Drugs and Poisons in Humans - A Handbook of Practical Analysis (Part 21)

Since morphine and codeine are fi nally excreted into urine in the conjugated forms with glucuronic acid [1–3], it is necessary to hydrolyze the conjugated forms of these compounds befo

© Springer-Verlag Berlin Heidelberg 2005

II.2.3 Morphine

and its analogues

by Hideyuki Yamada and Kazuta Oguri

Introduction

anti-tussives; 1 % powder of codeine or dihydrocodeine is commonly included in over-the-counter drugs of antitussives

> Figure 3.1 shows metabolic pathways of morphine, heroin and codeine Since morphine

and codeine are fi nally excreted into urine in the conjugated forms with glucuronic acid [1–3],

it is necessary to hydrolyze the conjugated forms of these compounds before GC/MS analysis

6-acetylmorphine is recommendable for diagnosis of heroin use, because of its relatively long half-life in the body [4]

For accurate diagnosis of a cause of death in an opiate poisoning case, the ratio of a free form to a conjugated form becomes important (see section 4 of this chapter) In such a case, an

of a conjugated form can be calculated by subtracting the amount of a free form from the total amount By HPLC, the simultaneous analysis of free and conjugated forms is possible without any hydrolysis; in the near future, LC/MS may become a main tool for analysis of opiates and their metabolites However, at the present time, GC/MS is being widely used for opiate analysis

For HPLC analysis of the conjugated forms of opiates, the authentic standards of mor-phine-3-glucuronide (M-3-G) and morphine-6-glucuronide (M-6-G) are necessary In U.S.A and Europe, it is easy to obtain these authentic compounds from commercial sources, but the import of these compounds to Japan is strictly controlled; easing of import of such compounds should be realized

GC and GC/MS analysis

Reagents and their preparation

hydro-chloride (Sankyo Co., Ltd., Tokyo, Japan) is dissolved in purifi ed water to prepare 1 mg/mL solution A 0.1-mL aliquot of this solution is mixed with 1.9 mL of purifi ed water for 20-fold dilution (the fi nal concentration in the form of the hydrochloride salt: 50 µg/mL), which is stored at –20 °C

Main metabolic pa

of morphine hydrochloride (Shionogi & Co, Ltd., Osaka, Japan and other manufacturers)

is dissolved in purifi ed water to prepare 1 mg/mL solution; 0.1 mL of this solution is mixed

3.33-fold and 10-fold to prepare 6 and 2 µg/mL solutions, respectively, which are also stored at –20 °C For preparing the standard solutions to be used for a calibration curve of 6-acetylmorphine hydrochloride or codeine phosphate (Shionogi & Co.), the same dilution procedure is followed 6-Acetylmorphine hydrochloride can be synthesized by the method previously reported [6]

water in a 100-mL volume glass beaker with stirring in an ice bath Aft er the temperature

of the solution is cooled to room temperature, it is transferred to a 100 mL-volume volu-metric fl ask together with the water solution which has been used for washing the above

solution

about 70 mL purifi ed water in a 100-mL volume glass beaker with stirring in an ice bath

until its saturation with stirring (about 30 g necessary) Upon saturation, the volume of the

at room temperature

of methanol and 2 mL purifi ed water are passed through a Bond Elut Certify cartridge (Var-ian, Harbor City, CA, USA) just before use For a new cartridge, each solution can fl ow by natural pressure through it without any aspiration When the fl ow stops, it is aspirated for a

should not be passed through the cartridge completely; the cartridge should not be dried

Analytical conditions

USA)

GC/MS conditions; instrument: an HP5890 GC-HP 5989A MS Engine (Agilent Technolo-gies, Palo Alto, CA, USA); column (oven) temperature: 200 °C (0.5 min) →5 °C/min →260 °C

fl ow pressure: 6 psi; ionization mode: EI; electron energy: 70 eV

GC and GC/MS analysis

Two extraction methods, the liquid-liquid extraction and the solid-phase extraction, are avail-able; the latter gives cleaner extracts for GC and GC/MS analysis

i Liquid-liquid extraction

50-mL volume glass centrifuge tube with a ground-in stopper, and heated at 100 °C for

level of the water bath should be slightly above the surface level of the hydrolysis solution

in the tube As a blank test, 2 mL urine obtained from a healthy subject is also treated as above For quantitative experiments, in addition, a 0.1-mL volume each of solutions at

urine; these samples are also processed in the same way as above

ii Aft er cooling to room temperature, 0.1 mL of the IS solution is added to the hydrolyzed solution A 3-mL volume of 5 M NaOH is added to the solution for neutralization, followed

solu-tion should be checked with a test paper (Whatman, type CF); if the pH of the solusolu-tion shift s from 9, it should be readjusted to pH 9.0 by adding the above ammonium buff er solution

iv Aft er centrifugation at 3,000 rpm for 5 min, the organic (lower) layer is carefully trans-ferred to another 50-mL volume glass centrifuge tube of the same type with a pipette,

mixed well

stream of nitrogen with warming the tube at 30–40 °C

solution is injected into GC or GC/MS

vii For the unconjugated (free) forms of morphine, codeine and 6-acetylmorphine, the extraction

of the solution is confi rmed to be 9 with a pH test paper; if not, an appropriate amount of the

isopropanol (9:1, v/v), and the following procedure is performed according to the above iv–vi

ii Solid-phase extraction

solution is comfi rmed to be 8–9 with a test paper If not, the pH of the solution is adjusted

cartridge slowly taking more than 2 min to adsorb an opiate

(pH 4) and methanol, and dried for 2 min by aspiration with a vacuum manifold, followed

by washing with 3 mL methanol and drying for 5 min again

wa-ter (80:20:2, v/v) through the cartridge, into a 10-mL volume glass centrifuge tube with a

evaporated to dryness under a stream of nitrogen with warming at 30–40 °C

that described in the step vi) of the above liquid-liquid extraction section

vii For the unconjugated (free) forms of morphine, codeine and 6-acetylmorphine, the extrac-tion is made as follows A 2-mL volume of urine is diluted 2-fold with purifi ed water and mixed with 0.1 mL of the IS solution and 0.2 mL of 2 M Tris-HCl buff er solution (pH 8.1)

Assessment and some comments on the methods

Qualitative analysis is performed by fi nding a peak appearing at the same retention time

as that of the authentic standard aft er trimethylsilyl (TMS) derivatization; it is also

iden-tifi cation is made by comparing a mass spectrum obtained from a test specimen with that obtained from the authentic standard A total ion chromatogram (TIC) of the authentic

in > Fig 3.3 Quantitation is performed by selected ion monitoring (SIM) using the peak

height or area ratio of a test compound to IS; the ratio is applied to a calibration curve, which has been prepared in advance, to calculate the concentration of the test compound in a

speci-⊡ Figure 3.2

TIC of TMS derivatives of morphine and its analogues by GC/MS Heroin is not derivatized

The peak of the TMS derivative of ethylmorphine is not included in this chromatogram; but it is eluted at 9.4 min under the same conditions.

GC and GC/MS analysis

⊡ Figure 3.3

quantitation)

By SIM of GC/MS, a low level of morphine less than 100 ng/mL can be detected and quanti-tated; however, when a low level of morphine is detected, special care should be taken, because

of the following reasons Low levels (0.1–25 pmol/g tissue) of morphine exist in various ani-mals including humans as an endogenous compound; the levels are signifi cantly increased by

poppy seeds are being used for various foods; when the foods including the seeds are eaten, an appreciable amounts of morphine (including its conjugates) are reported to be excreted into urine [9] Because of these reasons, it seems reasonable to set a cutoff value of morphine in urine to be 300 ng/mL However, caution is needed, because a morphine concentration higher than the cutoff level can be detected in some urine samples of subjects who have eaten a food containing poppy seeds [9] It should be noted that an amount of codeine comparable to that

of morphine are also included in the poppy seed- containing foods [10]

is, therefore, impossible to detect heroin itself from urine to prove its abuse 6-Acetylmorphine is alternatively being analyzed as an indicator of heroin abuse; however, even with this metabolite, its half-life is only about 40 min, which is much shorter than that of unconjugated morphine (3.7 h) [4] Even if the cutoff value of 6-acetylmorphine is set to be as low as 0.8 ng/mL, it is being consid-ered necessary to analyze a urine specimen sampled not later than 5 h aft er the heroin intake [4]

HPLC analysis

Reagents and their preparation

and their preparation”, above of GC and GC/MS analysis section

is dissolved in about 80 mL purifi ed water, followed by the adjustment of its pH to 9.4 with

stored at room temperature

Mass spectra of TMS derivatives of the authentic morphine and its analogues A: morphine-TMS; B: 6-acetylmorphine-TMS; C : codeine-TMS; D: ethylmorphine-TMS; E: nalorphine-TMS A 50-µL

volume of BSA is added to 20 µg each of opiates for derivatization [see the section 2-3)-(1)-vi)];

a 0.4-µL aliquot each is injected into GC/MS.



HPLC analysis

• 5 mM Ammonium sulfate solution (pH 9.4): a 66-mg aliquot of ammonium sulfate is sub-jected to the above procedure, and stored at room temperature

dissolved in about 400 mL purifi ed water, followed by adjustment of its pH to 2.1 using

adding purifi ed water, and stored at room temperature

dissolved in about 80 mL of purifi ed water, followed by adjustment of its pH to 2.1 using

purifi ed water, and stored at room temperature

in about 70 mL purifi ed water in a volumetric fl ask, and left overnight to dissipate bubbles Purifi ed water is gently added to the above solution to prepare 100 mL solution, mixed well and stored at room temperature

2.1)/ace-tonitrile (76:24, v/v, 1 L): a 152-mL volume of 0.5 M phosphate buff er solution and 19 mL

of the above 0.2 M SDS solution are placed in a 1-L volume glass graduated cylinder; the

fi nal volume of the solution is adjusted to 760 mL by adding purifi ed water, well mixed and transferred to a 1-L volume Erlenmeyer fl ask A 240-mL volume of acetonitrile of HPLC grade is measured with the above empty graduated cylinder, and added to the solution in the Erlenmeyer fl ask Aft er mixing, the mobile phase solution is passed through a fi lter

in a clean glass container with a stopper at room temperature A required amount of the above solution is transferred to another glass container, and degassed by immersing the container in an ultrasonic cleaner for 10 min just before use

50 mM phosphate buff er solution (pH 2.1) and 10 mL acetonitrile are placed in a graduated cylinder, and the volume of the solution is adjusted with purifi ed water to 100 mL and mixed well

buff er (pH 2.1)–10 % acetonitrile and 5 mL purifi ed water are successively passed through

use For the handling method, see “Reagents and their preparation”, of the GC and GC/MS analysis section of this chapter

Analytical conditions

HPLC conditions; instrument: a Hitachi 655 HPLC instrument (Hitachi Ltd., Tokyo, Japan) equipped a wavelength-variable UV monitor (Hitachi 655A); mobile phase: 5 mM-SDS con-taining 100 mM phosphate buff er (pH 2.1)/acetonitrile (76:24, v/v); fl ow rate: 1.2 mL/min; detection wavelength: 220 nm

Procedure

A modifi cation of the method developed by Svensson et al [11] is shown below

i A 5-mL volume of 0.5 M ammonium sulfate solution (pH 9.4) is added to 2 mL blood plasma or 4 mL of 2-fold diluted urine When required, the mixture solution is passed

speci-men obtained from a healthy subject and the urine specispeci-mens spiked with 0.1 mL each of

0.5 mL purifi ed water

iv Opiate compounds (free and conjugated forms) trapped in the cartridge are eluted with 1.2 mL methanol

into HPLC

Assessment and some comments on the method

Qualitative analysis is made by the comparison of the retention time of a peak of a specimen with that of the authentic standard and also by the confi rmation of the absence of such a peak in

For quantitation, the peak area or height of a test compound in a specimen is applied to an

accu-rate quantitation, but the authors have not studied on it Ethylmorphine may be usable as IS like

in the case of GC/MS analysis With a UV detector, the good linearity of morphine could be confi rmed in a range not lower than 20 ng/mL; the detection limit was reported to be 5 ng/mL [11] With an electrochemical detector, the analysis of opiates is reported to be achievable with even higher sensitivity (detection limit less than about 1 ng/mL) [12] However, since the elec-trochemical detection is based on the oxidoreductive potential of the hydroxyl group of the phenol moiety, it is not suitable for analysis codeine or M-3-G For cautions and cutoff values upon detection of trace levels of morphine, the readers can see the 2-4) section of this chapter

could be obtained in a range over 30 ng/mL, and the detection limit was 1–3 ng/mL [13]

Toxic and fatal concentrations

Fatal doses of morphine and heroin (in the form of their hydrogen chloride salts, per os) were

reported to be 70–500 mg and 10–600 mg, respectively [15], with great variation according to individuals As one of the reasons for the increase in fatal doses, the resistance to an opiate acquired by its repeated use can be mentioned In a chronic opiate-dependent patient, several grams of an opiate do occasionally not cause death

Toxic and fatal concentrations

As a result of analysis of 203 fatal cases with morphine and heroin, three parameters were reported to be related to the fatality; they are a blood concentration of free morphine (not lower

blood (not lower than 37 %) and a total morphine concentration in the brain (not lower than

taking place within 3 h aft er morphine intake, a phenomenon that the ratio of free morphine to its total amount in blood is not lower than 44 % is said to be characteristically observed [16]

Notes

a) Nalorphine at the same concentration can be also used as IS In the authors’ experience, it

is less stable and its solution should be prepared just before use If a deuterium-labeled morphine is available for use, it is most desirable for MS analysis

b) Convenient thick glass-made devices for assisting elution of extraction cartridges are com-mercially available ( GL-SPE Vacuum Manifold, GL Sciences, Tokyo, Japan and some man-ufacturers in USA); it enables the attachment of 12 cartridges, and the simultaneous wash-ing, elution and drying of the cartridges can be made under reduced pressure

HPLC chromatogram (detected at 220 nm) of the authentic morphine and its related compounds A: 6-sulfate; B: nor3-glucuronide; C: 3-glucuronide; D: morphine-6-glucuronide; E: codeine glucuronide; F: morphine The opiates and their conjugated forms,

10 µg each, were dissolved in 4 mL purified solution, and analyzed according to the procedure described in the section 3-3).

⊡ Figure 3.4