Keio Journal of Medicine

Vol. 16, No. 1. March 1967

SIMPLE, SENSITIVE AND RAPID METHODS FOR

THE DETECTION OF MORPHINE IN URINE

TETSUO OKA and EIKICHI HOSOYA*

Department of Pharmacology, School of Medicine, Keio University, Tokyo, Japan

(Received for publication February 20, 1967)

INTRODUCTION

From the scientific and criminal points of view, it is an important and inter-
esting problem to detect very small amounts of morphine in urine. From old
times, therefore, numerous methods have been devised by many investigators
(Pierce and Plant, 1932; Wolff et al., 1033; Oberst, 1938-39; Gross and Thomp-
son, 1940; Stolman and Stewart, 1949; Goldbaum and Kazyak, 1952; Jatzkewitz,
1953; Woods et al., 1954; Mannering et al., 1954; Fujimoto et al., 1954; Jatz-
kewitz, 1954; Stevenson and Rapoport, 1955; Feldstein and Klendshoj, 1956;
Paerregaard, 1957; Tompsett, 1960; Morgan, 1961; Cochin and Daly, 1962;
Paulus et al., 1962a, 1962b; Kazyak and Knoblock, 1963; Mule, 1964; Hosoya,
1965; Akera and Hosoya, 1965). But none of them was satisfactory enough
because of the difficulties in extracting very small amounts of morphine from
relatively large amounts of urine, whereas the identification of the urine extract
can be done well by paper-partition, thin layer and gas chromatography.
The authors tried to solve this problem by utilizing the specific adsorptive
nature of charcoal and to establish methods which may meet satisfactorily the
demands from field workers as well as from laboratory investigators.

METHODS

I. Materials and Tools.

Urine: Human urine of healthy young male, to which 5, 10, 20, 30,•c,

100μg of morphine hydrochloride as free base was added, was used. The urine

* Professor of Pharmacology .

23
24TETSUO OKA and EIKICHI HOSOYA

of non-tolerant dogs too, who were injected 100mg/kg of morphine hydrochloride

subcutaneously 4 hours in advance, was used.
Charcoal: Activated charcoal for chromatography (WAKO Pure Chemical
Industries, Tokyo, Japan) was arranged from 50 to 200 mesh with sieves, dipped
in 1N HCl solution overnight and poured into a column. The column was washed
with distilled water, glacial acetic acid and distilled water in the order of these.
Then, the charcoal in column was put into an evaporating dish, heated to dryness
at 120℃ in an oven for several hours, preserved in a desiccator and used when

necessary.
Thin layer chromatography: Kieselgel-G "Merck" was used for thin layer
chromatography. The following four solvents were used for development. (1)
Methanol, (2) Dioxane: 28% Ammonia water, 60: 5, (3) n-Butanol: Glacial
acetic acid: Water, 4:1:2, (4) n-Butanol: n-Propanol: 1/10N NH4OH,
2:1:1.
Paper chromatography: Toyo filter paper No. 51 (Toyo Roshi Kaisha, To-
kyo, Japan), prepared for chromatography, was used. Descending chromatograms
were developed for about 16 hours in chromat-cabinet. The following two
solvents were used. (1) n-Butanol: Glacial acetic acid: Water, 4:1:2, (2)
n-Butanol: n-Propanol: 1/10N NH4OH, 2:1:1.
Column: Two kinds of glass columns were used usually. One was 40cm
high and 6cm I.D. and was used only for the pretreatment of charcoal. The
other was 30cm high and 1cm I.D. and 1g of charcoal was poured in to this
column. If the volumes of urine were more than 50ml, larger columns were used
in few cases.
Reagents: All reagents used were reagent grades.

II. Procedures.
Two procedures are postulated, that is, (A) Simple procedure and (B)
Standard procedure.

(A) Simple procedure.

1. Pass the urine through a charcoal column and let morphine adsorb on
the charcoal: It must be noticed that air gases adsorbed on charcoal had to be
driven in vacuum desiccator before carcoal is poured into the column. Fifty
milliliter of urine was poured into a column containing 1g of charcoal. The
current runs through the column were adjusted at the speed of about 5ml/min.
Adjustment of pH of the urine was unnecessary.
2. Wash the charcoal column by 50ml of water.
 DETECTION OF MORPHINE IN URINE25

3. Elute morphine adsorbed on charcoalby glacialaceticacid: Both free

and bound morphine were elutedwith 20ml of glacialaceticacid.
4. Evaporate the aceticacid by heating:20ml of eluantwas evaporated to
dryness in about 10 minutes by heating and ventilation.Small parts of bound
morphine were hydrolizedto free morphine at this stage.
5. Dissolveaceticacid residue with small volumes of distilled water: Ac-
cording to the amounts of the residue,from 0.1 to 0.5ml of water were used.
6. Spot on thin layer: The more amounts were spotted at the original
points,the better resultswere obtained usually. However, in some instances,
residue in the solutionwas too much to be spottedso that they disturbedthe
identification.
7. Develop thin layer chromatcgraphy: Solvent (1) and solvent (2) had
advantages that the developmentaltime was short and identification was com-
pleted quickly. But the spot due to bound morphine did not move from the
originalpoint and was difficult to detect. Solvent (3) and (4) took about 60
minutes for development,but they fitfor the detectionof bound morphine. Free
morphine was easilydetectableby any one of these four solvents.At leasttwo
solventsamong the four were used in our experiments.
8. Spray iodoplatinatereagent (Munier and Macheboeuf, 1949): By com-
paring Rf and colorwith the spots due to authenticsubstances,identification
can be done without difficulty.
9. Where gas chromatography can be used,aceticacid residueat the end of
4th stage of thisprocedurehad betterbe dissolvedin 0.1-0.5ml of benzyl alcohol
and put into gas chromatography. Detectionand identification can be done easily
and rapidly.

(B) Standard procedure.

1. The residuein evaporating dish,that had been gained at 4th stage of
(A), was dissolvedin four ml of water. The solutionwas hydrolizedin boiling
water for 15 minutes with equal volume of conc.HCl solution.
2. The hydrolysatewas adjusted to pH 2.5 with 5N NaOH and four
volumes of chloroform (containing10% isopropanol)were added to it. Then the
mixtures were shaken vigorouslyfor fiveminutes with mechanical shaker.
3. After separatingand discardingthe organic solvent,the water layerwas
adjusted to pH 9.0 and four volumes of chloroform (containing 10% isopropanol)
were added to it. Then the mixtures were shaken for five minutes.
4. After separating and discarding the water layer, the organic solvent was
evaporated to dryness by heating and ventilation.
26 TETSUO OKA and EIKICHI HOSOYA

5. The residue was dissolved in 0.4ml of methanol (containing 10% isoamyl

alcohol) and thereafter the same procedures as A-6 to A-8 were done.

RESULTS

1. The adsorptive nature of charcoal to free and bound morphine were

shown in Table 1.

Table 1
The Adsorptive Nature of Charcoal to Free and Bound Morphine

2. Influence of pH change on adsorptive ability of charcoal: The adsorptive

ability of charcoal concerning free and bound morphine did not change at pH 5, 7

and 9 of urine and in 1N HCl solution.

3. The limit of detectable amounts of morphine in urine by two procedures

are presented in Table 2.

Table 2
The Limit of Detectable Amounts of Morphine in 50-100ml of Urine by
Two Procedures with Thin Layer (TLC) and Paper Chromatography (PC)

4. Dog urine to which 100mg/kg of morphine hydrochloride was injected

subcutaneously and collectedfour hours after morphine administration , showed
the existence of free (Rf 0.68) and bound morphine (Rf 0 .22) on paper chromato-
 DETECTION OF MORPHINE IN URINE 27

gram by simple procedure and one spot of free morphine by standard procedure
using No. 1 developing solvent as Figure 1 shows.

Fig. 1 Paper chromatogram of the urine

of dog administered morphine,
treated by respective procedures.
a: Simple procedure. b: Standard proce-
dure. c: Free morphine. d: Bound mor-
phine. Paper chromatogram developed des-
cendingly with the solvent mixture, n-buta-
nol, glacial acetic acid and water (4, 1,
2) and sprayed by iodoplatinate reagent.

5. Using the urines of five non-tolerant human adults to whom 10mg of

morphine hydrochloride was injected subcutaneously and from whom urines were
collected every four hours continuously for 72 hours, showed clearly the existence
of free morphine till 24 hours and of bound morphine till 52 hours after morphine
administration respectively by the combination of two procedures.

DISCUSSION

As for the establishment of new method, the following four factors should
be brought into consideration; i.e., simplicity, sensitivity, specificity and accuracy.
1. Simplicity: No particular apparatus, reagents and solvents nor special
technic are necessary except the case of gas chromatography, if any. The
time necessary for the detection of morphine in six urine samples is 120 minutes
by simple procedure, 180 minutes by standard procedure, when the identification
28 TETSUO OKA and EIKICHI HOSOYA

is done by thin layer chromatography. In normally equipped laboratory, 40

urine samples can be treated in 8 hours by one worker by simple procedure and
20 urine samples can be treated in 8 hours by standard procedure.
2. Sensitivity: 50μg of morphine (as free base) in 50ml of urine can be

detected by simple procedure. Ten microgram of morphine in 50ml of urine can

be detected by standard procedure. These are the values by thin layer chromato-
graphy. Sensitivity will be doubled when paper chromatography is used, although
the time necessary for detection is longer (Table 2).
3. Selectivity: The final identification of morphine is done by thin layer,
paper or gas chromatography. Accordingly, the selectivity depends mostly upon
the chromatographic nature of the solvents. Chlorpromazine which shows a
similar spot by iodoplatinate reagent is not adsorbed on charcoal and is excluded
at early stage. Codein, pethidine and nalorphine too are adsorbed on charcoal
but they can be distinguished from morphine by Rf values.
4. Accuracy: Recovery of free and bound morphine in urine by charcoal
is found to be high (Table 1). The detectable amounts of morphine by simple
procedure are changed a little according to the density of urine, but by standard
procedure they are almost unchanged.

As mentioned already, charcoal is the good tool for the adsorption of mor-
phine in urine, especially of large amounts. Moreover, the charcoal method will
be able to apply to gas chromatography, fluorescent spectrophotometry, spectro-
photometry and also to radioactivity methods.
One hazard which have not been settled yet in charcoal method, is that the
interfering substances increase when large amounts of urine is used.

SUMMARY

New methods utilizing the adsorptive nature of charcoal were proposed for
the detection of very small amount of morphine in urine. The advantages of
the methods are (a) simplicity, i.e., no particular apparatus, reagents and
compounds nor special technic are necessary throughout the procedures (b)
sensitivity, 50μg of morphine in 50ml of urine can be detected by simple

procedure and 10μg of morphine in 100ml of urine are detectable by standard

procedure (c) rapidity, treatment of six urine samples is completed in 120

minutes by simple procedure and 180 minutes by standard procedure. These
advantages may satisfy, the demands depending on their aims from field workers
as well as from laboratory investigators.
 DETECTION OF MORPHINE IN URINE 29

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