MED5069P: Project
Optimising the enzymatic hydrolysis of morphine glucuronides in blood and urine.
Dissertation submitted in partial fulfilment of the requirement for the MSc (MedSci) in Forensic Toxicology.
Forensic Medicine and Science
School of Medicine
University of Glasgow
Word Count: 9875
Date: 22.07.2016
Contents
ACKNOWLEDGEMENTS. 4
ABSTRACT. 5
FIGURES, TABLES AND APPENDICES. 6
INTRODUCTION.. 8
1.1 Background. 8
1.1.1 Heroin. 9
1.1.2 Metabolism.. 10
1.1.3 Analysis of morphine. 12
1.2 Concerning the literature. 14
1.2.1 Enzyme hydrolysis. 14
1.2.2 Optimising the conditions. 17
1.3 Conclusion. 18
1.4 Aims and objectives. 19
1.4.1 Aims. 19
1.4.2 Objectives. 19
METHODS. 20
2.1 Chemicals and equipment. 20
2.1.1 Chemicals for buffer solutions. 20
2.1.2 Drug and metabolite standards. 20
2.1.3 β-Glucuronidase enzymes. 20
2.1.4 Blank blood. 20
2.1.5 Equipment. 20
2.1.6 Instrumentation. 21
2.2 Solution Preparation. 21
2.2.1 Standards. 21
2.2.2 Buffers and eluting solutions. 21
2.2.3 β-Glucuronidase enzymes. 21
2.3 Enzyme hydrolysis. 21
2.4 Solid phase extraction (SPE). 22
2.5 Derivatisation. 23
2.6 GC-MS. 23
2.7 Data analysis. 24
RESULTS AND DISCUSSION.. 26
3.1 Method development. 26
3.2 Enzyme hydrolysis in blood. 29
3.2 Conversion in urine. 33
3.3 Temperature dependence. 35
3.4 Further work. 39
CONCLUSION.. 40
REFERENCES. 41
APPENDICES. 44
6.1 Appendix I 44
6.2 Appendix II 45
6.3 Appendix III 49
6.4 Appendix IV.. 53
ACKNOWLEDGEMENTS
I would like to thank my supervisors, Dr Hamnett and Dr Morrison, for their continued support and guidance throughout the project. Thanks also to all the PhD students in the Forensic Medicine and Science department for all their help and advice.
ABSTRACT
FIGURES, TABLES AND APPENDICES
Figure 1: Chemical structure of morphine (C17H19NO3)……………………………………………….. 8
Figure 2: Chemical structure of diacetylamorphine……………………………………………………. 9
Figure 3: Statistics from the National Records of Scotland showing the implication of heroin and/or morphine in drug related deaths in Scotland in 2014…………………………………………………………………… 10
Figure 4: Chemical structures demonstrating the metabolism of heroin within the body. 11
Figure 5: Two glucuronidation pathways of morphine……………………………………………….. 12
Figure 6: The reaction pathway for the enzymatic hydrolysis of M6G through the use of β-glucuronidase 14
Figure 7: Taken from Yang et al. 24 these result show the promising performance of IMCSzyme 16
Figure 8: Taken from Romberg’s paper26, this figure illustrates the optimal pH for each strain of enzyme 18
Figure 9: Un-extracted calibration curve…………………………………………………………………. 26
Figure 10: Extracted calibration curves for blood and urine………………………………………. 26
Figure 11: Ion chromatograms for morphine CAL5 in blood………………………………………. 28
Figure 12: Ion chromatograms for morphine CAL1 in blood………………………………………. 28
Figure 13: Figure illustrating the difference between the 3 and 6 positions in morphine..31
Figure 14: The % conversion of the two morphine metabolites within blood and urine…. 35
Figure 15: Gas chromatogram for the sample M3G P. vulgata incubated for 3 h at 60oC. 36
Figure 16: Gas chromatograms for M3G P. vulgata samples incubated at A – 37oC and B – 45oC. 37
Table 1: Taken from Wang et al.21 this table illustrates that P. vulgata shows the best efficiency for the conversion of morphine glucuronides………………………………………………………………………………………. 15
Table 2: This table shows the labels used for each individual combination of specific enzyme and morphine metabolite……………………………………………………………………………………………………………. 22
Table 3: The volumes used of a 5 µg/ml morphine working solution to obtain the 5 calibrator concentrations required………………………………………………………………………………………………………………. 23
Table 4: Parameters used for GC-MS analysis………………………………………………………….. 24
Table 5: Ionisation mode and settings used for GC-MS analysis………………………………….. 24
Table 6: The mean recoveries of morphine after SPE from blood and urine samples…… 27
Table 7: The % conversions in blood of both M3G and M6G when using four different strains of β-glucuronidase…………………………………………………………………………………………………………………………… 29
Table 8: Internal control data for hydrolysis in blood………………………………………………… 30
Table 9: The % conversions of both morphine metabolites in urine…………………………….. 33
Table 10: Internal control data for hydrolysis in urine……………………………………………….. 34
Table 12: % conversions for each sample are displayed when hydrolysis was carried out at 37 and 45oC 38
Table 13: Internal control data for preliminary temperature study…………………………….. 38
Appendix I: Raw data for calibration curves……………………………………………………………………44
Appendix II: Raw data for hydrolysis in blood…………………………………………………………………45
Appendix III: Raw data for hydrolysis in urine…………………………………………………………………49
Appendix IV: Raw data for preliminary temperature variation study………………………………53
1. INTRODUCTION
1.1 Background
Known botanically as Papaver somniferum, the poppy plant is notorious for its production of opium. Opium can be a liquid, solid or powder, but is generally described as the dried latex of the poppy, a yellow/brown powder containing a mixture of compounds called ‘alkaloids’. The major alkaloid present is morphine (Figure 1). In 1805 morphine was extracted from opium for the first time by a German pharmacologist, Friedrich Wilhelm Adam Serturner.1 The name originates from Greek, meaning ‘God of dreams’ after it was recognised as having potent pain relieving and sleep inducing properties. Morphine was hailed as a ‘miracle drug’ and soon became a common pain relief medication. This remains the case within modern medicine; a common use of morphine is before and after surgery to relieve severe pain. Other compounds are also found in opium and within medicine, these are collectively termed ‘opioids’. However, specifically within the field of forensic toxicology two terms are used to describe these compounds. Opiates are naturally occurring alkaloids within opium, whilst opioids are synthetic compounds with properties similar to opiates, e.g. methadone. The use of both opiates and opioids is increasingly widespread. In 2012, according to the Centre for Disease Control and Prevention in the USA and Canada, more than 259 million opioid prescriptions were written.2
Figure 1: Chemical structure of morphine (C17H19NO3)
The frequent clinical use of these drugs inevitability leads to concerns regarding the potential for their use recreationally. Many opioids/opiates are said to produce feelings of euphoria and as a consequence are heavily abused.3 This is a concern not only within the medical services but also within forensic toxicology. As the number of cases in which these drugs are involved increases, toxicologists need to ensure excellent analytical procedures are in place to guarantee accurate data collection and interpretation.4
1.1.1 Heroin
One of the most commonly abused opioids is heroin, a semi-synthetic drug derived from morphine. It is the acetylated form of morphine and is scientifically known as ‘diacetylmorphine’ (Figure 2). In the 1960s when heroin was first seen on the drug market, it was 100% pure diacetylmorphine.5 Now the term ‘heroin’ relates to a mixture of diacetylmorphine and other acetylated impurities, along with additives used to bulk out the powder in a profit making strategy. Recently within the UK, the purity of heroin has remained fairly constant and is said to be, on average, around 40% pure.5
Figure 2: Chemical structure of diacetylmorphine (C21H23NO5). It can be seen that the two hydroxy groups present in morphine at positions 3 and 6 have undergone acetylation.
Specifically within Scotland the abuse of heroin is a major problem. Revised on the 15th March 2016, the National Records of Scotland produced a statistical report into ‘Drug-related deaths in Scotland in 2014’.6 Six hundred and thirteen deaths due to drugs were reported for the year and, of these, heroin and/or morphine were implicated in, or potentially contributed to, 309 (over 50%). This figure was a substantial increase on the statistics of previous years (Figure 3) indicating that the problem is far from improving. Subsequently, these numbers illustrate the increasing need for the field of forensic toxicology to maintain and improve the accurate analysis of heroin and morphine within biological samples.
Figure 3: Produced from statistics from the National Records of Scotland. The implication of heroin and/or morphine in drug related deaths in Scotland is shown to be very significant. In the most recent study (2014), 50% of all deaths were thought to be attributed to one or both of these drugs.
It is for this reason that the research project detailed within this thesis was carried out. A thorough investigation into optimising part of the procedure for the analysis of morphine within biological samples was performed. This is due to the increasing numbers of samples received containing these drugs which necessitates the need to maintain and advance analytical procedures in morphine/heroin analysis.
1.1.2 Metabolism
Heroin is a highly unstable drug. It has a half-life of only 2-5 minutes and is rarely, if ever detected in post mortem biological samples.7 It is through an understanding of the metabolic pathway of heroin that post mortem forensic analysis can identify its presence.
Within the body, heroin is initially broken down into the intermediate 6-monoacetyl morphine (6-MAM). This also has a short half-life and is further metabolised to morphine (Figure 4). 6-MAM is only occasionally detected within post mortem blood samples, but can be detected within urine samples. Its presence within any biological sample is a marker of heroin use since the metabolite is unique to heroin. However, the main metabolite of heroin is morphine and as a consequence morphine analysis is the focus of this report.8
Figure 4: Chemical structures demonstrating the metabolism of heroin within the body. Initially the acetyl group at position 3 is hydrolysed to a hydroxy group to give the compound 6-MAM. This is then further metabolised into morphine through hydrolysis at position 6.
Morphine undergoes glucuronidation within the body to produce two metabolite conjugates known as morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) (Figure 5). M6G is pharmacologically active and acts as a µ-opioid receptor agonist. It is thought that most of the analgesic effects of morphine are due to M6G rather than morphine itself.9 Conversely, M3G does not exhibit any analgesic activity, although it has been linked to allodynia and seizures within humans.10 According to Chau et al.11 it is generally accepted that the urine recovery from an intravenous dose of morphine leads to 55, 10 and 8% of M3G, M6G and unchanged morphine, respectively. The remainder is attributed to normorphine, morphine-3-sulfate and morphine-3,6-diglucuronide. The focus of this research was on the main glucuronidation metabolic pathway. However, sulfation is still a significant process and has been linked to the intermediary metabolism within fetal-life.12
Figure 5: This figure illustrates the two glucuronidation pathways of morphine, yielding A – M6G and B – M3G. The glucuronic acid molecule is added at position 3 or 6.
1.1.3 Analysis of morphine
Gas Chromatography-Mass spectrometry (GC-MS) is a common analytical tool used in post mortem sample analysis within forensic toxicology. The drugs within a sample are separated through chromatography, and then identified using mass spectrometry. It was introduced in 1959 by Rolande Gohlke and now, along with Liquid Chromatography-Mass Spectrometry (LC-MS), it is the gold standard for drug analysis.13
Although both LC-MS and GC-MS have been used to carry out opiates analysis, the focus in this research remains on GC-MS analysis. The technique is highly suitable and does not suffer from problems often associated with LC-MS.
In general, GC-MS opiates analysis screens for free morphine rather than the two glucuronide conjugates. This is due in part, to the lack of glucuronide reference standards and issues regarding the stability of the metabolites within the mass spectrometer.14 If morphine is left as free morphine and conjugated morphine it remains impossible to identify the total level of morphine within the deceased. If this were the case, the quantitative analysis would be much lower than the real value since the morphine glucuronides would remain undetected. A previous study showed that from a sample of 56 patients’ urine, 43% tested positive for morphine glucuronide conjugates only, whilst just 11% tested positive for the parent drug only.15 Consequently, it has become common practice to carry out a sample pre-treatment step in order to convert morphine glucuronides back to free morphine. This not only yields a more accurate morphine level within the deceased but also gives rise to the opportunity to calculate the percent free morphine.16 This is very useful in terms of providing more information regarding the speed of death. Typically, a percent free morphine of between 51 and 75% is related to a very rapid death following heroin abuse.16
Specific levels of free and total morphine within the blood are more difficult to interpret. There are no definitive toxic, fatal or therapeutic levels due to the phenomenon of tolerance. This relates to the ability of an individual to diminish their response to a drug through chronic use. Every individual will have a unique level of tolerance to morphine/heroin which contributes to these difficulties in data interpretation. A study of 10 morphine fatalities in which no other drugs were detected demonstrated total morphine concentrations in the range of 0.2 – 2.3 mg/l (mean 0.7 mg/l). In contrast, cancer patients receiving morphine treatment at doses of 10 – 2540 mg were reported to have free and total morphine concentrations in the ranges <0.001 – 1 mg/l and 0.02 – 30 mg/l, respectively. All these levels are theoretically therapeutic, however the ranges span a concentration far higher than those seen in the fatal cases previously mentioned.17
The sample pre-treatment used to convert morphine glucuronides back to morphine for the determination of the total morphine concentration, consists of initiating the hydrolysis of the bonds between the morphine and glucuronic acid molecules. It is this procedure that is the centre of the investigation detailed in this thesis.
1.2 Concerning the literature
Within the department of Forensic Medicine and Science in the University of Glasgow, the current process for hydrolysing morphine glucuronides is through the use of the enzyme β-glucuronidase. When added to the sample and left overnight at 37oC, the enzyme breaks the bond between the glucuronic acid and morphine molecules, producing free morphine (Figure 6).
Figure 6: The reaction pathway for the enzymatic hydrolysis of M6G through the use of β-glucuronidase. This yields free morphine and glucuronic acid.
The use of enzymes for hydrolysis is one of two common methods. The other is acid hydrolysis. Acid hydrolysis is a much harsher treatment and, although proven to be more efficient, is less frequently used because of this. It can lead to the hydrolysis of other compounds within a sample, specifically 6-MAM or acetylcodeine and can convert these to morphine which could adversely impact data.18 Subsequently, this research was directed at enzyme rather than acid hydrolysis.
1.2.1 Enzyme hydrolysis
The process of enzyme hydrolysis for the analysis of total morphine has been used since the early 1970s. The literature that has been reviewed indicates that there are a number of different strains of β-glucuronidase that can be used for metabolite hydrolysis. These include, but are not limited to: Helix pomatia (H. pomatia), Escherichia coli (E. coli), Patella vulgata (P. vulgata), abalone and bovine liver.
In 1993 Jennsion et al.19 produced a quantitative report of the success of using β-glucuronidase from P. vulgata. Through a stringent 14 week research programme they illustrated that the conversion of M3G to free morphine was comparable with results previously obtained through acid hydrolysis, with a conversion rate of over 90%. They also demonstrated through the lengthy project, that the run-to-run reproducibility was of an acceptable standard.
A comparison between E. coli and H. pomatia was carried out as part of an investigation by Hackett et al.20 They found that in general H. pomatia was more effective than E. coli. However, good results were still obtained for both, with a 90% conversion rate of M3G in each case. Conversely, the conversion of M6G when using either enzyme was significantly lower. Although the conversion of morphine to M3G within the body does predominate, the far lower conversion of M6G through enzyme hydrolysis could lead to misleading results. The most likely consequence would be an under-estimation of total morphine within a biological sample. Accordingly, this paper indicated the need for further research looking at improving the hydrolysis of M6G.
More recently, Wang et al.21 looked at comparing P. vulgata with E. coli and H. pomatia strains of β-glucuronidase. Again, their work focused not only on the differing enzymes, but also on any differences in hydrolysis between the two morphine metabolites. This paper was one of the first to indicate the potential of the P. vulgata strain of β-glucuronidase. This strain resulted in the highest conversion of the two metabolites (Table 1). The discrepancy between the conversion of M3G and M6G was again demonstrated in this study, suggesting that individually optimised techniques for each metabolite might be required for a more effective total morphine analysis.
Table 1: Taken from Wang et al.21 this table illustrates that P. vulgata shows the best efficiency for the conversion of morphine glucuronides. In addition, it indicates a large difference in the conversion of M6G and M3G.
H. pomatia
E. coli
Acid
P. vulgata
2 h
16 h
2 h
16 h
Standard
Mean
SD
Mean
SD
Mean
SD
Mean
SD
Mean
SD
Mean
SD
M3G (%)
100
4
94
2
33
11
50
13
0
0
0
0
M6G (%)
98
5
12
1
0
0
0
0
0
0
0
0
Another strain of β-glucuronidase that has recently appeared in some papers is that obtained from abalone. Malik-Wolf et al.22 stated that abalone is a more cost-effective enzyme and investigated whether it was a viable substitute for the more commonly used strains detailed above. Through the study of both opiate hydrolysis and other drugs of abuse, they presented a very detailed report. The conversion of morphine metabolites using β-glucuronidase from abalone was shown to be the same when compared with previous procedures using H. pomatia strains of β-glucuronidase. Abalone was therefore shown to be a suitable alternative. However, the report did not detail the effectiveness of the abalone strain for both M3G and M6G conjugates, choosing to solely investigate the more readily hydrolysed M3G metabolite. Consequently, further work is required, particularly since other reports have shown promising results that indicate abalone β-glucuronidase could cause a reduction in the amount of chromatographic interference the hydrolysis step produces.23
Although just a small procedure within the realms of forensic toxicology, research continues to be carried out into enzymatic hydrolysis pre-treatment steps. This year a publication regarding the hydrolysis step evaluated some of the β-glucuronidase enzymes already mentioned, along with a novel recombinant enzyme, IMCSzyme®. Yang et al.24 compared four strains of enzyme (Figure 6) and found that the new ICMSzyme exhibited a significant improvement on metabolite hydrolysis, specifically for codeine-6-glucuronide (C6G) and M6G. Furthermore, their results suggested that the discrepancy between the hydrolysis rates of M3G and M6G was greatly reduced when using this novel enzyme. They demonstrated that for both metabolites a similar conversion rate could be achieved with ~60% conversion of M6G in 1 h. This is a vast improvement on any M6G conversions previously reported.
Figure 7: Taken from Yang et al. 24 these results show the promising performance of IMCSzyme, a recombinant enzyme, in the hydrolysis of a number of glucuronides. These were M3G, M6G, C6G, hydromorphone-3-β-D-glucuronide (H3G), oxymorphone-3- β-D-glucuronide (O3G), buprenorphine-glucuronide (BG) and norbuprenorphine-glucuronide (NBG).
1.2.2 Optimising the conditions
Beyond the varying strains of β-glucuronidase, the conditions at which the hydrolysis procedure is carried out have also been seen to influence the efficiency of the hydrolysis step. A detailed review of the literature revealed that the most influential parameters include incubation temperature, pH, enzyme concentration and incubation time.
Hackett et al.20 looked at ‘Optimizing the hydrolysis of codeine and morphine glucuronides in urine’. Part of the investigation focused on the effects of incubation temperature and length on enzyme hydrolysis. Through the choice of three times: 4, 16 and 24 h they found that for M3G the percentage hydrolysis was fairly time-independent, at roughly 90% for each length. However, in the case of M6G, the hydrolysis was time-dependent and was maximal at 16 h. Furthermore, the hydrolysis of this metabolite remained incomplete.
It may be thought that the temperature of the procedure would be limited to around 37oC due to the possibility of denaturing the enzyme. However, literature has been produced which suggests higher temperatures can be used. As far back as 1982, Combie et al.25 stated that one source of β-glucuronidase from the common limpet had the highest enzyme activity at 65oC. There are obvious benefits to carrying out the hydrolysis step at higher temperatures, since this will normally increase the rate of reaction. This would thereby lower the required incubation time and in turn, reduce sample through-put times within forensic departments.
Again a paper from the 1980s looked into the initial design of a hydrolysis step.26 Through the combination of varying both the strain of enzyme and also the procedure conditions, the paper provided a large introduction into the depth of variation possible within this small step of morphine analysis. The data showed that pH had the greatest influence on percent hydrolysis. Looking at Figure 8, for each strain of enzyme the activity increases with increasing pH to a maximal point and then significantly drops away after this. Interestingly, the optimal pH values vary for each strain of enzyme. This illustrated the importance of optimising the process individually for each different strain of β-glucuronidase.
Figure 8: Taken from Romberg et al.26, this figure illustrated that the optimal pH is different for each strain of enzyme and that the pH as a parameter has a marked effect on overall percent hydrolysis. Abalone (-+-), H. pomatia (-□-) and E. coli (-*-).
So far, all the literature has dealt with the hydrolysis of morphine glucuronides within urine. There is to date only one paper which looks at the process within blood. Sanches et al.18 aimed to look at determining opiates in whole blood and vitreous humour. They used a multivariate statistical analysis technique called response surface methodology (RSM) which allowed the simultaneous optimization of a number of parameters. Using β-glucuronidase from H. pomatia they looked at the impact of varying incubation temperature, incubation time and enzyme concentration. Their investigation showed that the optimal conditions for enzyme hydrolysis were a 4 h incubation time at 37oC with the addition of 20,000 U/ml β-glucuronidase. These conditions gave an overall efficiency of 75%. They noted that the efficiency increased as enzyme concentration increased. In addition, and of particular interest, was the very short incubation length of 4 h which appeared to be sufficient for complete hydrolysis to occur. This was a significantly shorter time than currently used within the department and suggests improvements in the procedure can be made. However, it must be noted that this paper only looked at the hydrolysis of the M3G metabolite, and, as we have seen earlier, it is the M6G metabolite that appears to be the more difficult to hydrolyse.
1.3 Conclusion
Knowledge of the metabolic pathways of heroin and morphine has led to the awareness that a sample pre-treatment step is required prior to morphine analysis, to produce results that give the total morphine concentration within biological samples. This pre-sample treatment normally utilises β-glucuronidase enzymes in the hydrolysis of the bonds between morphine and glucuronic acid molecules. From a thorough review of the literature, a number of strains of enzyme have been identified that can be used, and comparisons showing clear differences between them have been made. Furthermore, there are a number of publications detailing the effects of altering certain conditions during the treatment. There is, however, only one publication that focuses on the pre-treatment step within blood rather than urine. It is therefore the primary focus of this project to report on the pre-treatment hydrolysis step within blood samples to further the knowledge within this field.
1.4 Aims and objectives
1.4.1 Aims
To identify the best strain of β-glucuronidase for enzymatic hydrolysis of morphine metabolites, M3G and M6G.
To optimise the conditions for the hydrolysis process.
1.4.2 Objectives
Use four different strains of β-glucuronidase and obtain % conversion data to indicate the most effective strain.
Through the variation of a number of parameters, identify the best conditions to carry out enzyme hydrolysis after the enzyme that shows the most promising conversion rate has been identified.
2. METHODS
2.1 Chemicals and equipment
2.1.1 Chemicals for buffer solutions
VWR Chemicals, Disodium hydrogen orthophosphate (anhydrous) Lot: F1437681 641; VWR Chemicals, Sodium dihydrogen orthophosphate monohydrate Lot: A629321 602; VWR Chemicals, Sodium acetate (tri-hydrate) Batch: 10G060026; Glacial acetic acid MSc Chem039; SUPLECO N,O-Bistrifluoroacetamide (BSTFA) and trimethylsilyl chloride (TMCS) 99:1 Lot: LC15930V 33148; Methanol Batch: L1606102.
2.1.2 Drug and metabolite standards
Cerilliant, 100 µg/ml Morphine M-030 Lot: FE08061401 Exp: Sept 2019; Cerilliant, 100 µg/ml Morphine-d3 M-003 Lot: FE03061501 Exp: Apr 2020; Lipomed, Morphine-3-β-D-glucuronide 1 mg/ml Lot: 10.2B9.2L4; Lipomed, Morphine-6-β-D-glucuronide 1 mg/ml Lot: 57.1B38.4L3
2.1.3 β-Glucuronidase enzymes
Sigma-Aldrich, β-glucuronidase (Helix pomatia) type HP-2 Lot: 5LBM8544V ≥ 100,000 U/ml; Sigma-Aldrich, β-glucuronidase from limpets (Patella vulgata) Lot: 5LBN3052V ≥ 85,000 U/ml; Roche, β-glucuronidase from Escherichia coli K12 Lot: 10439731 ≥ 254,000 U/ml; Sigma-Aldrich, β-glucuronidase from bovine liver type B-1 Lot: 5LBF1457V ≥ 1,000,000 U/g.
One enzyme unit (U) was defined as the amount of enzyme that catalyses the conversion of 1 micro mole of substrate per minute, at specified conditions and substrate concentration.27
2.1.4 Blank blood
Blank blood was received from the blood service and preserved using a saline solution (9.5 g NaCl in 1 L) at a 50:50 ratio. Red blood cell in additive solution, A Rh D Positive G101615 118586M and A Rh D negative G101615 017543D; Sigma-Aldrich Sodium chloride Lot: U04371.
2.1.5 Equipment
Clean Screen extraction columns, sorbent type CSDAU Lot: 511330-MW Exp: 05/2017.
2.1.6 Instrumentation
Agilent Technologies 7890A GC system coupled with a 7683B series injector attached to a 5975C inert xL MSD with triple-axis detector. For data analysis the pre-installed software, ChemStation (Enhanced ChemStation, MSD ChemStation E.02.02.1431) was used.
2.2 Solution Preparation
2.2.1 Standards
Methanol was used to dilute all reference standards of, morphine, morphine-d3 and morphine metabolites, to the required working solution concentrations.
2.2.2 Buffers and eluting solutions
Phosphate buffer (0.1 M, pH 6) – 0.425 g disodium hydrogen orthophosphate (anhydrous) and 3.035 g sodium dihydrogen orthophosphate monohydrate were dissolved in deionised water. The pH was adjusted to 6 using 1.0 M potassium hydroxide. This was then transferred to a 250 ml volumetric flask and made up to the mark with deionised water.
Acetate buffer (0.1 M, pH 4.5) – 3.4 g sodium acetate (tri-hydrate) was dissolved in deionised water. The pH was adjusted to 4.5 using glacial acetic acid and this mixture was transferred to a 250 ml volumetric flask and made up to the mark using deionised water.
Dichloromethane/iso-propyl alcohol/ammonia (DCM/IPA/NH3) – DCM, IPA and NH3 were mixed in the ratio 78:20:2.
2.2.3 β-Glucuronidase enzymes
For use in the hydrolysis process, the enzymes were diluted down to 10,000 Units in 1 ml of deionised water. Enzyme solutions were prepared fresh on the day of use.
2.3 Enzyme hydrolysis
Four strains of β-glucuronidase enzymes were investigated:
Patella vulgata
Helix pomatia
Bovine liver
Escherichia coli
1 ml of blank blood or 0.5 ml of blank urine was spiked with 100 µl of a 5 µg/ml working solution of either M3G or M6G. To this, 0.4 ml of one strain of β-glucuronidase was added. This process was repeated until all combinations of metabolite and enzyme were prepared (Table 2). The samples were then capped and vortexed for 5 s.
Table 2: This table shows the labels used for each individual combination of specific enzyme and morphine metabolite.
LABEL
M6G P. vulgata
M6G H. pomatia
M6G E. coli
M6G Bovine
M3G P. vulgata
M3G H. pomatia
M3G E. coli
M3G Bovine
The initial part of the project looked at confirming which strain or strains of β-glucuronidase were the most effective for the conversion of the two morphine metabolites. After vortexing, both blood and urine samples were left to incubate overnight at 37oC in an incubator.
The second part of the project aimed to optimise the temperature at which the hydrolysis was carried out. This investigation was only carried out in blood. For this procedure the samples were prepared as before and incubated for 3 h at three different temperatures: 37, 45 and 60oC.
2.4 Solid phase extraction (SPE)
Following enzyme hydrolysis the free morphine was extracted using SPE.
The required number of test tubes were labelled with the appropriate calibrator or sample identification. To each test tube, 5 ml of a 0.1 M pH 6 phosphate buffer/deionised water (1:2) mix was added along with 100 µl of a 1 µg/ml morphine-d3 internal standard (I.S).
To each calibrator a specific volume of a 5 µg/ml morphine standard working solution was added (Table 3). After this, 1 ml of blank blood was added to the calibrators and 1 ml of each sample was added to the corresponding labelled test tubes. In the case of urine only 0.5 ml was added. All tubes were then capped and vortexed for 5 s after which they were centrifuged for 10 min at 3000 rpm.
Table 3: This table shows the volumes used of a 5 µg/ml morphine working solution in order to obtain the 5 calibrator concentrations required.
Calibrator
Concentration / mg/l
Volume of working solution needed / µl
CAL1
0.025
5
CAL2
0.05
10
CAL3
0.1
20
CAL4
0.2
40
CAL5
0.5
100
For SPE the CleanScreen cartridges were pre-conditioned by the following protocol: 3 ml methanol was added, followed by 3 ml deionised water and finally, 1 ml 0.1 M pH 6 phosphate buffer. The samples were then transferred onto the cartridges and left to flow through completely. 3 ml of deionised water was then added. Following this, 2 ml of 0.1 M pH 4.5 acetate buffer and then 3 ml methanol were added. Finally the samples were left to dry for 10 min under vacuum and eluted off with 2.5 ml DCM/IPA/NH3.
2.5 Derivatisation
Following SPE, the samples were evaporated to dryness using a heating block set to 30oC under a flow of nitrogen. Once completely dry 50 µl of BSTFA with 1% TMCS was added to each vial. The samples were capped and vortexed for 5 s and the reaction left to proceed at 90oC for 15 min. Once complete the samples were cooled and transferred to 1.5 ml autosampler vials.
2.6 GC-MS
A method was set up for GC-MS analysis of morphine. Table 4 shows the parameters that were used:
Table 4: Parameters used for GC-MS analysis
Injection volume
1 µl
Inlet temperature
250oC
Oven programme
150oC and ramped to 300oC at 10oC/min and hold for 5 min.
Column
Agilent HP-DB5 5% phenyl methyl siloxane 30 m x 250 µm x 0.25 µm
Table 5: Ionisation mode and settings used for GC-MS analysis
Ionisation mode
Selective ion monitoring (SIM)
Dwell time
0.05 span 0.1
SIM settings
Morphine: 429, 414, 236
Morphine-d3: 432, 417
The ions shown in bold (Table 5) were the quantifier ions and the others were used as qualifier ions.
2.7 Data analysis
To quantitate the levels of morphine within each sample a calibration was set up for each matrix using the ChemStation software. This used the peak area ratio (PAR) between morphine (m/z 429) and morphine-d3 (m/z 432), where morphine-d3 acted as the I.S.
To calculate percentage efficiency of the hydrolysis procedure the following equation was used:
Quantitative data relating to the accuracy and reproducibility of the experiments was achieved through using standard deviation (SD), percentage coefficient of variation (% CV) and % accuracy. The equations for each are shown below:
Where µ = mean and N = total number of data sets
3. RESULTS AND DISCUSSION
3.1 Method development
Initial experiments focused on optimising and confirming the GC and MS parameters. This produced a working method for the identification of morphine. Once established (Section 2.6) an un-extracted calibration curve (Figure 9) and extracted calibration curves for both blood and urine matrices were produced (Figure 10).
Figure 9: Un-extracted calibration curve. This illustrated the method set on the GC-MS was satisfactory and the derivatisation of morphine worked well. The R2 value of 0.997 was adequate and the equation of the straight line was y = 12.34x – 0.0803.
Figure 10: Extracted calibration curves for blood and urine. For blood R2 = 0.997 and y = 9.9632x + 0.0556 and for urine R2 = 0.997 and y = 10.463x + 0.1199.
From the calibration curves, the final method for GC-MS analysis was decided upon and a robust procedure was set in place. All R2 values were > 0.99 which indicated an appropriate experimental procedure had been determined. Furthermore, the chromatograms produced, of which two are shown (Figures 11 and 12), illustrated a high quality analysis was carried out. Both the quantifying and qualifying ion peaks for morphine and morphine-d3 were narrow and there was little background interference. The excellent quality of the chromatograms provided evidence that accurate quantifications would be carried out throughout the investigation.
From the un-extracted and extracted calibration curves, the mean % recovery of morphine from both blood and urine using SPE was calculated (Table 6). The calculated recovery was in line with that published in previous literature and showed that SPE was an appropriate extraction method.28 As expected, the recovery was slightly better in urine than in blood. Urine is almost 95% water and unlike blood does not contain other proteins and lipids which can interfere with the extraction process. It should be noted that to obtain this quantitative data the first calibration PAR was removed. This was due to a poor signal for the un-extracted calibrator which resulted in unrepresentative % recoveries for both blood and urine (Appendix I).
Table 6: The mean recoveries of morphine after SPE from blood and urine samples are given. The % recovery of morphine and the repeatability were marginally improved in urine when compared to blood. % CV values for both matrices were adequate.
Matrix
Mean Recovery / %
% CV
Blood
87.3
15.3
Urine
96.7
8.73
Figure 11: Ion chromatograms for morphine CAL5 in blood. A very clean chromatogram was obtained with a clear peak for morphine and morphine-d3 shown. Retention times of 16.127 and 16.109 min were observed for morphine and morphine-d3, respectively.
Figure 12: Ion chromatograms for CAL1 in blood. In comparison to CAL5 there was a much smaller response for morphine which was as expected. All peaks were narrow which again indicated a good analysis procedure.
3.2 Enzyme hydrolysis in blood
Table 7 shows the % conversion of the two main morphine metabolites for each strain of β-glucuronidase. All raw data can be found in the Appendix II.
Table 7: The % conversions of both M3G and M6G through the use of four different strains of β-glucuronidase showed that the E. coli strain was the most effective for M6G, the bovine liver strain was not effective at all and for the conversion of M3G, P. vulgata, H. pomatia and E. coli all performed to a very similar degree.
% Conversion
Sample
1st repeat
2nd repeat
3rd repeat
Average
% CV
M6G H. pomatia
10.4
9.6
11.6
10.5
9.1
M6G P. vulgata
16.9
16.4
14.5
15.9
7.9
M6G E. coli
34.1
33.8
28.9
32.3
8.9
M6G Bovine
0.0
0.54
4.0
1.5
143.0
M3G H. pomatia
24.3
23.7
23.4
23.8
2.0
M3G P. vulgata
24.1
27.1
24.0
25.1
7.1
M3G E. coli
27.6
26.2
22.2
25.3
11.0
M3G Bovine
1.5
2.4
1.4
1.7
32.8
The three repeats were run as individual experiments and not as duplicates, thereby providing an indication of the reproducibility of the procedure. The statistical analysis (% CV) showed that overall there was a high level of precision between the three separate runs. Traditionally, within the field of forensic toxicology, a % CV value of ≤ 15% is deemed appropriate. Here that was the case in all but two samples, M6G bovine and M3G bovine. However, since the hydrolysis of the metabolites was very low when using bovine liver β-glucuronidase, the high % CV values were not a significant concern.
Beyond reproducibility, the accuracy of the procedure was monitored through the use of a spiked sample as an internal control. Each repeat incorporated a sample at a known concentration of 0.25 mg/l. Table 8 shows the determined concentrations of morphine for each spike after GC-MS analysis, along with the corresponding % accuracy. The results obtained demonstrated that the accuracy of each process was very high. This lends weight to the findings and conclusions drawn from the investigation.
Table 8: For each repeat run the quantitation of the spiked sample after analysis showed good agreement with the real value of 0.25 mg/l.
Repeat
Spike concentration / mg/l
% Accuracy
1
0.246
-1.6
2
0.257
2.8
3
0.242
-3.2
Overall, the % conversions were much lower than expected. Such low conversions would be a significant problem within forensic toxicology departments due to the occurrence of false negatives, or poor representations of the amount of heroin or morphine taken by an individual. Within the literature far higher conversions have been obtained. However, as noted in Section 1.3, there is currently just a single paper published that concerns the hydrolysis of morphine glucuronides in blood. Sanches et al.18 reported a 75% conversion of M3G using H. pomatia and incubating for 4 h at 37oC with an enzyme concentration of 20,000 U/ml. In this investigation the enzyme concentration used was only 10,000 U/ml. This lower concentration may be one reason for the noticeably poorer % conversions. Furthermore, it is difficult to make a definitive comparison with just a single paper. It remains necessary for more research to be carried out regarding the hydrolysis of morphine glucuronides within blood, for a better evaluation of the overall experimental performance to be made.
Within Section 1.1.2 it was ascertained that morphine is metabolised into two main compounds; M3G and M6G. The results showed that the two morphine metabolites did not act in the same way during the hydrolysis process. Reports of this have already been seen and the evidence suggests M6G is less readily hydrolysed than M3G.21, 26 The most accepted explanation for this, centres around the influence of the benzene ring within morphine. In M3G the glucuronic acid group is attached to the benzene ring, whilst in comparison, the glucuronic acid group in M6G is attached to a cyclohexene ring (Figure 13). The chemical nature of a benzene ring means that the anion produced during the hydrolysis of M3G is more stable. If the anion is stabilised to a higher degree then the reaction is more favourable and so can occur more readily and hence M3G is hydrolysed at a quicker rate. However, within this investigation, the highest conversion of all the samples was seen for M6G rather than M3G. For each repeat the E. coli strain of β-glucuronidase gave the highest conversion for M6G, at an average of 32.3%. Already reported is the preferential conversion of M6G by E. coli compared to H. pomatia in urine.20 However, it is difficult to explain the apparent better conversion by E. coli β-glucuronidase of M6G than M3G considering the same conditions were used for each sample. The manufacturer (Sigma-Aldrich) state that the enzyme activity of E. coli β-glucuronidase is retained better through hydrolysis than for other similar strains which may be more sensitive to changes in concentration of glucuronides. This difference could potentially give some explanation to the results obtained here, although the metabolite concentrations were identical in each sample so the effects of this would have been limited. Consequently, this demonstrates a pressing need for more research to be carried out regarding the E. coli strain of β-glucuronidase, to identify whether it always preferentially hydrolyses M6G. If this were the case, it would be influential to forensic department protocols in increasing the conversion of the active metabolite which has, until now, shown much poorer conversions.
Figure 13: Morphine molecule, illustrating the difference between the 3 and 6 positions. The benzene ring at position 3 makes hydrolysis at this position more favourable.
In contrast to the E. coli strain of β-glucuronidase, the comparable performance of the other three strains in the conversion of the two metabolites was more in line with the literature and what was expected. It was clear that β-glucuronidase from bovine liver was very poor at hydrolysing morphine glucuronides. The average % conversions of M3G and M6G were just 1.7% and 1.5%, respectively. These decidedly low conversions could have been due, in part, to the incubation temperature (37oC) which was lower than the internal body temperature of a cow (38.6oC) and so would not have been optimal.29 Other parameters may also have played a role, for example pH, but it appears to be probable that the activity of the strain was just not as high as the others. Consequently, it can be concluded that β-glucuronidase from bovine liver is not an appropriate choice for the pre-treatment hydrolysis step in morphine analysis.
Looking more closely at H. pomatia and P. vulgata, there was little recognisable difference between the performances of these two strains of enzyme. There was a marginally better conversion of M6G by P. vulgata than H. pomatia, yet the difference was limited for M3G and it cannot be stated confidently that one was significantly better than the other. We noted the report earlier by Wang et al.21 which detailed the conversion of M3G and M6G by P. vulgata and H. pomatia β-glucuronidase. The quantitative data here, presented a significant improvement in the conversion of the M6G metabolite in comparison. Specifically the conversion of M6G by H. pomatia was far better than Wang et al.21 reported. This suggests the potential suitability of these enzymes for analytical use. However, Wang et al.’s 21 experiments were carried out in urine not blood and the inconsistencies may accordingly be due to the differences between the matrices. Furthermore, in similarity with β-glucuronidase from bovine liver the low conversions seen for H. pomatia and P. vulgata strains of β-glucuronidase could have been due, in part, to the non-optimised conditions. In his work looking at ‘The biology of limpets’, Branch stated that the internal body temperature can vary from 5 to 34oC when exposed at midday.30 In the case of H. pomatia, the internal body temperature of a snail is also very adaptable`.31 This could mean an incubation temperature of 37oC was not optimal for these two strains of β-glucuronidase. A study concerning the effects of temperature on hydrolysis was carried out as part of this project and the results are detailed subsequently (Section 3.3).
As already discussed, the conversion of M6G by E. coli β-glucuronidase was the most promising. In continuation, the conversion of M3G by the same strain of enzyme was comparable with H. pomatia and P. vulgata. This led to the conclusion that this strain of enzyme would appear to be the most effective for the hydrolysis of morphine glucuronides in blood. This is particularly encouraging due to the economic advantage of E. coli. Its cost per ml is significantly lower than that of P. vulgata and H. pomatia and it is also sold at a higher U/ml concentration which means smaller quantities can be purchased. However, M3G is the main metabolite of morphine and therefore present at the highest concentrations within case samples. The comparatively poorer conversion of this metabolite by E. coli β-glucuronidase compared to M6G needs to be rectified to ensure the maximum conversion of both morphine glucuronides is reached.
3.2 Conversion in urine
The next stage in the project aimed to identify any differences between the hydrolysis of morphine glucuronides within urine compared to blood. Furthermore, since the majority of the literature has so far been carried out in urine, this allowed for better comparisons to be made between the literature and the data obtained through this investigation. Due to the extremely low performance of β-glucuronidase from bovine liver in blood samples, this strain of enzyme was not included in this part of the study. All raw data can be found in Appendix III.
Table 9: The % conversions of both morphine metabolites within urine are shown in this table. Similar to the results seen within blood, the M6G E. coli sample showed the best conversion.
% Conversion
Sample
1st repeat
2nd repeat
3rd repeat
Average
% CV
M6G H. pomatia
10.2
3.0
3.4
5.5
74.0
M6G P. vulgata
7.7
2.7
2.7
4.4
65.6
M6G E. coli
21.2
25.8
25.3
24.1
10.6
M3G H. pomatia
19.7
16.9
15.5
17.4
12.4
M3G P. vulgata
19.8
19.6
17.9
19.1
5.25
M3G E. coli
17.7
17.8
18.3
17.9
1.65
Repeats 2 and 3 were run as duplicates and hence had a closer correlation than the 1st repeat which was run individually. The % CV values were adequate for four of the samples, but were very high in the case of the M6G H. pomatia and M6G P. vulgata samples. Seemingly the conversion was much lower in the 2nd and 3rd repeats. This discrepancy was surprising since the lower conversions were not seen for M3G when using these two strains of enzyme or within the other samples. Spiked urine controls were used to monitor the accuracy of each run. Table 10 shows the results of the quantitation post analysis for each internal control. The first run showed a slightly poor % accuracy. This could have been the reason for the high % CV values identified already, and is in line with the higher % conversions seen for some samples in this repeat. Further runs are required to obtain lower and more appropriate % CV values across all of the samples. Unfortunately due to time constraints this could not be carried out, but future work would incorporate this.
Table 10: Spikes were used to monitor the accuracy of the experiment. Repeat 1 shows a slightly poorer % accuracy than the spike for runs 2 and 3 which was more in line with the level desired.
Repeat
Spike concentration / mg/l
% Accuracy
1
0.280
12
2 & 3
0.242
-3.2
The overall % conversions were again very low. % conversions as high as > 90% and > 80% of M3G and M6G, respectively, have previously been reported in the literature.20, 22 The low % conversions obtained here could have been due in part, to not having optimised the conditions for each strain of β-glucuronidase. Section 1.2.2 presented several studies that demonstrated the effects of varying specific parameters on the efficiency of morphine glucuronide hydrolysis. However, this lack of optimised parameters would not have been expected to cause such a large reduction in the % conversion. Again due to time constraints, more work in trying to improve the % conversion could not be carried out. This would be an area of importance in any further work.
Even more unexpected was the lower % conversions within urine than blood (Figure 14). Section 3.1 illustrated the % recovery of morphine from urine was slightly higher than that from blood. Subsequently, a higher concentration of morphine would have been expected in this matrix. However, that was not the case. It is difficult to give a definitive explanation for this, but there are reports that endogenous glucuronidase inhibitors can be present in some urine.32 If that were the case then it could have been a factor which reduced the overall conversion in urine.
What was of particular interest was the similarity in the relative performance of each strain of β-glucuronidase within urine and blood (Figure 14). In both matrices the M6G E. coli sample gave the highest conversion, averaging 24.1% and 32.3% in urine and blood, respectively. This lent significant strength to the argument that the E. coli strain of β-glucuronidase is very promising for the conversion of the M6G metabolite. Moreover, in terms of the H. pomatia and P. vulgata strains of β-glucuronidase, the relative performance was similar within both matrices. The conversion of M3G differed by no more than 2% for each enzyme and was consistently higher than M6G. There was a slightly larger difference between the conversions of M6G by the two enzymes, where in blood P. vulgata β-glucuronidase was more effective, and in urine H. pomatia β-glucuronidase produced a slightly more efficient conversion. Nevertheless, the results for both enzymes were still relatively similar and there was a discrepancy of no more than 5% between each enzyme.
As yet, a direct comparison between the hydrolysis of morphine glucuronides in blood and urine has not been carried out. These results demonstrated that although the comparable efficiency of each enzyme remained relatively alike, the overall % conversion varied between the two matrices. Therefore, it might be necessary to look at altering certain parameters, for example enzyme concentration, to try to bring the conversion in urine more in line with that in blood.
Figure 14: A direct comparison was made between the % conversion of the two morphine metabolites within blood and urine. For all samples the conversion was better in blood.
3.3 Temperature dependence
Studies have previously been carried out that indicated the incubation temperature for the hydrolysis pre-treatment step can heavily influence the degree of conversion. So far, the research has focused almost entirely on the hydrolysis of morphine glucuronides within urine. It was the aim of this project to ascertain whether similar effects were seen within blood samples. Unfortunately, due to time constraints only a preliminary investigation could be carried out, and the data reported here needs repeating to verify it. Nonetheless, initial results and corresponding conclusions were gathered.
Three different incubation temperatures were used: 37, 45 and 60oC and all raw data can be found in Appendix IV. It was initially the aim to continue to use an overnight incubation. However, due to time restraints and other individuals using the incubator the incubation time was reduced to 3 h. The first run was carried out at 60oC. At this temperature the blood samples became very congealed. This meant the extracts were extremely dirty and as a result, the chromatograms were too noisy for any quantifiable peaks to be identified (Figure 15). This suggests that although sometimes effective within urine, such a high temperature is not optimal for morphine analysis within blood samples. A repeat extraction to attempt to produce a cleaner sample could be applied, but this would be neither time nor cost efficient within a busy forensic toxicology department.
Figure 15: The gas chromatogram for the sample M3G P. vulgata incubated for 3 h at 60oC. The sample was too dirty for any peaks to be identified. This was the case for all samples incubated at this temperature.
For the incubation temperatures of 45 and 37oC the chromatograms were much cleaner (Figure 16). The background noise was slightly higher within the 45oC sample as would be expected, but the peak was still very narrow and an accurate quantitation could be carried out. Table 12 shows the % conversion of each metabolite carried out at 45 and 37oC. No data was available for the M3G E. coli sample at 37oC since the chromatogram for this sample was very poor and there was not time to run a repeat analysis. The % conversions for all other samples were higher when incubated at 45oC. This is suggestive of an optimal hydrolysis temperature higher than that currently used within the Forensic Medicine and Science department at the University of Glasgow. In addition the % conversion for M3G H. pomatia, M3G P. vulgata and M3G E. coli when incubated at 45oC for just 3 h was only marginally below the % conversion of the same samples when incubated overnight at 37oC, which were, 23.8, 25.1 and 25.3%, respectively (Section 3.2). This could lead to savings in both time and money by running the hydrolysis at a slightly higher temperature for a much shorter incubation time. However, there was a discrepancy between the two investigations. The M6G conversions were much lower in this preliminary temperature study. This needs to be verified and explained before any substantial changes are made to methods already in place.
B
A
Figure 16 Gas chromatograms for M3G P. vulgata samples incubated at A – 37oC and B – 45oC. Both showed clean narrow peaks which gave accurate quantitation. There was slightly more background noise in the sample incubated at 45oC.
Again, the E. coli strain of β-glucuronidase showed the best performance for the conversion of morphine metabolites. However, in contrast with what was reported in the previous section, the M6G E. coli samples did not show the highest conversion. The accepted trend from the literature of improved conversion of M3G compared with M6G was observed. Overall, no single enzyme appeared to perform better at the higher temperature than any other. For M6G the highest increase in % conversion was seen when using P. vulgata, whereas for M3G it was H. pomatia. Although of course it was not possible to know for definite, due to the lack of data for E. coli. Subsequently, the only real conclusion that can be drawn is that the higher temperature appears to provide a more optimal environment for enzymatic hydrolysis.
Table 12: % conversions for each sample are displayed when hydrolysis was carried out at 37 and 45oC. The conversion was better at the higher temperature. No data could be obtained for M3G E. coli at 37oC due to a poor chromatogram being produced.
% Conversion
Sample
37oC
45oC
M6G H. pomatia
2.3
4.0
M6G P. vulgata
2.8
8.4
M6G E. coli
7.7
9.8
M3G H. pomatia
13.2
18.3
M3G P. vulgata
20.3
22.3
M3G E. coli
–
23.4
Spiked samples were again used as internal controls to monitor the accuracy of each procedure (Table 13). For the runs at 37 and 60oC there was excellent accuracy in the quantified spike concentrations. However, the quantification of the spike in the 45oC run was relatively poor and did not confirm an accurate analysis had been carried out. In general, within forensic toxicology an internal control within ±20% of the real value is considered adequate. This was not the case and so the procedure needs to be repeated to validate the initial trends identified.
Table 13: Calculated spike concentration post analysis showed accurate runs for 37 and 60oC, but a poor quality run when incubated at 45oC
Incubation Temperature / oC
Spike Concentration / mg/l
% Accuracy
37
0.253
1.2
45
0.195
-22
60
0.253
1.2
Sanches et al.18 reported the optimisation of hydrolysis within blood. However, they used the RSM for optimization. This meant that they did not report on the individual influence of temperature. They did state that a decrease in the hydrolysis rate was observed with an increase in temperature and attributed this to enzyme degradation. Here the first report looking directly at the effects of varying temperature on morphine glucuronide hydrolysis within blood has been produced.
3.4 Further work
Due to some discrepancies already identified between repeat analyses, it would be the intention to carry out further investigations to confirm the findings reported here. The variation in the performance of E. coli between the two incubation times needs to be confirmed. In addition, it was the original aim (Section 1.4) to optimise a number of conditions for morphine glucuronide hydrolysis. Therefore, further work would include studies looking at the influence of pH, enzyme concentration and incubation length on the hydrolysis within blood samples.
There is still no research published which looks at the hydrolysis of morphine glucuronides within post mortem blood samples. Here, only blank ante mortem blood was used for the investigation. Post mortem blood is often much more difficult to work with. It was shown through the temperature study the effects congealing of the blood had on the GC-MS analysis and this phenomenon would likely be a problem when using post mortem samples. Consequently, the next step is to use case samples to monitor the conversion of morphine metabolites and the extraction of morphine following hydrolysis.
Finally, there have been a number of papers that have reported the success of using LC-MS to analyse samples for morphine metabolites directly, rather than using the hydrolysis pre-treatment step. Generally these methods utilise LC-MS-MS. The hybrid technique increases the sensitivity, with one report claiming to identify the metabolites in the low ng/ml range.33 If this is the case, then the ability to detect the levels of morphine metabolites directly would remove any of the uncertainties that have been established through this investigation when using enzymatic hydrolysis. Consequently, this should become an area of interest in the continuing research into accurate morphine analysis.
4. CONCLUSION
A thorough investigation was carried out to ascertain the most efficient strain of β-glucuronidase for the hydrolysis of morphine glucuronides, M6G and M3G. β-Glucuronidase from bovine liver was very poor and would not be a suitable option for use in morphine analysis. The E. coli strain was identified as being the most effective for the conversion of M6G in both blood and urine. For the conversion of M3G the results were less definitive and a similar average % conversion was seen for H. pomatia, P. vulgata and E. coli. E. coli was thus recognized as the most promising strain of β-glucuronidase overall.
Through the use of both blood and urine matrices the first comparison of hydrolysis within the two was made. It was concluded that although there was a lower % conversion of both metabolites in urine, the respective performances of the individual strains of β-glucuronidase within the two matrices were very similar.
The preliminary investigation into the effect of temperature for the hydrolysis of M6G and M3G within blood, demonstrated that incubating at 45oC was optimal. At 60oC severe congealing of the blood made the samples too dirty to analyse.
Finally, further work was identified including: validation, analysis in post mortem blood and direct metabolite analysis. Ultimately this would add to the results documented within this project and continue to aid the improvement of morphine analysis within the field of forensic toxicology.
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6. APPENDICES
6.1 Appendix I
All raw data for the un-extracted and extracted calibration curves is given here:
6.2 Appendix II
All raw data is given for the analysis of the samples within blood. A chromatogram is also included for the sample M6G E. coli.
6.3 Appendix III
All raw data is given for the analysis within urine. A chromatogram of the M6G E. coli samples is also included.
6.4 Appendix IV
All raw data is given for the chromatograms obtained during the temperature study.
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