Protocol for Analysis of Doxepin

by Floriza Michelia (260110110069)

The protocol for performing an analysis in a particular specimen, especially biological

samples differs depending upon the goal or questions to be answered. It can be specifically
for drug use, drug dependence, overdose situations of medication or abused drugs, chronic
exposure, or drug toxicity. Analysis, actively or passively performed, all are matters that
could be answered through a specific analysis.
Analysis of doxepin that cause several case of poisoning provides information which
needed to understand based on dose-response relations. The specimen from body matrices
usually used for toxicological analysis of clinical and forensic interest are blood, urine, saliva,
hair, breath, sweat, any tissues or fluids is considered suitable for the particular analysis
requested. Here, methods for analysis are described by various protocol depends on the
biological samples.
I.

Blood
a. Chemicals and reagents
All chemicals were analytical grade with purity greater than 98% and
were supplied by Sigma. Doxepin, sulpiride and SKF525A standards (1
mg/mL in ethanol) were obtained from Institute of Forensic Science Ministry
of Public security P.R.C.
b. Sample Preparation and Extraction
One mililiter of blood were brought with distillate water to a volume of
3 mL, and then homogenized in a 10 mL tube of centrifuge (3000xg, 10
minutes). Prior to vortex for mixed, internal standard solution (SKF525A0 was
added for final concentration of 3 g/mL. The solution was brought to pH of
10 with the addition of NaOH 2 M. To each sample, 5 mL of ethyl ether was
added.
After horizontal agitation during 10 minutes and centrifugation at 3000
rpm for 5 minutes, the organic extract was transferred into a glass tube then

evaporated to dryness at 40C under a gentle stream of air. The residues were
reconstituted by 50L of ethanol, one micro liters were injected into the
GC/MS and two micro liters were injected into the LC-ESI-MS/MS.

c. Instrumentation and MS/MS conditions

The doxepin analyses were performed with a Thermo-Fisher gas
chromatography/mass spectrometry system (GC/DSQ). Capillary column
DB5-MS (30 m 0.25 mm 0.25 mm) were used for analysis. Carrier was
helium at the flow rate of 1 ml/min. Injector temperature, 280C, injection
mode was split, split ratio, 50:1. Transfer line temperature, 250C. Ion source
temperature, 250C. The column temperature program used for doxepin was:
initial temperature 100C for 1 min, 20C/min to 280C, maintained for 4 min.
The instrument was used in full scan EI (70 eV) mode, scanning in the range
between 40650 m/z.

II.

Urine
Urine is one of the most common proteomic samples in diagnostics and also in
clinical analysis because of its nature that easily attainable. However, due to its
highly variable contents, and the presence of various proteins in low abundance or
modified forms, urine is considered one of the most difficult samples to work
with.
According to International Union of Pure and Applied Chemistry, urine is the
human fluid which contains water and metabolic products and is excreted by the
kidneys, stored in the bladder and normally discharged by the way of urethra.
a. Materials
Ultraviolet analyses were perfomed with a Cary Model 11-A recording
spectrophotometer.

Reagents
Spectroquality n-hexane.Potassium permanganate, saturated aqueous
solution.

Standards.
Stock solution was prepared by dissolving 100 mg of doxepin
hydrochloride per 100 milliliters in water. We diluted the stock with
human urine to provide working standars.

b. Method
Five milliliters of urine are pipetted into a 40-ml centrifuge tube
followed by 1.5 ml of the saturated KMnO4 solution. The tube is shaken to
mix thoroughly and allowed to stand for 5 mm. The KMnO4 oxidizes doxepin
to its corresponding ketone, theN, N-dimethyl--y-propylammne side-chain
being replaced by =0.
Doxepin ketone is extracted from the oxidized urine sample by shaking
for 10 mm with 10 ml of n-hexane. The tubes are centrifuged to sharply
separate the phases, and the hexane extract is removed for ultraviolet analysis.
Hexane extracts are scanned in the ultraviolet region from 260 to 300 nm in
spectrophotometer cells with a 20-mm light path. Spectroquality n-hexane is
used as the solvent blank. Absorbance at 295 nm (trough) is subtracted from
the absorbance at 266 nm (peak) to obtain net absorbance.

III.

Hair
In recent years hair has become a fundamental biological specimen for drug
testing, alternative to the usual samples such as blood and urine. The major
practical advantages of hair testing are larger detection windows (form 3 days to
years), depending on the length of the hair shaft, compared to those of urine or
blood (hours to 2-4 days for most drugs); evaluation of long term history to short
term history; and the sample collection is non-invasive, it is easy to be performed
under conditions that prevent adulteration and substitution.
a. Sample
Sample (approximately 200 rag, 1 cm in length) were collected by
clipping hair fromthe same area on the back of the head approximately 1 cm
fromthe skin. Samples were placed in plastic bags until analyzed. Negative
hair samples were washed in deionized water, dried at room temperature, and
pulverized.

b. Standards and Controls

The doxepin and desmethyldoxepin standard stock solutions (1
mglmL) were diluted with methanol to concentrations of 10, 2, and 1 pg/mL.
The doxepin-d3 standard stock solution (100 IJg/mL) was diluted with
methanol to 10 pg/mL. Six-point standard curves were prepared for doxepin
and desmethyldoxepin by using 50-mg aliquots of the negative hair spiked
with methanolic solutions of both drugs to achieve the following
concentrations of standard hair preparations: 0.25, 0.50, 1.0, 5.0, 10.0, and
20.0 ng/mg.
In addition, two levels of controls were prepared. The low control (2
ng/mg) were made by adding 50 IJL of both 2-1Jg/mL doxepin and
desmethyldoxepinsolutions to 50 mg of pulverized negative hair samples. The
high controls (15 ng/mg) were prepared by adding 75 IJL of the lO-pg/mL
solutions of both doxepin and desmethyldoxepin to 50 mg of pulverized
negative hair.

c. Analytical Procedure
The patient's hair samples were washed in deionized water and allowed
to dry at room temperature. They were then pulverized, and 50-mg aliquots
were analyzed in triplicate. Twenty microliters of the 10-lJg/mL solution of
the internal standard (doxepind3) was added to the hair samples, standards,
and control preparations.
This was followed by the addition of 0.1M HCl (3 mL). The test tubes
were capped, vortex mixed, and incubated overnight (18 to 24 h) at 50~ The
tubes were removed from the heating block and allowed to cool down before
being centrifuged for 5 rain (400 x g). The supernatant was transferred to clean
tubes, 1 mL 1.93M acetic acid was added to each test tube, and they were
vortex mixed. Ten milliliters of deionized water was then added to each tube.
The HCX columns were conditioned as follows: methanol (3 mL),
deionized water (3 mL), and 1.93M acetic acid (1 mL). The samples were
added to the columns and slowly drawn through before drying 1-2 min. The
columns were washed with 3 mL of deionized water (dried 1-2 rain), 1 mL of
0.1M HCI (dried 1-2 min) and 3 mL of methanol (dried 5 min). The doxepin,

desmethyldoxepin, and doxepin-d3 were eluted by allowing 3 mL of the

methylene

chloride/isopropanol/ammonium

hydroxide

(78:20:2,vh~/v)

mixture to pass through the columns.

All samples were then evaporated to dryness under a stream of air. The
drug residue was reconstituted in acetonitrile (40 pL). The samples were
transferred to autosampler vial inserts, and the vials were crimped. The
samples were derivatizedby adding BSTFA with 1% TMCS (40 pL) with a
syringe and vortex mixed. After incubating for 15 rain at 60~ the samples
were cooled and analyzed using GC-MS.

d. Chromatographic Method
The injector was working in the splitless mode, and a 1-pL volume was
injected. The injector temperature was 270oCTheflow rate of the cartier gas
(helium) was 1.2 mL/min. The initial GC oven temperature of 130 oC was held
for 1 min, then it was increased at the rate of 12oC/min until it reached
280oC.This temperature was maintained for 3 min.
The MS ion source temperature was 230~ and the quadrupole
temperature was 150oCThe instrument operated inselected ion monitoring
(SIM) mode, and the followingions were monitored: for doxepin m/z 58and
279, for doxepin-d3 m/z 61 and 282, and for desmethyldoxepin m/z 116 and
337. The followingions were used for quantitation: m/z 58 for doxepin,rn/z 61
for doxepin-ds, and rn/z 116 fordesmethyldoxepin. The dwell tirnes were 50
ms form/z 58, 61,279, and 282 ions and 100 ms for m/z116 and 337 ions.

IV.

Tissues
a. Sample
Human Milk

b. Methods
Diagnosed as having a major depressivedisorder. Treatment with
oxazepam initiated bythe obstetrician was unsuccessful and caused some
drowsiness in the infant. On admission to a psychiatric inpatient unit 30 days
postpartum she was treated with 150 mg doxepin at night. The acute episode

began to resolve after 2 weeks,after which she was discharged and then
treatedas an outpatient for some 6 months.
Doxepin and N-desmethyldoxepinconcentrationsin the milk and
plasma were analysedbyhigh performance liquid chromatography(h.p.l.c.). An
aliquot of biological fluid (1 ml)was placed in a polypropylene tube, 200
ngofamitriptyline was added as internal standard andthe sample was
alkalinised by the addition of 0.1ml 1 M NaOH. The sample was extracted
with 10ml hexane containing 1% isoamyl alcohol byshaking for 5 min.
After centrifugation, 9 ml ofthe organic phase was transferred to a
cleanpolypropylene tube and the compounds ofinterest were back extraced by
shaking with 0.2ml of 0.05 M HCl. After a further centrifugation,the organic
phase was aspirated and discardedand 50 ,ul aliquots of the acidic extract
wereinjected onto the h.p.l.c. column was used with a mobile phase of
40%acetonitrile in an aqueous solution of 0.01%H3PO4 and 0.01% NaCl
(flow rate = 2 ml miMf1).Peaks were detected by their ultraviolet
absorbanceat 210 nm.
Known concentrations ofdoxepin and N-desmethyldoxepin in both
milkand plasma were put through the above procedureand test samples were
quantified from aplot of peak height ratio (drug/intemal standard)vs drug
concentration. The intra-assay coefficientof variation for doxepin in plasma
andmilk was tested at 50 and 200 ,ug 1-1 and rangedfrom 1.3 to 3.6% (n = 5);
coefficients

of

variationfor

N-desmethyldoxepin

at

similar

plasmaconcentrations ranged from 0.6 to 3.8% (n = 5).Inter-assay coefficient

of variation, assessedfrom the slope of the standard curve on differentdays was
6.2% for doxepin and 8.7% for Ndesmethyldoxepinin milk (n = 4) and 6.2%
fordoxepin and 8.4% for N- desmethyldoxepininplasma (n = 7).

References
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