydesipramine
(IV) following acetylation under aqueous conditions. Imipramine
is underivatized but under the basic conditions used is subse-
quently extracted into the organic solvent.
analysis of imipramine, desipramine, 2-hydroxyimipra-
mine, and 2-hydroxydesipramine. This procedure,
combined with GC-NPD and GC-MS has been uti-
lized to study metabolism of imipramine in vitro
by CYP2D6 expressed in human cell lines (Coutts et al.,
1993; Su et al., 1993). The fact that acetic anhydride
derivatizes phenolic hydroxyl groups under both
aqueous and anhydrous conditions but derivatizes alco-
hol hydroxyl groups only under anhydrous conditions
has value in structural elucidation. By making use of
this knowledge and comparing mass spectral frag-
mentation patterns of antidepressants and potential me-
tabolites before and after derivatization with acetic an-
hydride, under both aqueous and anhydrous conditions,
identification of the location of hydroxyl groups in the
metabolites of drugs such as the tricyclic antide-
pressants can be greatly facilitated. This strategy
JPM Vol. 31, No. 3
June 1994:141-148
has been employed to examine metabolites of such
drugs as imipramine, trimipramine, and iprindole in
1990, 1991a,b; Hussain et al., 1991), and investigate
CYP2D6-catalyzed formation of metabolites of amphet-
amines and antidepressants in vitro (RT Coutts et al.,
unpublished data).
Several antidepressants and antipsychotics are exten-
sively metabolized, and recent reviews have stressed the
importance of considering metabolites in the overall
therapeutic actions and side effects of psychotropic
drugs (Midha et al., 1987; Potter and Manji, 1990;
future studies on metabolism of such drugs.
In summary, the use of acetic anhydride, under both
aqueous and anhydrous situations, as a derivatizing re-
mistry, pharmacology, and biological psychiatry. Acety-
lation under aqueous conditions has proved to be a rapid
and effective means of improving the extractability of
amine- and phenol-containing neurochemicals and drugs
from biological fluids and aqueous supernatants of tis-
sue homogenates; in doing so, it also offers an addi-
tional rapid clean-up stage in assay procedures. In some
instances (e.g., with some GC-NPD and GC-MS
assays), the acetylated derivatives can be used directly
for analysis. However, after acetylation, primary amines
can be derivatized further with perfluoroacylating re-
properties for subsequent analysis by GC-ECD or GC-
MS. In addition, esters formed by acetylation of phenols
can subsequently be hydrolyzed selectively to free the
phenolic function for reaction with a perfluoroacylating
reagent in a later stage of an assay procedure. Reaction
with acetic anhydride (under aqueous and anhydrous
conditions) is also proving to be a useful technique for
investigating detailed drug metabolism, and it is antici-
pated that this application will be important in future
research.
Funds for research conducted by the authors have been provided by the
Medical Research Council of Canada, the Alberta Mental Health Research
Fund, and the Alberta Heritage Foundation for Medical Research. Dr. A.
Holt gratefully acknowledges fellowship support from Ciba-Geigy Phar-
maceuticals. The authors are grateful to Ms. Sally Omura for typing this
manuscript.
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