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 130oC 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).

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