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The use of microwave-assisted extraction (MAE) in screening of decomposed bone tissue for model drugs of abuse is described. Rats received 50mg/kg (i.p.) pentobarbital (n =2), 75mg/kg (i.p.) ketamine (n =2) or 16mg/kg (i.p.) diazepam (n =1), or remained drug-free (control). Drug-positive animals were euthanized within 20min of drug administration. Animal remains were allowed to decompose in a secure outdoor environment to the point of complete skeletonization. Bones (tibiae, femora, vertebrae, ribs, pelvi, humeri and scapulae) were collected and pooled (according to drug) in order to minimize effects due to inter-bone differences in drug distribution. Bones were crushed and cleaned of marrow and residual soft tissue in alkaline solution or phosphate buffer with ultrasonication. Cleaned bones were then ground and underwent MAE in phosphate buffer (pH 6), methanol or a methanol:water mixture (1:1, v/v) at atmospheric pressure in a domestic microwave oven, or passive extraction in methanol. Bone extracts (control and drug-exposed) containing methanol were evaporated to dryness before reconstitution in phosphate buffer (pH 6) and subsequent analysis by ELISA, while bone extracts containing only phosphate buffer were assayed directly by the same ELISA protocol. Measured absorbance values were expressed as the decrease in absorbance, measured as a percentage, relative to the corresponding drug-free control bone extract. The semi-quantitative nature of the ELISA assay allowed examination of the effects of extraction solvent and bone sample mass on the assay response for each drug examined, and subsequent comparison to assays of extracts obtained through passive methanolic extraction of various bone tissues. Overall, the time required for maximal extraction varied with extraction solvent and bone mass for each drug investigated, with significant extraction occurring with all solvent systems examined. MAE may represent a substantially faster extraction system than passive extraction, with significant extraction recovery observed within 1min of exposure for all samples examined. The implications of these results in the context of the available literature on drug analysis in skeletal tissues are discussed.
Study | Sample matrix | Extraction solvent | Drug(s) detected | Extraction time (h) |
McIntyre et al. [4] | Mid-femoral sections--human | Methanol | Tricyclic antidepressants, nontricyclic antidepressants benzodiazepines, antipsychotics | 18 |
Horak and Jenkins [8] | Mid-femoral slivers--human | Methanol or water | Citalopram | 24 |
Guillot et al. [7] | Bone (unspecified anatomic source)--mouse | 0.1N HCl | Morphine | 12 |
Raikos et al. [10] | Femoral fragment--human | 3N HNO3 | Morphine | 24 |
Watterson et al. [1,2,5] | Diaphyseal and epiphyseal femoral sections--rat | Methanol | Ketamine, diazepam | 72 |
Wohlenberg, et al. [3] | Cancellous vertebral Bone--Human | Methanol | Nortriptyline | 15 |
Gorczynski and Melbye [6] | Bone (unspecified anatomic source)--mouse and rats | PBS | Midazolam, diazepam, lorazepam | Unspecified |
Table 1 - Summary of reported extraction methodologies for toxicological analysis of skeletal tissues, with time used for extraction.
Drug | R MAE (PBS) vs. MAE (MeOH) | R MAE (PBS) vs. passive extraction (MeOH) | R MAE (MeOH) vs. passive extraction (MeOH) |
Pentobarbital | 1.9 | 1.3 | 0.69 |
Ketamine | 1.8 | 2.1 | 1.2 |
Diazepam | 1.4 | 2.6 | 1.8 |
Table 2 - Semi-quantitative comparison of extraction recovery: ratio of sum of incremental %decrease in absorbance from successive rounds of MAE relative to sum of incremental %decrease in absorbance from successive rounds of passive extraction.
1
Introduction
When skeletonized remains are encountered in death investigations, conventional sample matrices, such as blood, are scant or completely unavailable. Therefore, bone tissue may be the only source of toxicological information. Although a growing body of research has been performed in this area [1-10], with drugs such as ketamine [1,2], tricyclic antidepressants [3,4], benzodiazepines [4-6], antipsychotics [4] and morphine [7] being detected in bone or bone marrow, understanding of the implications and limitations of drug measurements in skeletal tissues remains poor. For example, drug and metabolite uptake into bone is poorly understood, as is the time course of drugs within those tissues. Only a handful of studies have considered the distribution of drug within a bone (i.e., trabecular vs. cortical bone) [1,2,5]; drug distribution between different bones (e.g., femur vs. vertebrae) has not been studied under controlled conditions.
Drug recovery from mineralized bone may be challenging, and remains poorly characterized. Review of the literature shows that drug isolation from bone has been attempted using a number of different methods, including passive methanolic extraction of transverse slices from the mid-femoral region [4], bone slivers [8], or ground bone [1,2,5], Soxhlet extraction [9], and acid digestion [7,10]. Typically, the methods reported thus far have been time-consuming, typically requiring 12-72h of incubation time (Table 1). In laboratory studies of drug distribution between different tissue types, which typically make use of experimental animals, it would not be unreasonable to expect to generate 5-10 different samples per animal under a given set of experimental conditions (e.g., drug, dose, etc.). Clearly then, use of passive extraction or digestion methods represents a significant limitation to analytical throughput.
One approach to expedite this process is microwave-assisted extraction (MAE). MAE is well established and commonly used in environmental analysis, with reported extractions of polyaromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and organochlorine pesticides (OCPs) [11,12] from a variety of substrates of environmental interest (e.g., sediments). Microwave heating is also used for digestion of heavy metals [13]. Variables such as time, pressure, temperature and moisture content have been found to affect extraction efficiency [14,15].
In ordinary passive extraction, heat is transferred to the vessel, which heats the solvent in order to desorb constituents from the matrix, which then diffuse into and become dissolved in the bulk solvent. Microwave heating makes use of the absorption of microwaves by the solvent and/or the test substance. Microwaves, which can range from 300MHz to 300GHz (2450MHz is reserved for commercial or domestic microwave ovens [15]), are a form of electromagnetic radiation composed of oscillating perpendicular magnetic and electric fields. Interaction of polar molecules with these fields results in dipolar rotation in order to facilitate molecular alignment with the applied field. However, due to the oscillatory nature of the field, molecular dipoles ultimately undergo rotational oscillations, creating friction which is released as heat. Ionic solutions undergo a second heating mechanism that proceeds by resistive heating of the solution following movement of the dissolved ions in the presence of the oscillating field. Ultimately, the passive (conductive/convective) heating process is by-passed, and heating may be applied in a focused manner and almost instantaneously to the matrix undergoing extraction. The resultant molecular motions result in desorption of bound analyte, and subsequent dissolution in the surrounding solvent [15,16].
The ability to absorb microwave energy is material dependent. Energy absorption is related to the dielectric loss factor, which, in turn, is a function of the permittivity of the material. Generally, polar substances have a larger dielectric loss factor than non-polar substances, and thus tend to absorb microwaves much more efficiently. Depending on the choice of extraction solvent and the nature of the sample, heating can be focused directly and almost exclusively on the sample itself and/or to the surrounding solvent.
The application of MAE in forensic toxicology has been extremely rare. Franke et al. discussed the use of MAE to facilitate the extraction of drugs into an organic solvent mixture [17] and reported substantial increases in extraction recovery in some cases. In this study, we examined the utility of MAE for the extraction of model drugs from ground bone, as part of an effort to streamline methodologies to improve the efficiency of studies of drug disposition in skeletal tissues. The objective of this work was to examine the effects of target drug, solvent, irradiation time and sample mass on the time required for drug recovery, and to compare this methodology against a passive methanolic extraction strategy which has been used in our laboratory previously [1,2,5]. This work involved the extraction of three model drugs (ketamine, diazepam and pentobarbital) from pooled, ground bone tissues obtained from acutely exposed animals following a period of significant decomposition. These drugs were chosen for their forensic relevance as well as their varying chemical properties (e.g., acid-base character). We used an open-vessel/atmospheric pressure configuration and a domestic microwave oven in the extraction process. Tissue extracts were analyzed by automated ELISA. We have shown recently [1,2,5] that ELISA is a very sensitive and reliable method for screening of skeletal tissues for drug exposure. In this application, ELISA is valuable in that it provides a semi-quantitative response, which facilitates estimation of optimal extraction conditions, while allowing for parallel analysis of samples.
2
Methods
2.1 Chemicals
Methanol used in drug extraction was HPLC grade and purchased from EMD chemicals (Gibbstown, NJ) Drug standards (Cerilliant, Round Rock, TX) were obtained as 1mg/ml methanolic solutions and diluted as required. All other chemicals were reagent grade and were obtained from EMD chemicals (Gibbstown, NJ).
2.2 Animals and drug administration
Adult male Wistar were given 16mg/kg diazepam (n =1), 75mg/kg ketamine (n =2) or 50mg/kg pentobarbital (n =2). Rats were euthanized with CO2 within 20min. The remains were placed in secure cages, and were left to decompose outdoors at a rural Ontario site during late summer (August-September), with full exposure to sunlight and other weather features (precipitation, etc.). The mean maximum and minimum daily temperatures were 23.8 and 11.8°C, respectively, and the total rainfall was 55.8mm. Decomposition was allowed to proceed naturally to the point of nearly complete skeletonization (only skin, fur and bones remaining), and required roughly 3 weeks. Bones, including femora, tibiae, vertebrae, pelvi, humeri, scapulae and ribs were collected. Drug-free animals were also prepared in this fashion, to provide negative control tissues.
2.3 Bone preparation
Ketamine-exposed, pentobarbital-exposed and control bones were immersed in a 0.5M NaOH:NaCl (50:50) solution and cleaned by ultrasonication until no soft tissue remained (approximately 1.5h for femoral, tibial and pelvic bones and 3h for vertebrae). Solution was replaced once for leg bones and three times for vertebrae. Diazepam-exposed bones were cleaned with a 0.5M phosphate buffer, pH 8.5 (PB8) solution because of observed degradation of the ELISA response following exposure of the drug to the alkaline solution with ultrasonication. Bones were immersed in PB8 and underwent ultrasonication for approximately 2h. The solution was replaced twice.
Once clean, the bones were washed twice with distilled water and once with acetone. Bones were then dried under a gentle stream of argon at 50°C. The different bones were then pooled (according to drug) and pooled bones were then ground into fine particles using a domestic grinder, resulting in a total mass of approximately 9g of each of the drug-exposed tissues. Glass screw-cap tubes (20ml) were used as extraction vessels. A single 1/8in. hole was drilled in the top of each cap and a glass capillary was inserted into the hole and held in place with Teflon tape, in order to serve as a vent.
2.4 Passive extraction
Samples (0.5g) of each bone were accurately weighed into screw-cap test tubes, and 2ml of methanol was added to each tube. Samples were incubated on a hot plate at 50°C (±1-2°C). For each sample, at successive, defined intervals (1, 6, 12 and 24h), the solvent was removed and replaced with clean solvent in order to monitor the rate of extraction. Following each solvent recovery, bones were washed twice with 1ml of methanol and the methanol washes were pooled with the originally recovered fraction. All recovered methanol fractions were then evaporated under a gentle stream of argon at 50°C. The samples were reconstituted in 2ml of 0.1M phosphate buffer, pH 6 (PB6).
2.5 Stability of drugs to microwave exposure--ELISA
The solvents examined in the extraction of each drug were PB6, methanol:water (MeOH:H2O, 1:1, v/v) and methanol (MeOH). The stability of the ELISA response to ketamine, pentobarbital and diazepam under microwave irradiation in each of these solutions was examined. Solutions of ketamine (50ng/ml), pentobarbital (50ng/ml) and diazepam (2.5ng/ml) were prepared in each solvent and transferred to capillary screw-cap test tubes. A household Danby microwave oven DMW1153W (1100W, 2450MHz) equipped with a turntable was used for the extraction. Solutions were then irradiated for 1, 2, 3, 4 and 5 cycles, where each cycle consisted of successive 10s intervals lasting for a total of 3min. Between successive 10s irradiation intervals, the tubes were swirled to ensure good mixing of solvent, and to liberate any dissolved gases in order to prevent boiling. MeOH and MeOH:H2O were evaporated under argon at 50°C, and then reconstituted in 1ml PB6 for analysis.
2.6 Stability of drugs to microwave exposure--gas chromatography/mass spectrometry
The stability of the drugs examined to microwave irradiation in PB6 and methanol was also examined by gas chromatography/mass spectrometry (GC/MS), in order to further investigate whether the drugs were decomposing under microwave irradiation. Three solutions (1ml) of ketamine (400ng/ml), pentobarbital (400ng/ml) and diazepam (400ng/ml) were prepared in each solvent and transferred to capillary screw-cap test tubes. Chlorpheniramine (200ng/ml) was used as an internal standard for the ketamine and diazepam analyses, while secobarbital (200ng/ml) was used as an internal standard for the pentobarbital analysis. Each solution was irradiated as described above in successive 10s intervals lasting for a total of 0min (control), 7min or 15min. MeOH was evaporated under argon at 50°C, and then reconstituted in 1ml PB6 for analysis.
For ketamine and diazepam, the solution pH was raised to approximately 10 with 4M NaOH, and 5ml ethyl acetate:toluene (1:1, v/v) was added. The mixture was rotated for approximately 1h. The organic phase was recovered, and evaporated to dryness under argon (50°C). The residues were reconstituted in ethyl acetate (50μl) and analyzed by GC/MS. For pentobarbital, 5ml ethyl acetate was added to the buffer solution and the mixture was rotated for approximately 1h. The organic phase was recovered, and evaporated to dryness under argon (50°C). The residues were reconstituted in 50μl of 0.2M trimethylphenylammonium hydroxide in methanol (United Chemical Technologies, Bristol, PA), which was then injected directly into the GC/MS to facilitate flash methylation of the barbiturates. The GC/MS used was a PerkinElmer Clarus 600 (PerkinElmer LAS, Shelton, CT), equipped with an Elite 5-MS column (30m×0.25mm) and operated in the electron impact ionization mode, using helium as the carrier gas at a flow rate of 1ml/min. The injection port temperature was programmed, with an initial temperature of 60°C, which was held for 3min after an injection of 5μl. The injection port temperature was then increased 270°C, with the split vent open and a split flow rate of 50ml/min. The initial column temperature was 60°C, which was held for 2min, increased directly to 160°C, and then increased linearly at a rate of 10°C/min to a final temperature of 300°C, where it was held for 3min. Each drug was examined using selected ion monitoring (ketamine: t R 10.0min; m /z180 , 167, 152; diazepam: t R 15.3min; m /z 283, 256 , 221; chlorpheniramine: t R 11.3min; m /z203 , 167, 152; pentobarbital: t R 8.0minm/z 184, 169, 112; secobarbital: t R 8.3min; m /z 196, 181, 169 ; ions for area comparison in bold). The response ratio was determined as the peak area ratio of the appropriate ions from the analyte and internal standard, where the peaks examined were required to have t R values within 1% of the expected values. The stability of each drug was examined by comparison of the response ratios of a given drug after being exposed to microwave radiation for different periods of time (0, 7 or 15min). The detection limits for this method were approximately 15, 20, and 15ng/ml for ketamine, diazepam and pentobarbital, respectively. At concentrations of 25 and 100ng/ml, the precision (%CV) response ratios was 13% and 3%, respectively, for ketamine, 17% and 6%, respectively, for diazepam, and 12% and 3%, respectively, for pentobarbital.
2.7 Effect of extraction solvent
Samples (0.5g) of each bone were accurately weighed into MAE tubes, and 2ml of MeOH, MeOH:H2O or PB6 was added to each tube. Samples were irradiated for 10s intervals, separated by 30s intervals during which the solvent was allowed to cool to ensure that the boiling point was not reached. After six 10s cycles (1min), the supernatant solvent was removed and transferred to a regular test tube. Further (2-4) 1min cycles were undertaken, for a maximum of 5min of irradiation. MeOH and MeOH:H2O were evaporated at 50°C under a continuous flow of argon. Samples were then reconstituted in 2ml PB6 for ELISA analysis. For samples which used PB6 as the extraction solvent, the PBS was analyzed directly with ELISA.
2.8 Effect of sample mass
Bone samples (0.5, 1 and 2g) derived from control and drug-positive animals were accurately weighted into capillary screw-cap test tubes. PB6 (3ml) was added to each sample. Samples underwent microwave irradiation for 3min, in cycles of 10s. Solvent was transferred to a test tube and set aside. Another 3ml of PB6 was added to screw-cap tubes and the above irradiation pattern was repeated. The above process of solvent recovery and replacement was repeated until samples had undergone a maximum of 15min of irradiation.
2.9 Enzyme-linked immunosorbent assay (ELISA)
Bone extracts were assayed for the appropriate drug with commercially available ELISA kits (Immunalysis Corp., Pomona, CA) for barbiturates (group assay), ketamine or benzodiazepines (group assay), as per the manufacturer's instructions. Immunoassay was automated using a ChemWell® 2910 Automated EIA and Chemistry Analyzer (Awareness Technologies, Palm City, FL) Sample (10μl) was mixed with 100μl of enzyme conjugate in the antibody-coated microwells, maintained at an operating temperature of 25°C. The plate was shaken for 10s and incubated for 1h. The solution delivery probe was washed with 1N HCl to remove any traces of enzyme conjugate. The wells were then washed six times with 100μl wash solution (0.025M phosphate buffer (pH 7), containing 0.025% Tween 20). After aspiration of the wash solution, 150μl of enzyme substrate (3,3',5,5'-tetramethylbenzidine, TMB) was added to each well and allowed to incubate for 30min. The reaction was stopped by the addition of 50μl of 1N HCl stop solution to each well. The absorbance of each well was measured at 450nm.
3
Results
3.1 Performance characteristics of the ELISA method for detection of pentobarbital, ketamine and diazepam exposure--precision, concentration dependence of response and cross-reactivity
For the purpose of this work, measured absorbance values were considered directly, and also transformed to give the decrease in absorbance, relative to drug-free controls. The following formula was used to determine the percent decrease in absorbance[Formula omitted. See PDF]where A is the absorbance of a given sample and Actrl is the absorbance of a matrix-matched sample of the same treatment. Consideration of %DA values has been adopted in earlier work [1,2,5] as a means of controlling for the effects of endogenous cross-reactants and differences in the ELISA response to variations in tissue types. We continue to use it here for both consistency with our experimental approach, and to make the graphical description of data more intuitive.
The precision of replicate analyses (% coefficient of variation of measured absorbance values) of matrix-matched spiked bone extracts ranged from 2 to 11% for the diazepam assay, 0.5-5% for the pentobarbital assay and 0.9-6% for the ketamine assay. Limits of detection for the assays were approximately 0.25, 2.5 and 2.5ng/ml for diazepam, pentobarbital and ketamine, respectively.
3.2 Concentration dependence of ELISA response
Standard solutions of diazepam (0-10ng/ml), ketamine (0-200ng/ml) and pentobarbital (0-200ng/ml) were prepared in 1ml solutions of bone extracts derived from drug-free animals and analyzed by ELISA. Fig. 1 illustrates the concentration dependence of all three drug assays.
3.3 Passive extraction in methanol
Drug-positive bone and drug-free control bone samples (0.5g) underwent passive methanolic extraction for purposes of comparison relative to the microwave-assisted extraction methods used. Fig. 2 presents the relative decrease in absorbance after 1, 6, 12 and 24h of passive extraction for bone samples derived from animals exposed to pentobarbital, ketamine or diazepam, respectively.
3.4 Stability of assay response to drugs exposed to microwave radiation
The effect of microwave radiation on the stability of the ELISA response to each drug was examined for each solvent type investigated. The data are summarized in Fig. 3. The data show that there was no clear loss in assay response to any of the drugs examined. Further examination of drug stability to microwave irradiation through the GC/MS assay showed that the response ratios of each drug after 7 or 15min total irradiation time did not differ significantly from those of the control samples, which contained drug at the same concentration but did not undergo irradiation. Overall, these data indicate that the drugs examined here are stable under the conditions applied (i.e., choice of solvents, irradiation times, microwave power, etc.)
3.5 Effect of extraction solvent: comparison of phosphate buffer, methanol and methanol:water mixture
The effect of the extracting solvent was examined. Phosphate buffer (PB6), methanol (MeOH) and a methanol-water mixture (MeOH:H2O, 1:1, v/v) were used to extract 0.5g of drug-positive and drug-free control bone. Extraction continued in rounds of 1min until 5 cycles had been completed or the assay signal reached the limit of detection (Actrl --3S.D.) for each solvent. For diazepam and ketamine, the extraction proceeded more quickly using MeOH and MeOH:H2O, than for PB6. However, signal magnitude was greater for the PB6 solvent system, which may be associated with a greater net drug recovery. In the case of pentobarbital, extraction was similar for all solvents. Extraction profiles can be seen in Fig. 4.
3.6 Effect of bone mass on number of extraction cycles
Samples of control and drug-positive bone (0.5, 1 and 2g) were extracted in 3ml of PB6. Irradiation was done in cycles of 3min. Extraction continued until the relative decrease in absorbance of a specific sample reached the limit of detection for at least one of the samples (i.e., for the 0.5, 1 or 2g sample). In the case of diazepam, the detection limit was not approached for any of the samples after 5 rounds (15min), and so the extraction of diazepam from bone samples of different mass (0.5, 1 and 2g) was attempted with MeOH. Fig. 5 illustrates the effect of sample mass on the extraction time.
4
Discussion
Drug screening in skeletal tissues is an inherently complex process. The use of these tissues as an analytical matrix would typically only occur in cases where there is little or no other alternative; when decomposition processes have rendered conventional samples (blood, soft tissues) unavailable. Such decomposition processes are generally accompanied by a variety of environmental stresses, such as water exposure, scavenging and temperature fluctuations. Understanding the effects of these variables is tremendously challenging as they are very difficult to control.
In this work, we made use of animal models (rats) in order to facilitate control over drug exposure history, drug dose and postmortem environment. One difficulty with the use of small animal models is the sensitivity requirements for determination of trace quantities of drugs in small tissue masses. ELISA is a very suitable approach for this experimental application, as it provides for highly sensitive and semi-quantitative parallel analysis of numerous samples. It is acknowledged that the primary limitation of ELISA is the potential for interference from cross-reacting substances, including drug-metabolites or compounds endogenous to the sample matrix, and that a higher degree of analytical selectivity may be achieved through methods such as GC/MS. However, such methods are less sensitive, and the selectivity limitation was accounted for somewhat through the expression of ELISA response as the relative decrease in absorbance, measured as a percentage of matrix-matched, drug-free control tissues, as described in equation (1). This corrects, to some extent, for the presence of cross-reacting endogenous compounds that may be present in both the control tissues, and those derived from drug-positive animals. Indeed, we have exploited the sensitivity of ELISA to selectively detect acute drug exposure for each of the compounds investigated here [1,2,5,9] in fragments of individual bones of acutely exposed rats.
4.1 Considerations for experimental design
As part of efforts to improve analytical sensitivity and sample preparation time, the goal of this study was to examine the effect of some experimental factors (e.g., extraction solvent, bone mass, irradiation time, etc.) in microwave-assisted extraction of ground bone on the analytical response. This requires a homogeneous sample for analysis under these different conditions. As a solid matrix, bone presents a particular challenge in this regard, since we noted in previous work [1,2,5,9] that there may be variation in analytical response based on the tissue type sampled (i.e., cortical vs. trabecular bone and bone vs. marrow). Consequently, we chose to collect a variety of different bone types, which incorporate both cortical and trabecular bone, and to pool them for analysis. Recovery of spinal and pelvic bone from freshly euthanized animals is difficult due to the significant amount of soft tissue, so allowing the animals to decompose to the point of skeletonization facilitated collection of these tissues, and also provided an application that was more relevant to forensic casework, since skeletal tissues are most likely to undergo toxicological analysis when there are no conventional samples (e.g., blood and liver) available. Finally, to facilitate maximal drug recovery and to create as homogeneous a sample as possible, the pooled bones were ground into a fine powder and mixed extensively.
In order to minimize drug contributions from residual marrow and soft tissue, the bones were vigorously cleaned prior to pooling and analysis, as we have described in earlier work [1,2,5,9]. It is acknowledged that this process may lead to some drug loss from within the bone itself, and that this loss cannot be resolved from the removal of drug-laden soft tissues or marrow. Further, it remains unclear whether drug recovered from the cleaned bone and subsequently detected has been absorbed directly into osteocytes, or is situated within the microscopic cannaliculi that exist within the mineralized matrix. The process of decomposition, through the liquefaction of soft tissue, may facilitate further deposition of drug into the microporous network of the bone. Regardless of the source of the recovered drug, it seems that MAE provides some driving force for rapid dissolution of drug retained within this tissue into the extraction solvent for subsequent analysis.
4.2 Stability of drugs to microwave radiation
In order to verify that decreases in the %DA values observed following successive irradiation cycles were due to progressive extraction of the drug from the bone sample as opposed to breakdown of the drug induced by the microwave radiation, the stability of the ELISA response to standard solutions of each drug in PBS, methanol and methanol:water (1:1) was examined (Fig. 3). The data showed that the ELISA response was stable to irradiation for all drugs and solvent systems examined. However, due to the potential for cross-reactivity in immunoassay, it is possible that some chemical decomposition occurred and the decomposition products displayed significant cross-reactivity with the immobilized antibodies. Consequently, GC/MS was used to assay standard solutions of each drug examined, prepared in PBS or in methanol, following microwave irradiation for 7 or 15min, in 10s intervals, as well as similar solutions which did not undergo irradiation. The results of those experiments showed no significant differences in analytical response for samples irradiated for different periods of time, suggesting that no significant breakdown of the drugs occurred as a result of the exposure to microwave radiation described here.
4.3 Extraction parameters
As can be seen in Fig. 4, there is a noticeable effect of the extraction solvent on the apparent rate of extraction and resultant signal intensity. In the case of diazepam and ketamine, drug extraction appears to proceed more quickly in the MeOH and MeOH:H2O. For the extraction in PB6, diazepam extraction was still not maximal after 5 cycles of irradiation (60s in duration). As shown in Table 2, it appears that the use of PB6 as an extraction solvent provided greater extraction yield. In the case of pentobarbital extraction, the solvent effect was not as prominent with respect to time required for maximal extraction, but the use of PB6 appeared to provide some advantage of in terms of relative recovery.
In addition, the effect of mass on the extraction time was examined. As can be seen in Fig. 5, the number of cycles needed for maximal extraction increased with sample mass. In the case of diazepam, 15min was not sufficient for maximal extraction from 0.5, 1 or 2g bone. For ketamine extraction, 15min was not adequate for the maximal extraction from the 2g sample. Therefore, as would be expected, increased extraction time is necessary for larger bone samples.
Clearly, the optimal extraction procedure may be expected to be drug-dependent. Under the conditions examined here, pentobarbital was extracted from 0.5g of bone within 3min of PB6, while diazepam and ketamine required longer irradiation times for maximal recovery from the 0.5g sample. Also, when studying the effect of bone mass, pentobarbital was the only drug that appeared to have been maximally extracted from 2g of bone within 15min. Conversely, the observed %DA values in assays of extracts for diazepam from 2g bone samples did not seem to display any sensitivity to the number of irradiation cycles initially, which was attributed to the high sensitivity of the benzodiazepine ELISA and the non-linear nature of response with respect to diazepam concentration. Subsequent dilution of the samples resulted in the expected profile. Another uncharacterized effect is the relative rate of extraction of diazepam metabolites that are known to have a high-degree of cross-reactivity with the ELISA used [5]. In this work, the aim was to illustrate the effects of various operational variables associated with MAE on the overall performance of the extraction, and so resolution of these cross-reacting metabolites is relatively unimportant in this case, but would be of obvious importance in the application of this methodology in forensic casework, where the relative quantities of parent drugs and metabolites may provide valuable information.
Overall, the variables examined here would affect the approach taken if applied in forensic casework. Obviously, extraction methods would require a separate validation for each solvent used, and the extraction time would need to be selected according to the longest time necessary for maximum drug extraction of all potential drugs under consideration. The volume of solvent used should also be examined as an extraction parameter.
4.4 Extraction recovery: MAE vs. passive extraction
The concentration dependent nature of the ELISA methodology allows for a semi-quantitative comparison of the recovery of two preparation methodologies. Examination of Fig. 1 shows that the %DA parameter is approximately proportional to drug concentration over a limited range. For samples in this pseudo-linear range, two different extraction methodologies may be compared in terms of which provides the greater recovery through the ratio of the sum of incremental %DA values:[Formula omitted. See PDF]
Values of R comparing MAE using PB6 or methanol as an extraction solvent to passive extraction were determined for each drug, after any dilutions necessary to yield sample concentrations into the pseudo-linear region, and are summarized in Table 2. The data in Table 2 show that there was a tendency towards higher extraction recovery through MAE in PBS than from either MAE or passive extraction in methanol for all three drugs, with the effect appearing to be more pronounced with ketamine and diazepam. Overall, while it may be that both the optimal extraction solvent system and the magnitude of the benefit of MAE will be drug-dependent, direct extraction into PB6 or other phosphate buffers should be considered as they may provide high recoveries with rapid extractions. Again, it is important to remember that this approach should be treated as being semi-quantitative only, due to the potential for cross-reactivity effects.
4.5 Benefits of microwave-assisted extraction
The use of MAE offers a substantial advantage in terms of extraction time, and may provide improvements in extraction efficiency. The data in Table 1 summarize some of the sample preparation approaches used for toxicological analysis of bone as described in the literature. Overall, the approaches used to date have involved lengthy extraction steps, using a Soxhlet or passive extraction configuration, which typically required many hours (6-72h). Here, passive extraction of drug from ground bone required 6h or more (Fig. 2). Interestingly, the data in Fig. 2 show an unexpected result wherein the passive extraction of diazepam showed a substantial increase between the 12 and 24h sampling periods. The analysis of this fraction was repeated with similar results, ensuring this was not a randomly anomalous immunoassay result. Given that the sample concentration in that sample was too low for GC/MS analysis, we cannot comment conclusively on the source of this effect, although one potential explanation may be that multiple sources of drug exist within the bone sample; one type being more loosely adsorbed to the bone surface and more readily diffusing under the extraction conditions, and another source which is more strongly bound within the matrix and which is slower in diffusing into the extraction solvent. Even in the absence of a conclusive mechanism, this phenomenon should be borne in mind and extra analysis of second extractions from a given bone sample may be used to verify that maximal extraction has in fact been achieved [1,4].
Overall, MAE may reduce the time required for maximal extraction to as little as 5min, depending on the drug in question (and, most likely, the associated dose), extraction solvent and bone mass. Furthermore, while extraction into phosphate buffer may be sub-optimal for a particular drug in terms of extraction time, the use of buffer as an extraction solvent facilitates immediate subsequent ELISA or extraction procedures (e.g., solid-phase extraction, liquid-liquid extraction), eliminating the need for an intermediate solvent evaporation step. Even under sub-optimal conditions where maximal recovery is not achieved, sufficient drug may be extracted within 1-3min to give a positive response on ELISA. If samples are positive, further extraction can be continued and samples can be pooled and confirmed. This could lead to a reduction in unnecessary extraction of negative samples and a significant reduction in the use of organic extraction solvents.
4.6 Future work
The data presented here suggests that MAE may constitute a valuable methodology for the efficient toxicological analysis of skeletal tissue samples. While the work here made use of animal models, as is common in toxicological research involving bone tissues, this methodology may find utility in forensic analysis of human bones. Laboratory-grade microwave ovens facilitate the use of larger extraction vessels (e.g., 50ml) which can accommodate the larger sample masses that would be required for human bone analysis. The use of closed-vessels similar to those used in environmental chemistry could prove useful to the extraction of drugs from skeletal tissues, as that configuration typically provides for significantly elevated solvent temperatures, in excess of their normal boiling points [12,14-16].
5
Conclusions and summary
Overall, experimental parameters such as temperature, pressure and sample moisture content should be optimized for each drug. An important consideration for future work is the extraction behaviour of drugs and their metabolites, as the ratio of parent drug to a given metabolite may serve as an indicator of the pattern of drug use (i.e., recent exposure, acute vs. chronic exposure, etc.). In so doing, MAE should prove to be a useful tool for the extraction of drugs from skeletal tissues, and its utility may be extended to the rapid extraction of drugs from other solid matrices of forensic value, such as hair.
Acknowledgement
The authors wish to acknowledge the Natural Sciences and Engineering Research Council of Canada for financial support of this work.
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