Forensic Science International 187 (2009) 34–41

A validated hybrid quadrupole linear ion-trap LC–MS method for the analysisof morphine and morphine glucuronides applied to opiate deaths

Kerry Taylor *, Simon Elliott 1

Regional Laboratory for Toxicology, Sandwell and West Birmingham Hospitals NHS Trust, City Hospital, Dudley Road, Birmingham B18 7QH, UK

A R T I C L E I N F O

Article history:

Received 22 August 2008

Received in revised form 6 February 2009

Accepted 12 February 2009

Available online 17 March 2009

Keywords:

Morphine

Heroin

LC–MS

SPE

A B S T R A C T

A hybrid quadrupole linear ion-trap mass spectrometer using an electrospray ionisation ion source

coupled to a HPLC system has been used to develop a method which can accurately measure morphine,

morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) in plasma, whole blood and post-

mortem blood following solid-phase extraction. The method can also qualitatively detect various other

opioids and related compounds including: codeine, dihydrocodeine (and metabolites), noscapine,

papaverine and 6-acetylmorphine (6-AM). The method has been favourably compared to an existing

laboratory method using a now discontinued radio-immunoassay technique. The advantage of

measuring the glucuronides directly rather than following deconjugation by b-glucuronidase has also

been shown. Detection and quantification of compounds was achieved using multiple reaction

monitoring (MRM) incorporating the use of deuterated morphine and M3G as internal standards.

Precision and accuracy was determined to be less than 10% at both high and low levels for all analytes

and the calibration curve was deemed linear over an acceptable range. Recovery in blood was greater

than 90% and ion suppression/enhancement was shown to be less than 15%. This method was applied to

over 130 post-mortem cases involving the use of heroin, prescribed morphine and codeine. The range of

concentrations of morphine, M3G and M6G was large (particularly in heroin and prescribed morphine

cases), reflecting the many different factors involved with therapeutic use or fatal opiate poisonings,

including tolerance associated with regular use, variable dose regimens and co-administration of other

drugs. Detection of other constituents of the opium poppy such as noscapine and papaverine and

metabolites of diacetylmorphine in the blood (6-AM) was useful in determining the source of the

morphine (i.e. illicit heroin) and the rapidity of death after administration.

Crown Copyright � 2009 Published by Elsevier Ireland Ltd. All rights reserved.

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1. Introduction

Heroin (diacetylmorphine) is one of the most commonly abuseddrugs in the world and morphine and diamorphine (diacetylmor-phine) are some of the commonest drugs prescribed in themanagement of pain relief. As a result, toxicologists haveinvestigated these drugs for many years, particularly in post-mortem cases where the toxicological significance of morphine isan important factor. Concentrations associated with fatal opioidand opiate poisonings are highly variable and notoriously difficultto interpret. This is due to a number of factors including: thedevelopment of tolerance, the presence of other drugs and in somecircumstances, the position of the body can exacerbate respiratory

* Corresponding author at: (ROAR) Forensics Ltd., Toxicology, Malvern Hills

Science Park, Geraldine Road, Malvern, Worcestershire WR14 3SZ, UK.

Tel.: +44 1684 585282; fax: +44 1684 574276.

E-mail address: kerry.taylor@roarforensics.com (K. Taylor).1 (ROAR) Forensics Ltd., Malvern Hills Science Park, Geraldine Road, Malvern,

Worcestershire, WR14 3SZ, UK.

0379-0738/$ – see front matter . Crown Copyright � 2009 Published by Elsevier Irelan

doi:10.1016/j.forsciint.2009.02.011

depression produced by the activation of the m-opioid receptor [1].Various studies have reported blood concentrations of morphine indeaths attributed to the drug that overlap significantly withconcentrations associated with non-fatal users [1–4]. Some studieshave attempted to assess the concentration ratio of morphine to itsglucuronide metabolites in relation to the survival time betweendrug administration and death, such assessment requires theaccurate measurement of morphine and its glucuronide metabo-lites [4,5].

The metabolism of heroin/diamorphine and morphine is welldocumented. Diacetylmorphine is rapidly deacetylated to 6-acetylmorphine (6-AM) followed by further deacetylation tomorphine, with diacetylmorphine and 6-AM having relativelyshort half-lives of approximately 2–6 and 6–25 min, respectively[3]. The primary elimination pathway of morphine involves theaddition of glucuronic acid at the 3- or 6-carbon of the moleculeresulting in the formation of morphine-3-glucuronide (M3G) andmorphine-6-glucuronide (M6G). M6G has been proven to exhibitactivity at the m-opioid receptor to a potency that may surpassmorphine whereas M3G is virtually inactive [6,7]. Another minor

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K. Taylor, S. Elliott / Forensic Science International 187 (2009) 34–41 35

route of metabolism is the conversion to inactive normorphine,constituting 5% of the initial dosage, there is also evidence of adiglucuronide moiety termed morphine-3,6-diglucuronide, how-ever the presence of this metabolite has only been documented inurine [8]. Measurement of morphine and its major metabolites,M3G and M6G have been routinely carried out by various methodsincluding radio-immunoassay and GC–MS to determine a ‘‘free’’ to‘‘total’’ morphine concentration ratio [5]. As well as measuringmorphine directly (‘‘free’’ or unmetabolised morphine), thisapproach employs the use of glucuronidase enzymes or acidhydrolysis to cleave the glucuronic acid portion of the molecule tomeasure ‘‘total’’ morphine (i.e. unmetabolised and metabolised). Ifan individual dies relatively quickly after the administration ofheroin/morphine there would be little time for the metabolism tooccur and thus there would be very little 3- and 6-morphineglucuronide present. As there would mainly be only unmetabo-lised morphine present, the ‘‘free’’ and ‘‘total’’ morphine concen-trations would be very similar (i.e. a ‘‘free’’ and ‘‘total’’ ratio close to1.0). However, these methods do not distinguish between M3G andM6G. Studies in urine have also shown a significant difference inglucuronidase efficiency and specificity, with M6G cleaved to amuch lesser extent than M3G [9]. Acid hydrolysis appeared to bemore efficient at both positions of the morphine glucuronidemolecule but is more suitable for urine and ante-mortemspecimens, as opposed to more viscous post-mortem blood [10].In order to negate the need for deconjugation, a number oftechniques have been published that measure M3G and M6Gdirectly, such as HPLC with fluorescence detection and HPLC withdual electrochemical and spectrophotometric detection [11,12].More recently, various papers have described the quantification ofmorphine and glucuronides by HPLC–MS, which offers signifi-cantly more sensitivity and specificity [13–21]. Despite this,simultaneous extraction of morphine and it glucuronides meta-bolites remains problematic in some matrices. HPLC and GCmethods have used various extraction techniques (e.g. liquid–liquid or solid-phase extraction) to quantify morphine and itsglucuronides in urine, plasma and ante-mortem whole blood.However, there are very little data concerning the quantification ofmorphine and its glucuronides in post-mortem blood [11,12]. Thismay be due to the difference in sensitivity required for bloodmeasurement in addition to the issue of the efficient extraction ofglucuronides from post-mortem whole blood. Solid-phase extrac-tion columns are particularly affected by high viscosity andclotting in post-mortem samples.

This paper describes an accurate, specific and validated methodfor the quantification of morphine, M3G and M6G using solid-phaseextraction and a hybrid quadrupole linear ion-trap LC–MS. Unlikeother published methods this involves a much reduced run time andlower volume of sample [22]. The method also incorporates thequalitative detection of various other opiate and opioid relatedcompounds such as codeine, codeine glucuronide, noscapine,papaverine dihydrocodeine and dihydrocodeine glucuronide, whichcan provide additional interpretative advantages. The method wasalso compared against an existing laboratory radio-immunoassaytechnique with assessment of the efficiency of glucuronidase usedfor glucuronic acid cleavage in post-mortem blood. The method hasbeen applied to over one hundred forensic cases involving heroin,morphine and codeine use.

2. Experimental

2.1. Instrumentation

High performance liquid chromatography with mass spectrometry (LC–MS) was

performed using an Applied Biosystems MDS/SCIEX 2000 QTRAP1 (Warrington,

UK) with an Agilent 1100 Series HPLC system consisting of a degassing unit, binary

pump, auto-sampler and diode-array detector (Wokingham, UK). A Phenomenex

Synergi Polar 150 mm � 2 mm column, protected by a 4 mm � 3 mm Phenomenex

Synergi Polar guard column was used for the analysis (Macclesfield, UK). The

column temperature was maintained at 30 8C in a Dionex STH 585 column oven

(Camberley, UK) with post-column flow splitting 1:2 between the QTRAP1 and the

diode-array detector to reduce the flow rate at the ion source. An electrospray

source was used in positive mode to produce ions which then entered the QTRAP1

for multiple reaction monitoring (MRM) with information-dependent Enhanced

Product Ion (EPI) scanning utilising the linear ion-trap aspect of the equipment. The

following parameters were used; source temp = 400 8C, curtain gas = 20, gas 1 = 30

units, gas 2 = 70 units, ion spray voltage = 5400 V, collision gas = high, declustering

potential = 40 V, entrance potential = 5 V, scan rate = 4000 amu/s, MRM dwell

time = 40 ms.

2.2. Reagents and standards

Ammonium formate, formic acid, ammonium carbonate and b-glucuronidase

enzyme (from Helix pomatia) was obtained from Sigma–Aldrich–Fluka (Poole, UK).

HPLC-grade acetonitrile and methanol were supplied by Rathburns Chemicals Ltd.

(Walkerburn, UK). Pure methanolic reference standard solutions of morphine,

morphine-d3, M3G, M3G-d3, M6G were purchased from LGC Promochem

(Teddington, UK). Reference standards of 6-AM, noscapine, papaverine, normor-

phine, codeine, norcodeine, oxycodone, hydrocodone and hydromorphone were

purchased from Sigma–Aldrich–Fluka (Poole, UK). A dihydrocodeine reference

standard was kindly donated from Napp Pharmaceuticals (Cambridge, UK).

Morphine, M3G and M6G reference standards were used to prepare a 500-mg/L

combined standard in equine plasma. This was diluted with blank plasma to

produce calibration standards at 250, 100, 50, 25 and 10 mg/L. Additional standards

of 2.5 and 5 mg/L were prepared for the determination of limit of detection.

Morphine, M3G and M6G quality control plasma standards were prepared at

concentrations of 10 and 100 mg/L. In addition to neat (undiluted), all post-mortem

blood samples were diluted 3-fold with equine plasma for matrix matching and to

reduce potential blocking of the solid-phase column. Further dilution with plasma

was performed if required in order to be within the linear calibration range.

Morphine-d3 and M3G-d3 deuterated standards were used to prepare a combined

1000 mg/L water solution to be used as an internal standard during solid-phase

extraction.

2.3. Extraction method for post-mortem blood

2.3.1. Sample preparation

300 mL of each calibration standard, QC standard and post-mortem blood

samples were diluted with 1 mL of 0.5 M ammonium carbonate buffer (pH 8). 50 mL

of the prepared deuterated internal standard solution was added. Samples were

vortexed briefly and 1 mL of the solution was loaded onto the conditioned solid-

phase cartridges.

2.3.2. Solid-phase extraction method

The method was based on a Varian Bond Elut C18, 6 mL, 200 mg SPE column.

Column conditioning was performed using 2 mL of methanol followed by 2 mL of

water and finally 1 mL of 0.5 M ammonium carbonate buffer. After initial sample

preparation (above), 1 mL of the sample or standard was loaded onto the column

and allowed to elute at approximately 1 mL/min. The column was washed with

5 mL of 0.005 M ammonium carbonate buffer and then flow dried under vacuum for

5 min. Elution was achieved with 1 mL of 70:30 acetonitrile:water. The eluent was

collected and evaporated to dryness under air at 45 8C and reconstituted with

100 mL of LC–MS mobile phase (96 Phase B:4 Phase A). This was transferred to a

HPLC vial insert for injection. The injection volume was 20 mL.

2.4. Chromatographic conditions

The LC–MS mobile phase consisted of Phase A buffer (containing 1 mM

ammonium formate and 0.1% formic acid—pH 2.7) and Phase B solvent (containing

70% acetonitrile, 1 mM ammonium formate and 0.1% formic acid—pH 3.8).

Extracts of the morphine, M3G and M6G plasma standards and QC standards

were analysed with 3% acetonitrile isocratic conditions (96% Phase A) with a run

time of 5 min at a flow rate of 0.6 mL/min. The typical elution times were morphine

(2.80 min), M3G (1.63 min) and M6G (2.75 min), this gave a capacity factor (k0) of

greater than 1 for all analytes which is a similar k0 found in other published methods

[21]. Although a k0 of less than 2 can result in more pronounced ion suppression or

enhancement, any contribution to ion suppression/enhancement has been assessed

as part of method validation. In order to elute additional opiate/opioid drugs, post-

mortem blood samples were analysed with a gradient solvent system. This started

with 3% acetonitrile for 3 min (as for the standards) and then increased to 65%

acetonitrile (95% Phase B) after 8 min. This was quickly reduced to 3% acetonitrile

for equilibration and held for 3 min. This resulted in a total run time of 11 min.

2.5. LC–MS detection of analytes and identification criteria

MRM transitions were selected based upon analysis of pure reference standards.

Surviving ion monitoring (precursor–precursor or precursor monitoring) and

precursor–product ion transitions were used depending on the fragmentation of

Table 1MRM transitions used for LC–MS detection and LC retention times (relative to morphine-d3 and M3G-d3).

Compound Transition 1 (m/z) Transition 2 (m/z) Transition 3 (m/z) Typical retention time (min) Relative retention time

Morphine 286/286 286/201 286/165 1.90 0.89

Morphine-3-glucuronide 462/462 486/286 462/268 1.37 0.98G

Morphine-6-glucuronide 462/462 486/286 462/268 2.21 1.58G

Normorphine 272/272 272/121 272/165 1.71 0.80

6-AM 328/328 328/193 328/165 6.07 2.84

Noscapine 414/414 414/220 N/A 7.12 3.33

Papaverine 340/340 340/202 N/A 6.95 3.25

Codeine 300/300 300/215 300/165 5.71 2.67

Norcodeine 286/225 286/185 286/227 5.28 2.47

Codeine-6-glucuronide 476/476 476/300 N/A 5.70 4.07G

Dihydrocodeine 302/302 302/199 302/244 5.12 2.39

Nordihydrocodeine 288/200 288.0/213.0 288.0/185.0 3.25 1.52

Dihydrocodeine-6-glucuronide 478/478 478/302 N/A 5.60 4.00G

Dihydromorphine 288/288 288/213 288/185 1.76 0.82

Oxycodone 316/297 316/256 N/A 6.18 2.89

Hydrocodone 300/241 300/199 N/A 6.29 2.94

Hydromorphone 286/227 286/185 N/A 4.26 1.99

Internal standards

Morphine-d3 289/289 289/201 289/165 2.14 1.00

Morphine-3-glucuronide-d3 465/465 465/289 465/204 1.40 1.00G

K. Taylor, S. Elliott / Forensic Science International 187 (2009) 34–4136

each compound (Table 1). For identification, all transitions were required to be

present. The average abundance ratios between the precursor:product ion

transitions for each compound were determined. For morphine 286/286 was

compared with 286/201 and 286/165 and for the glucuronides 462/462 was

compared with 462/286 and 462/201. To ensure the ratios obtained from the

plasma standards can be applied to real cases, the ratios of transitions obtained in

genuine case blood were also calculated. For further identification criteria, when a

pre-determined intensity threshold was reached (>3000 cps), an Enhanced Product

Ion scan in the linear ion-trap performs a full fragmentation scan of the precursor

ion that can be compared against a library mass-spectrum. Identification was

supported using relative retention times (RRTs) to take into account any changes in

the chromatography which would result in differences in absolute retention time.

RRTs were calculated using morphine-d3 for non-glucuronide compounds and

M3G-d3 for the glucuronides. As both M3G and M6G had the same fragmentation

(and hence MRMs), sufficient retention separation was necessary for identification

(Fig. 1).

Fig. 1. Chromatogram showing the MRM transitions us

2.6. Method validation

2.6.1. Recovery

Recovery of the compounds from post-mortem blood and plasma matrices was

determined by comparing the relative intensities produced by standards spiked

post-extraction (i.e. compounds spiked into extracted ‘‘blank’’ post-mortem blood

and plasma extracts) with post-mortem blood and plasma standards spiked pre-

extraction [23]. Post-mortem blood and plasma are spiked at a concentration of

100 mg/L and extracted in replicate (n = 5).

2.6.2. Matrix effects

Although direct infusion of the analyte into the MS has been used to investigate

ion suppression/enhancement effects [24], this only shows the potential for such

effects rather than the actual impact on analysis (e.g. measurement). In this

situation, where a different matrix is used for calibration, it is more appropriate to

evaluate the differences between blood and plasma standards. To this end, whole

ed for the detection of morphine, M3G and M6G.

K. Taylor, S. Elliott / Forensic Science International 187 (2009) 34–41 37

donor blood was spiked at various concentrations for morphine, M3G and M6G (5,

10, 25, 50, 100 mg/L), extracted and measured against a plasma standard-based

calibration curve. Internal standards were used to reflect actual case practice. This

process was performed on multiple occasions (n = 5) and two MRMs were assessed

for each compound; 286/286 and 286/201 (morphine); 462/462 and 462/286 (M3G

and M6G). Ion suppression/enhancement was also determined by comparing post-

mortem blood and plasma spiked post-extraction with standards made up directly

in mobile phase (as detailed by Matuszewski et al.) [23]. This provides a % difference

relative to the analyte in unextracted mobile phase. Post-mortem bloods from five

different cases were used in order to determine any differences in matrix effects

from different blood sources.

2.6.3. Selectivity

A series of plasma standards which contained various opiate/opioid compounds

were extracted and analysed to ensure that only one chromatographic peak was

produced for each compounds’ specific MRMs. Standards spiked at a concentration

of 1 mg/L with dihydrocodeine, codeine, norcodeine, papaverine, noscapine,

normorphine, hydrocodone and oxycodone were used. To determine if a significant

amount of cross-talk occurred due to the in-source fragmentation of M6G to

morphine, blood and plasma were spiked with M6G only and analysed on the LC–

MS for the presence of morphine.

2.6.4. Linearity

Morphine, M3G and M6G calibration standards of 5, 10, 25, 50, 100, 250, 500 mg/

L were extracted and analysed on consecutive days. Calibration curves were

produced using least squares regression with a 1/x weighting factor.

2.6.5. Quantification LOD and LOQ

For the limit of detection (LOD), blank plasma standards with deuterated internal

standard were analysed on multiple days to determine three times the baseline

noise and standard deviation (n = 13). The calibration curve was used each time to

calculate the corresponding concentration. 10 mg/L standards (n = 10) were

analysed on the same day in order to ascertain the lowest concentration that

could be accurately and consistently measured to ascertain a viable limit of

quantitation (LOQ).

2.6.6. Accuracy and precision

Plasma standards at low (10 mg/L) and high (100 mg/L) concentrations were

analysed on one day and over many days to determine the intra- and inter-day

accuracy and precision values. Accuracy was determined by the percentage

deviation of the mean calculated concentration compared to the spiked

concentration. Precision was determined by calculating the coefficient of variation

(%CV) at each concentration level based on the mean concentration and the

standard deviation.

2.6.7. Stability

The stability of morphine, M3G and M6G in both plasma and blood was

determined by spiking plasma and post-mortem blood with morphine, M3G and

M6G at a concentration of 250 mg/L as a combined standard and separately and

then analysing them immediately and again after 11 months after being stored at

either room temperature (22 8C), in the fridge (4 8C) or the freezer (�20 8C).

The stability of the extract was investigated by extracting a post-mortem blood

sample and a plasma standard and analysing them. The extract was then left at

room temperature for 4 h and overnight and then re-analysed.

Table 2Validation data for the quantification of morphine, M3G and M6G.

Morp

286/2

Limit of detection (mg/L) 0.34

Limit of quantification (mg/L) 10

Inter-day mean: M n = 25, M3G n = 21, M6G n = 19 LQC (10 mg/L) 9.9

HQC (100 mg/L) 94.6

Inter-day accuracy (% deviation) LQC (10 mg/L) 4.0

HQC (100 mg/L) 3.4

Inter-day precision (%CV) LQC (10 mg/L) 5.2

HQC (100 mg/L) 4.9

Intra-day mean: M n = 10, M3G n = 10, M6G n = 10 LQC (10 mg/L) 9.6

HQC (100 mg/L) 93.3

Intra-day accuracy (% deviation) LQC (10 mg/L) �4.3

HQC (100 mg/L) 6.7

Intra-day precision (% CV) LQC (10 mg/L) 4.1

HQC (100 mg/L) 6.2

Recovery (%) Blood 91

Plasma 96

2.7. Comparison with radio-immunoassay (RIA)

The existing radio-immunoassay (DPC, California, USA) laboratory method for

the quantification of morphine and metabolites incorporated the enzymatic

hydrolysis of the glucuronides to measure total morphine which was compared to

the measurement of free morphine, without hydrolysis (limit of detection = 2.5 mg/

L for morphine). For comparative purposes, blood samples were analysed by radio-

immunoassay (with and without hydrolysis) and the LC–MS method described

(direct analysis of glucuronides, no hydrolysis). Product information indicated the

assay had a cross-reactivity of less than 1% for M3G and M6G.

For the determination of total morphine, 100 mL of post-mortem whole blood

was diluted 3-fold with equine plasma (200 mL). 200 mL was then diluted a further

2-fold with 200 mL pH 5 acetate buffer and 10 mL b-glucuronidase enzyme added.

This was incubated at 30 8C overnight in a water bath prior to analysis.

2.8. b-Glucuronidase efficiency

To assess the efficiency of the b-glucuronidase enzyme used for the hydrolysis of

the glucuronides, whole post-mortem blood (n = 17) was incubated with b-

glucuronidase at 30 8C overnight, extracted and analysed using LC–MS. The quality

and viscosity of the blood was also noted, in case this affected hydrolysis. Fresh

donor blood was also spiked with morphine and morphine glucuronides separately

and in combination at high (1000 mg/L) and low (100 mg/L) concentrations. These

were also incubated at 30 8C overnight with b-glucuronidase, extracted and

analysed using LC–MS.

3. Results and discussion

3.1. Validation

The method was validated for the quantification of morphine,M3G and M6G as shown in Table 2. All analytes were linear over therange 10–500 mg/L with a correlation coefficient (r2 value) of 0.999,no interferences occurred with any of the standards ran under themethod. Accuracy and precision was calculated at low and highconcentrations and was found tobe less than 10% for all analytes withthe exception of the 462/462 surviving ion monitoring of M3G andM6G. Therefore, for the purpose of analysis, this surviving ion wasused as an initial identification indicator only and was not used forquantitation of the glucuronides. In the case of morphine, both 286/286 and 286/201 were shown to have acceptable validationparameters so either could be used for quantification. Comparisonof the mass-spectral data indicated that deconjugation duringfragmentation occurred readily and may have produced inconsistentfragmentation for the 462 m/z precursor ion monitoring. This did notappear to be the case for morphine, with less spontaneousfragmentation of the 286 m/z precursor ion and more consistent201 m/z product ion formation.

The theoretical limit of detection was calculated for eachanalyte based on the blank plasma noise. For the MRMs chosen for

hine M3G M6G

86 286/201 462/462 462/286 462/462 462/286

0.81 2.73 0.35 3.33 1.41

10 10 10 10 10

9.7 9.9 9.9 10.9 9.8

95.3 101.5 100.1 97.8 97.9

7.0 2.0 2.0 13.0 6.0

3.4 1.5 0.7 0.6 4.6

7.1 21.1 5.3 25.7 5.3

6.6 8.0 5.7 11.3 6.5

9.3 9.1 10.4 10.4 9.8

90.3 97.9 99.0 101.0 95.2

�6.6 �9.5 3.9 4.4 �1.8

9.7 2.2 1.0 �1.0 4.8

6.5 13.2 4.7 10.8 8.6

4.3 8.8 5.3 6.0 6.3

92 N/A 90 N/A 104

84 N/A 91 N/A 105

K. Taylor, S. Elliott / Forensic Science International 187 (2009) 34–4138

quantification (286/286, 286/201 and 462/286), this gave limits ofdetection below 1.5 mg/L for all analytes. Identification was basedon the detection of all 3 MRM transitions for each analyte asdescribed in Table 1. Transition ion ratios were also calculated forall analytes. Tolerance was calculated to be less than 20% for bothtransition ratios for morphine, M3G and M6G. In order to satisfythis identification criterion, it was found that an analyteconcentration of 5 mg/L was required. However, above 50 mg/Lthere was often sufficient ion intensity for successful EPI formationto enable library matching. Production of a specific EPI provides asignificant advantage over existing methods which incorporatetandem MS only. Therefore, for casework, identification was basedon the presence of 3 MRM transitions, the corresponding ion ratiosand where possible, EPI scanning data compared against areference spectrum in a library.

Recovery of morphine, M3G and M6G in blood was found to begreater than 90% for all analytes and was greater than 84% inplasma (Table 2). Evaluation of the matrix effect and any possibleion suppression/enhancement indicated that all spiked post-mortem blood concentrations were within 5% of the expectedvalue at lower concentrations and were still within 15% at higherconcentrations. This indicated it was acceptable to measure bloodconcentrations based on a plasma calibration curve. Directinvestigation of ion suppression indicates that some suppres-sion/enhancement may occur in both blood and plasma. However,this was calculated at less than 15% for both blood and plasma. Anyion suppression or enhancement that did occur would be correctedby using deuterated analogues of the compounds of interest asinternal standards. Although there maybe some ‘‘cross-talk’’between compounds with a similar mass (e.g. dihydromorphineand morphine-d3), these compounds eluted at different times (i.e.dihydromorphine eluted at 1.76 min whilst morphine-d3 eluted at2.13) so any cross-talk would not affect quantification. It wasshown that negligible cross-talk occurred (<0.0002%) from theformation of morphine from M6G due to in-source fragmentationbased on analysis of a M6G only standard and evaluation for thepresence of morphine.

Morphine, M3G and M6G were shown to be stable over an 11-month period when stored at 4 8C and �20 8C in both blood andplasma. Some decrease in the concentration of M3G and M6G andan apparent increase in morphine concentration was observed inspiked blood and plasma stored at room temperature (22 8C) whichcould be the breakdown of M3G and M6G to morphine over time,but as plasma standards and post-mortem blood samples arealways stored in the fridge or freezer this was not considered asignificant issue for casework. With regards stability of extracts, noobservable decrease in concentration was seen in the post-mortemblood and plasma standards stored at room temperature for 4 h(e.g. if remaining on an auto-sampler prior to analysis). However, ifextracts are stored overnight at room temperature, significantdecreases in all analytes are seen. Therefore, samples are extractedand analysed on the same day and are not stored prior to analysis.

Table 3Efficiency of the glucuronidase enzyme for the deconjugation of M3G and M6G in pos

% proportion of M3G or M6G not cleaved by glucuronid

Morph H Morph L

M3G H M3G H M3G L M3G H

M6G H M6G H M6G L M6G L

Morphine-3-glucuronide 17 19 24.8 24

Morphine-6-glucuronide 32.3 45.1 69.2 48.6

H = high concentration spiked into blood (1000 mg/L); L = low concentration spiked inta n = 17.b n = 10.

3.2. Comparison with radio-immunoassay (RIA)

When free morphine concentrations determined by DPC radio-immunoassay and LC–MS were compared (n = 14), calculatedconcentrations were within 20% but with an overall high bias withLC–MS (data not shown). This could be due to an improvedextraction efficiency of the solid-phase method used for LC–MS ascompared with direct binding of the blood to the tubes as part ofthe radio-immunoassay technique. For the glucuronides, concen-trations of ‘‘total glucuronide’’ obtained by both methods werevery different by comparing ‘‘total morphine’’ concentrations afterenzyme hydrolysis (RIA) with combined M3G and M6G concen-trations using LC–MS. There was an average 85% differencebetween methods with a variance up to 100% in some cases, withRIA consistently underestimating the amount of glucuronidespresent. This was thought to be due to inefficiency of theglucuronidase enzyme and was studied further.

An interesting result occurred in a case of dihydrocodeine andprescribed morphine overdosage. The morphine and morphineglucuronide concentrations were far lower than those found by RIA(66% less for free morphine and 466% less for the glucuronides). Avery high concentration of dihydrocodeine was measured in theblood (37 mg/L) with dihydromorphine and dihydrocodeineglucuronide detected by LC–MS. Although the cross-reactivity ofdihydrocodeine with the RIA assay was stated as being less than0.15% according to the product leaflet, the cross-reactivity ofdihydromorphine was not quoted. The apparent greater concen-tration of morphine by RIA may therefore have been due to cross-reactivity with dihydrocodeine metabolites. The results of analysisof additional cases where only dihydrocodeine and metaboliteswere present by LC–MS but the apparent detection of morphine byRIA, indicated a potential for ‘‘false positive’’ findings using RIA. Asthe DPC radio-immunoassay method has since been discontinued,specific cross-reactivity studies with dihydrocodeine metabolitesare not possible. This indicates a significant advantage in usingselective LC–MS.

3.3. b-Glucuronidase efficiency

Table 3 shows the results of the analysis of post-mortemblood and spiked blood standards with enzyme hydrolysis (RIA)and without hydrolysis (LC–MS). For spiked blood, variablepermutations of glucuronide and morphine concentrations wereused to study potential inhibition of the hydrolysis reaction. Theresults showed that between 17 and 27% of M3G was not cleaved,regardless of the concentration and the proportion of M6G andmorphine present. For M6G, there appeared a slight decrease inefficiency at lower concentrations (100 mg/L) but between 32and 45% remained conjugated at higher concentrations(1000 mg/L), this supported the findings of other researcherswhen using urine [9,10]. The poor efficiency was even morepronounced in the post-mortem blood samples, it was found that

t-mortem blood samples and blood standards spiked at specific concentrations.

ase

M3G L M3G H Case blood

M6G H M6G H M3G H M6G H (mean proportion uncleaved)

26.5 24.3 16.9 8.1a (range 0.8–23.3)

39.2 39.5 34.6 44.3b (range 29.9–92.1)

o blood (100 mg/L)

Fig. 2. Extracted ion chromatogram of a post-mortem blood sample from a known heroin user showing the typical analytes detected.

K. Taylor, S. Elliott / Forensic Science International 187 (2009) 34–41 39

b-glucuronidase did not cleave 100% of M6G in any post-mortemblood samples analysed, with an average of 44% M6G leftuncleaved which did not seem to correlate to the viscosity orcondition of the blood. In contrast, M3G seemed to be

Fig. 3. Extracted ion chromatogram of a post-mortem blood sample from an

deconjugated to a much greater extent with complete hydrolysisin some cases, in cases where hydrolysis was incomplete, anaverage of less than 10% remained. Therefore, the results of thisstudy have particular implications for the practice of using

individual prescribed morphine showing the typical analytes detected.

Fig. 4. Extracted ion chromatogram of a post-mortem blood sample from an individual prescribed codeine showing the typical analytes detected.

K. Taylor, S. Elliott / Forensic Science International 187 (2009) 34–4140

glucuronidase enzyme to determine ‘‘total’’ morphine concen-trations whether analysed by RIA or GC–MS. This would producean inaccurate estimation of the ‘‘free’’ to ‘‘total’’ morphine ratioand may affect any correlation to the rapidity of death.Furthermore, published ratios utilising glucuronidase-deriveddata should not be applied to interpret ratios derived from directanalysis of M3G and M6G without hydrolysis.

3.4. Detection of other opiates/opioids in blood

Following validation, the solid-phase LC–MS methoddescribed has been successfully applied to 130 post-mortemblood specimens submitted for analysis by HM Coroner and thePolice. Of the samples analysed, 91 decedents (70%) were knownor suspected heroin users, 15 (12%) had been prescribedmorphine, 4 (3%) were codeine users and 20 (15%) had no drugor prescription history.

Depending on the source of the morphine detected in the cases,other opiates, opioids and related compounds can also be detectedby this method. In circumstances where a rapid death fromintravenous injection of heroin is suspected (e.g. syringe still inthe vein), it is common to detect constituents of the opium poppysuch as noscapine, codeine and papaverine in the blood (Fig. 2).The detection of these opium compounds is also useful indetermining illicit heroin use compared to medical diamorphineuse. In particularly rapid deaths, precursors of morphine may alsobe detected (e.g. 6-AM) and the ratio of morphine to itsglucuronides is usually large as there may not have been timefor a significant portion of the morphine to be metabolised.However, if the deceased is a regular user then M3G and M6G canaccumulate due to their longer half-life than morphine, or if thedeceased has survived for many hours post-dose, the ratio maybemuch smaller. In situations where there are significant glucur-onide concentrations, the presence of 6-AM or opium-derived

compounds could indicate recent use following chronic use.Unlike heroin cases, in instances of prescribed morphine, onlymorphine, M3G and M6G are detected with no additionalcompounds (Fig. 3). With codeine use, codeine glucuronide,morphine, M3G and M6G are also detected (Fig. 4). As thesecompounds may also be present as a result of illicit heroin use, theconcentration of codeine in relation to morphine should beevaluated, with morphine concentrations typically less than 10%of the codeine concentration [25]. Future application of thedescribed LC–MS method for the measurement of the relativeconcentrations of these various compounds could provide newdata for the interpretation of codeine deaths.

4. Conclusions

This paper presents a validated method to measure morphineand metabolites in post-mortem blood. The method also incorpo-rates the qualitative detection of other opiate/opioids and relatedcompounds, which may be relevant in determining the particulardrug(s) involved. The solid-phase extraction method used has beenfound to extract morphine and glucuronides from post-mortemblood of varying viscosity, even from clotted or decomposed blood.The ability to analyse glucuronides without the use of inefficienthydrolysis or the need for derivatisation, provides a distinctadvantage over existing techniques. Overall, the method provides avery useful and necessary approach to the investigation of thesedifficult cases types.

Acknowledgments

The authors would like to thank H.M. Coroners for theirpermission in publishing the data. The authors would also like tothank Dr Robin Braithwaite and Prof. Kevin Chipman, for theirsupport and advice during this work.

K. Taylor, S. Elliott / Forensic Science International 187 (2009) 34–41 41

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