High-performance liquid chromatographic determination of diacetylmorphine metabolites in urine and postmortem plasma

Determination of drugs of abuse in blood is of great importance in clinical and forensic toxicology. This paper represents a simple and rapid method for the determination of diacetyl morphine metabolites in urine and postmortem plasma specimens using reversed-phase high-performance liquid chromatography with ultraviolet spectrophotometric detection. The proposed method was based on using C18 column with mobile phase consisting of a mixture of acetonitrile-20 mM KH2PO4 diethylamine (10: 90: 0.1, v/v, pH 3.0 adjusted with phosphoric acid). Quantitation was achieved with UV detection at 280 nm using phenylephrine as internal standard. The metabolites were successfully extracted from addict urine and postmortem plasma with good recoveries and reproducibility.


Introduction
Heroin (diacetylmorphine) (DM) is one of the most commonly abused drugs in the world.Morphine and diacetylmorphine are some of the commonest drugs prescribed in the management of pain relief.As a result, toxicologists have investigated these drugs for many years, particularly in postmortem cases where the toxicological significance of morphine is an important factor.Toxicological analysis of opiates is of interest with respect to assessment of intoxications and culpability as well as to the cause of death in cases of clinical, pathological or forensic importance and, finally, also to drug abuse control and human pharmacokinetic or pharmacodynamic studies.In heroin-related deaths, the situation will be more complex due to heroin metabolism.After administration, DM is degraded enzymatically or by hydrolysis in blood, tissues and also in vitro to 6monoacetylmorphine (MAM) and further hydrolyzed to morphine (MR) at a slower rate (Lockridge et al., 1980).MAM conjugated with glucuronic acid to form MAM-3 glucuronide and excreted in urine also morphine itself is deactivated by glucuronidation to morphine-3 and 6-glucuronide (Colin Dollery, 1999).
Chromatography is the most widely accepted method for chemical separation and determination of the components in complex mixtures.HPLC is usually applied for amino acids, proteins, nucleic acids, hydrocarbons, carbohydrates, drugs, pesticides, antibiotics, steroids, metal-organic species and a variety of inorganic substances.From all mentioned above it was very important to conduct a simple and rapid HPLC method used in forensic laboratories for determination of diacetylmorphine metabolites.
In this study, a simple and rapid reversed phase isocratic HPLC method was developed for the determination of both diacetylmorphine metabolites (MR and MAM) in an addict urine and postmortem plasma using phenylephrine (PN) as internal standard, by direct injection for the urine samples and after pretreatment and cleaning up procedure for blood autopsy to remove the interfering substances which hinder the chromatographic analysis.
Centrifuge (Hettich, Germany) Werk Nr type was used in the cleanup procedure of blood samples.

HPLC conditions
The HPLC separation and quantitation were made on a 250 X4.6 mm (i.d.) Luna 5 µm C18 column (Luna, Phenomenex, USA).The mobile phase was prepared by mixing acetonitrile, 20mM KH2PO4, and diethylamine in a ratio10: 90: 0.1 v/v/v and adjusted to the apparent pH 3.0 using phosphoric acid.The flow rate was 1.0 ml min -1 .All determinations were performed at ambient temperature.The injection volume was 20 µL.The detector was set at 280 nm.
Data acquisition was performed on class-VP software.

Standard solutions
Stock standard solutions were prepared by separately dissolving DM, MR, MAM, and PN in methanol, to obtain concentration of 100 µg ml -1 .These stock solutions were stored at 4ºC.

For urine sample
Different volumes of MR and MAM stock standard were transferred to 10 ml volumetric flask, 1ml of blank urine was added and the solutions were diluted to 10 ml with the mobile phase to obtain concentration range of 0.01-50 µg ml −1 .All solutions contained 10µg ml -1 PN as Internal standerd.The solutions were filtered through 0.45µm disposable membrane filters.Triplicate 20 µl injections were made for each concentration of MAM and MR and chromatographed under the conditions described above.The peak area ratios (peak area of MAM and MR /peak area of PN) were determined for each concentration and plotted against the corresponding concentration to obtain the calibration graphs of MAM and MR.

Standard solutions and Extraction procedure for plasma samples
Standard solution of MR and MAM were further diluted 10-fold with drug-free human plasma to obtain the concentration range of 0.01-50 µg ml -1 .
The spiked plasma samples were kept in ice bath all time.
Into 2.5 ml Eppendorf tubes, equal volumes (0.5 ml) of acetonitrile and ethylacetate, were added to spiked plasma sample (1 ml).10µg ml -1 PN as internal standard, then vortexted for 20 second and centrifuged for 10 min at 3000 r.p.m and 4ºC.The upper layer was transferred to another tube filtered through 0.45 µm millipore filter, evaporated by nitrogen at ambient temperature, then reconstituted with 100 µL mobile phase; 20 µl were injected on the analytical column for analysis using the same analytical mobile phase.The peak area ratios were determined for MAM and MR and were plotted against the corresponding concentration to obtain the calibration graphs.

Case 1 history
A 27-year-old man (height 170 cm, weight 65 kg) was stopped in police station, by searching his pockets, a plastic bag filled with white to off-white powder was found and tested for heroin.Also a tramadol tablet strips were found in the inner pocket of his jacket.Seven recent needle puncture markers were seen in the right elbow.Urine sample was taken as a suspicion of drug abuse.

Case 2 history and autopsy findings
A 33-year-old man (height 175 cm, weight 78 kg) with a half-year history of heroin abuse, was found dead near his bed in a supine position.
A syringe was found near the body, and 0.55 g of an illicit, off white powder wrapped in aluminum foil, tested to be heroin.At autopsy, no significant preexisting natural diseases were found.Four recent needle puncture markers with numerous old ones were seen in both arms.There was evidence of aspiration of some gastric material.Additional findings were an interstitial lung edema, a dilation of the right atrium and an abnormal accumulation of blood of the internal organs.The autopsy was performed 12 h after discovery of the body and at most 24 h after death.

Urine sample
One milliliter of urine specimen was acidified with equal volume of orthophosphoric acid was added.
The mixture was sonicated for 30 min.This mixture was neutralized by addition of 1M sodium hydroxide and suitable dilution was carried out to 10 ml with the mobile phase to reach calibration range.Each sample solution contained 10 µg ml -1 PN as internal standard.
The solution was filtered through 0.45µm membrane filter.A 20µl was injected into HPLC, in triplicate for each solution and chromatographed under the conditions described above.The concentration of MAM and MR was calculated using regression equations.

Blood samples
Blood samples were collected at autopsy room of suspected case of heroin overdose.Prior to sampling, urine or a liquor sample was screened for abused drugs by chemical experts of forensic department.
After the chest and the abdominal cavities were opened, the vessels were ligated or cross-clamped prior to blood sampling by needle puncture.Blood was obtained from the thoracic aorta, the inferior vena cava (suprarenal, infrarenal), the superior vena cava, the subclavian and femoral veins, and from the right and left ventricles of the heart.Additionally, vitreous humor and liquor were collected.All blood samples were centrifuged (10 min, 3500 rev min -1 ), plasma was removed and frozen (-20 °C), and thawed just prior to extraction and analysis.
One milliliter of human plasma was acidified with equal volume of orthophosphoric acid, 10µg ml -1 PN as internal standard, and equal volumes (0.5 ml) of acetonitrile and ethylacetate, were added.This mixture was sonicated for 30 min.The mixture was neutralized using 1M sodium hydroxide.The extraction procedures described above were followed and the concentration of MAM and MR were calculated using the regression equations.

Deproteinization and extraction
In the determination of opiates in urine; hydrolysis pretreatment is usually incorporated to free opiates from mainly glucuronide conjugates depending upon the purpose of the assay.There are several hydrolysis procedures and, in the present work, urine samples were subjected to standard acid hydrolysis (Low et al., 1995).Hydrochloric acid, and orthophosphoric acid were used.
Since presence of MAM in urine distinguishes heroin use from morphine, and as MAM was known to be unstable (Moriya et al., 1997), preliminary test was made by incubating MAM with orthophosphoric acid for 30 min to insure whether this compound was deacetylated to morphine.Only a small amount of MAM was lost under these conditions which are compensated by the presence of internal standard.
This procedure was performed to insure hydrolysis of 3-glucuronide linkage without conversion of MAM to MR.On the other hand; hydrolysis using hydrochloric acid gives poor recovery to MAM.Also, use of phosphoric acid was preferable to hydrochloric acid because of the possible adverse effect of hydrochloric acid on the column material.
Hydrolysis of the conjugated MAM and MR was completed by using 1ml of orthophosphoric acid within 30 min at ambient temperature.
Most of the methods reported for determination of drugs in plasma and tissue involve protein precipitation with an organic solvent.Commonly used organic solvents are acetonitrile, methanol, trifluoroacetic acid, and ethylacetate.A combination of acetonitrile with ethylacetate was found to be the most beneficial for protein precipitation and extraction from postmortem blood.

Chromatographic condition
The analysis of postmortem specimens can provide special challenges for forensic toxicologists especially if this specimen were taken from an addict, who used to take a large collection of drugs.These drugs may be one of tranquillizers, hallucinogens, antidepressant, cannabinoids, and (or) opiates.These all give rise to a complex matrix with high interference level.Also, the simplicity of the analytical method and the availability of the analytical tools in every laboratory are considered as another challenge for the analyst mind.Simple mobile phase, short time of separation and rapid applicability are the target for toxicological analysis.
To optimize the HPLC assay parameters, the effects of acetonitrile concentration and pH of the mobile phase on the capacity factor (K \ ) were studied.were well separated.The average retention time ± SD for PN, MR, MAM and DM were found to be 3.81 ± 0.01, 4.60 ± 0.04, 6.05 ± 0.02 and 7.10 ± 0.14 min, respectively, for seven replicates.The peaks obtained were sharp and have clear baseline separation.The system suitability results are given in Table 1.
The performance of the proposed method on real samples was demonstrated by its application to human urine of an addict and postmortem blood sample provided by forensic medicine institution.

Linearity
The linearity of the proposed method was evaluated by analyzing seven concentrations of MR and MAM ranging between 0.01 and 50µg ml -1 in different biological matrices.Each concentration was repeated three times.The assay was performed according to experimental conditions previously established.
Calculations of calibration curves were based on the peak area ratios of MR and MAM to the internal standard, PN.The linearity of the calibration graphs was validated by the high value of the correlation coefficient and the intercept value, which was not statistically (p = 0.05) different from zero (Table 2).
Characteristic regression data for the method, obtained by least squares treatment of the results, are given in Table 2.

Precision
To judge the quality of the method, precision was determined.The precision of the method, expressed as CV (%), was determined by analysis of three different concentrations within the linearity range for MR and MAM in urine and plasma samples.Intraday precision was assessed from the results of five replicate analyses of each sample on the same day.
Inter-day precision was determined by analysis of the samples on five consecutive days.CV (%) for intraday and inter-day assays for human urine, and plasma
indicated good precision, as the CV% did not exceed 2.04%.The results obtained from determination of intra-day and inter-day precision are listed in Table 3.

Range
The calibration range was established by consideration of the practical range necessary to give accurate, precise, and linear results.Calibration ranges for the proposed HPLC method are given in Table 2.

Detection and quantitation limits
According to ICH recommendations (ICH, 1996), the approach based on the S.D. of the response and the slope was used for determining the detection and quantitation limits.The theoretical values were assessed practically and given in Table 2.

Selectivity and accuracy
Method selectivity and accuracy were achieved by addition of appropriate volumes of standard solutions of MR and MAM (with different concentrations compared to those used in the calibration curve but within the linearity range concentration) to human blank urine and plasma samples after spiking with a fixed concentration of PN as internal standard and analyzed according to the previous procedures described previously.Satisfactory results were obtained for MR and MAM, indicating the high selectivity and accuracy of the proposed method.

Robustness
The effect of percent of organic strength on resolution was studied by varying acetonitrile from 8 to 12 %.
The effect of the pH of the mobile phase on resolution

Stability
In anticipation of unexpected delays during analysis, it is important to have information about the stability of all solutions.When the stability of MR, MAM, DM and the internal standard in urine and plasma was tested, it was found the samples were stable for at least 2 h at room temperature and for 5 weeks at -20ºC.Therefore, samples are extracted and analyzed on the same day and kept in ice during the analysis period.
Urine and plasma samples spiked with MR, MAM and DM were evaluated for stability after freezing and thawing.The drugs were stable through at least three freeze-thaw cycles.

Conclusion
A simple, rapid, specific, sensitive and validated reversed-phase HPLC method using combined UV detection is described, suitable for the simultaneous measurement of MR and MAM in addict urine and postmortem blood specimens.The proposed method proved to be applicable not only to forensic toxicology where special requirements have to be met, but also to clinical toxicology.

Conflict of interest
The authors report no declaration of conflict of interest.
Postmortem changes will occur for all of the drugs of abuse.Heroin is converted within minutes to morphine through the intermediate MAM.All species are active pharmacologically, although both heroin and MAM are only present in blood and tissues.MAM conjugated with glucuronic acid to form MAM-3 glucuronide and excreted in urine.MR is removed from the body by metabolism to 3-and 6glucuronides and excretion in urine and bile.The glucuronic compounds of MR and MAM represents 52% of urinary metabolites of DM (2).
The mobile phase used for HPLC consisted of different components, acetonitrile, potassium dihydrogen phosphate buffer and diethylamine.In preliminary experiments, mixtures of acetonitrile and 20 mM potassium dihydrogen phosphate buffer (pH 3.0) were used.These preliminary experiments showed that good resolution of PN, MR, MAM and DM in the shortest analysis time was achieved by the use of 10 % acetonitrile.The influence of acetonitrile concentration of the mobile phase on the separation efficiency between PN, MR, MAM and DM as well as endogenous compounds from different biological samples was investigated at pH 3. It was found that, decreasing the concentration of acetonitrile to less than 5 % caused a severe increase in the retention time of DM peak that resulted in band broadening and excessive tailing, while increasing the concentration of acetonitrile more than 20 % caused a decrease in retention time which led to inadequate separation of the studied peaks and the biological matrices.Best separation could be obtained by using 10 % v/v acetonitrile.The influence of pH of the mobile phase on the retention behavior of PN, MR, MAM and DM as well as endogenous compounds of biological samples was investigated.It was found that variation of pH of mobile phase resulted in maximum K \ value at pH 5 with loss of peak symmetry for DM.At lower pH values (less than 2.5) poor resolution for PN, MR, MAM, DM and different biological matrix was observed.At pH 2.5-3.5, improved resolutions of the studied peaks were observed; however, at pH 3.0 optimum resolution with reasonable retention time was affected.The effect of the concentration of potassium dihydrogen phosphate in the mobile phase was studied between 10 and 40 mM.20 mM was found to give better resolution of PN, MR, MAM and DM.The addition of diethylamine to the mobile phase is essential, to prevent peak tailing and provide better separation especially for MAM and DM.Chromatogram of blank human urine and plasma (Figure 1a and b), showed no interfering peaks at the retention times of PN, MR, MAM and DM. Figure 2a and b shows a typical chromatogram for the samples prepared from human urine, and plasma containing PN, MR, MAM and DM where the drugs

Figure 1 :
Figure 1: Typical HPLC chromatograms obtained from analysis of (a) blank human urine and (b) blank human plasma.

Figure
Figure 3a and b shows a typical HPLC

Figure 3 :
Figure 3: Typical HPLC chromatograms obtained from analysis of real samples of (a) addict urine and (b) postmortem plasma.

Table 1 : The system suitability test results of the developed method for determination of PN, MR, MAM and DM.
The retention time of unretained peak is 0.902 min.a1, b1: are α and Rs calculated for PN -MR.a2, b2: are α and Rs calculated for MR-MAM.a3, b3: are α and Rs calculated for MAM -DM.

Table 3 : Intra-day and inter-day precision for the assay of MR and MAM in human urine and postmortem plasma.
Mean ± SD from five determinations was studied by varying pH from 2.8 to 3.2.Changing flow rate of the chromatographic method from 0.8 to 1.2.In all the deliberate varied chromatographic conditions (percentage organic strength, pH of the mobile phase and flow rate), the resolution between PN, MR, MAM, DM and other components in the biological matrices was not altered. a