morphine blood

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Determination of morphine in the plasma of addicts in using Zeolite Y

extraction following high-performance liquid chromatography

Mahmoud Ghazi-Khansari a,*, Rezvan Zendehdel a,b,Morteza Pirali-Hamedani b, Mohammad Amini b

a Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, P. O. Box 13145-784, Tehran, Iranb Department of Medicinal Chemistry, School of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran

Received 21 May 2005; received in revised form 27 June 2005; accepted 7 July 2005

Available online 8 September 2005

Abstract

Background: The measurement of morphine in biological samples has become a routine in many clinical and forensic toxicology

laboratories. A high-performance liquid chromatography (HPLC) method was developed for the determination of morphine in plasma.

Methods: Samples were extracted using Zeolite Y column followed by reversed phase HPLC with fluorescence detection. This method was

based on an ex-calibration procedure and was linear between 20 and 200 ng/ml of morphine. Blood from 10 male opiate addicts were

obtained from Rosbeh Hospital. All of the male smoked opiate (heroin, opium) and cigarettes.

Results: The mean total level 5 h after the last abuse was 152.4 ng/ml and 37.6 ng/ml at 1015 h. The method was reliable for morphine

determination in blood even after 5 half-lives after the last abuse.

Conclusions: This method is simple and rapid and may be useful for routine monitoring of plasma morphine concentration.

D 2005 Elsevier B.V. All rights reserved.

Keywords: Morphine; Plasma; Addict; Zeolite; HPLC

1. Introduction

Morphine exhibits well known analgesic properties

which render the drug useful for the treatment of chronic

pain such as cancer pain [1,2]. Urine is the preferred

biological fluid for the analysis of morphine. The disadvan-

tages of urine testing include concentration variations

attributable to changing fluid intake and a greater possibilityfor adulteration or substitution and another limitation is the

range of detection in urine which is


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plasma from subjects on morphine therapy is in the range of

880 ng/ml and in tolerated person could be as low as 50

ng/ml [4]. One of the major problems associated with HPLC

is the need of complicated extraction procedure to separatethe compound of interest from a biological matrix prior to

analysis, such as liquid liquid [15,16] or solid-phase

methods [17,18].

Zeolites are microporous crystalline solids with well-

defined structures. Generally they contain silicon, aluminum

and oxygen in their framework, and cations, and water or

other molecules within their pores. Zeolite Y has a 3-

dimensional pore structure with pores running perpendicular

to each other in the x, y, and z planes. The pore diameter of

Zeolite Y is known as 7.6 A [19]. Because of their unique

porous properties, zeolites are used in a variety of

applications. Recently zeolite has been used as purification,

size exclusion chromatography, molecular absorbing and

catalyses effects. In some circumstance zeolites were used

as size exclusion while not affecting the target analyzed,

which are excluded from the zeolite pores, removes most of

the interfering material [20]. Zeolite Y has been used for

catalyses effects and its molecular absorbing properties [21]

which resulted in using Zeolite Y for size-exclusion

chromatography.

2. Materials and methods

Ten addicts chosen from psychiatric department of

Rosbeh Hospital who smoke either heroin and opium were

used in this study. All were also cigarette smokers. They

were between 26 and 68 years old and were addicted for atleast 5 years. Patients were explained about the study

protocol and those who volunteered were enrolled. The

written informed consent was obtained from all patients.

The ethics of this study conformed to the ethics committee

of the Vice Chancellor for Research of Tehran University of

medical sciences.

Four milliliters of blood were collected in heparinized

tube. Blood were centrifuged and 1.0 ml of plasma was

obtained. Each plasma sample was coded and refrigerated

for later analysis. 0.5 ml of human plasma were added to

0.25 ml of zinc sulfate 0.7 mol/l, 2.5 ml of bicarbonate

buffer (pH = 10.5) and 6 ml of THF. The mixture was vortex

and centrifuged and the upper organic layer was transferred

to Zeolite Y column. The HPLC system consisted of a

model 6000A solvent delivery pump (Waters Assoc.,

Milford, MA); a Model 7125 injector equipped with a

100-Al loop (Rheodyne, Cotati, CA), a ABondapak C18Column (300 mm4 mm I.D., 10 Am), a model 474

fluorescence detector with an excitation wavelength of 235

nm and a 349 nm emission. The chromatography was

Table 1

Plasma morphine concentration among addicts

Number Agent

abused

Time after the

last abuse (h)

Age

(years)

Concentration of

morphine (ng/ml)

1 Opium 14 27 47.2

2 Opium 11 37 13.6

3 Opium 15 35 51.54 Opium 5 37 66.9

5 Heroin 4 32 74.6

6 Opium 3 36 155.4

7 Opium 3 38 193.8

8 Opium 2 35 190.8

9 Opium 2 68 233

10 Opium 48 26 < 10

Fig. 1. Regression plot used to determine concentration of morphine in

plasma.

Table 2

Mean of morphine concentration in plasma hours after abuse

Number The time after

abusing (h)

Number of

abusing

Mean of morphine

concentration (ng/ml)

1 1 3 4 193.25

2 4 6 2 70.75

3 13 15 2 49.6

Fig. 2. Chromatogram showing extracted plasma blank spiked with 1 Ag/ml

of morphine by THF.

Fig. 3. Chromatogram showing extracted plasma blank spiked with 500 ng/

ml of morphine by THF followed by Zeolite Y column.

M. Ghazi-Khansari et al. / Clinica Chimica Acta 364 (2006) 235238236


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performed at ambient temperature using a mobile phase

consisting of 5% methanol, 3% acetonitrile, 0.5 mmol/lsodium acetate, 0.012 mol/l potassium dihydrogen ortho-

phosphate and 0.148 mmol/l phosphoric acid in distilled

water. The flow rate was 1.2 ml/min.

3. Results

The calibration curves showed good linearity between

peak-height and concentration from 20 to 200 ng/ml

(y =0.026X+0.46; r2=0.99). Table 1 shows the morphine

concentrations in 10 addicted people. Morphine concen-

tration in plasma was not detectable in an individual who

abused opiate 48 h before sampling. The regression analysis

of linear calibration curves for plasma morphine is shown in

Fig. 1. The within-run coefficient of variance (%CV) of

morphine at analyte concentrations of 20, 50, 100 and 200

ng/ml (n =4 for each) and between-run CVs obtained from

independently prepared standards dilutions during 4 days

ranged from 7% to 15%. The mean blood morphine

concentration with respect to the time of abusing before

analysis is shown in Table 2.

Fig. 2 shows typical chromatogram of plasma sample via

extraction of morphine by THF using a mobile phase

consisting of 8% methanol, 5% acetonitrile, 0.5 mmol/l

sodium edetate and 0.012 mol/l potassium dihydrogenorthophosphate in distilled water. The flow rate was 1.2

ml/min with an excitation wavelength of 210 nm and a 340

nm emission as described by others [15]. There is an

unknown peak at 5.63 min that interferes with morphine at

interest concentrations.

To overcome this interference, several stationary and

mobile phases were investigated to establish the optimum

separation of morphine and interference peaks but an

efficient separation was not achieved. Therefore, we used

a column loaded with the Zeolite Y to achieve an improve

separation of morphine in plasma (Fig. 3). After collecting

the extract solution (THF phase), the zeolite column was

washed with 10 ml methanol to show that no morphine

remained in the column.

Fig. 4 shows chromatogram of methanol extraction from

zeolite column. The Zeolite Y retained the unwanted

interference of unknown compounds. The chromatographic

condition was the same as in Fig. 2. Our study showed that

there was not any specific interaction between morphine and

Zeolite Y; therefore, this could be attributing to the other

morphine analogues. Morphine was detected in plasma as

late as 15 h after smoking, with a maximum mean

concentration of 49.6 ng/ml (range 13.6 51.5) measured1315 h after the subjects smoked opiates. Detection limit

of this method was 10 ng/ml and can be detected as late as

48 h after the subjects smoked opiates. With this method,

morphine concentration can be analyzed after 5 half-times

of morphine. A typical HPLC chromatogram obtained after

analysis of plasma addicts sample of morphine is shown in

Fig. 5. The peak of morphine was resolved from the

interference peak.

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