Endogenous GHB in Segmented Hair Part II: Intra-individual Variation for Exogenous Discrimination.

The endogenous presence of gamma-hydroxybutyric acid (GHB) complicates the interpretation of results in cases where an exogenous dosing is suspected. Due to GHB's rapid metabolism and clearance following exogenous doses, hair has become a preferential matrix for confirmation of GHB exposure in drug-facilitated crimes. However, unlike blood and urine where an agreed-upon cut-off concentration for differentiation between endogenous and exogenous GHB has been made, there has been no consensus on a cut-off concentration for hair. This is due in part to the wide inter- and intra-individual variation that has been observed in endogenous GHB hair studies. A large (>50) population study of 214 donors was conducted to better understand these variations and to evaluate whether a cut-off concentration could be established for endogenous GHB in human hair. As seen in our previous study, the inter-individual variation was large, with concentrations ranging from <0.40 to 5.47 ng/mg. This range made an absolute cut-off concentration recommendation inappropriate, so an alternative approach for GHB discrimination was investigated utilizing the intra-individual variation. Male donors appeared to have greater intra-individual variation than female donors, yet it was noted that segment-to-segment variation along the length of hair had minimal change between individual donor's adjacent segments. Overall, 97.1% of the adjacent segment differences were within ±0.5 ng/mg. Therefore, instead of a recommended cut-off concentration, it appears that using adjacent segment concentration differences could be a strategy to assist in differentiating endogenous from single exogenous GHB exposure. In the absence of controlled dosing data, previously published segmented results from controlled and suspected dosing donors are examined using the adjacent segmental difference approach and the results compared to currently used ratio-based calculations.

Endogenous GHB in Segmented Hair Part I: Inter-individual Variation for Group Comparisons.

While earlier studies have attempted to resolve the challenges encountered when interpreting gamma-hydroxybutyric acid (GHB) concentrations in hair (primarily due to its endogenous presence), few have had large sample sizes. The first objective of this study was to evaluate the inter-individual variation of endogenous GHB concentrations. The second objective, to be detailed in another report, was to assess intra-individual variation and the impact on exogenous GHB discrimination. Over 2,000 hair segments from 141 women and 73 men (all processed hair 3-12 cm long) were analyzed in this study. The raw calculated range of endogenous GHB concentrations was <0.40-5.47 ng/mg with 97.5% of the segmental results calculated less than 2.00 ng/mg. Imputation, assuming a lognormal distribution, was applied to the data to include non-detect (ND) data (

Evaluating Endogenous GHB Variation in Hair with a Synthetic Hair Matrix.

The variation in drug concentrations in human head hair from 22 donors was measured using a synthetic hair matrix (SMx™ hair). This matrix is being reported for the first time as a calibrator for an endogenous substance. In comparison to authentic hair or melanin, the synthetic hair provided a reliable batch-to-batch source of liquid matrix similar in composition to authentic hair, but without detectable concentrations of endogenous gamma-hydroxybutyric acid (GHB). Using the synthetic matrix for calibrator samples, validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantitative method for GHB in human head hair was completed. Validation included the evaluation of the following parameters: accuracy, precision, calibration model, carryover, interferences, limit of detection (LOD), limit of quantitation (LOQ) and processed sample stability. The method was valid over a range of 0.4-12 ng/mg, and its LOD and LOQ were both experimentally estimated to be 0.4 ng/mg. After validation, the variation in endogenous GHB concentrations across multiple donors and locations in the vertex posterior region of the human head were evaluated. Results for 11 non-GHB users showed minimal variability (average 3.0% RSD) across the vertex posterior for hair samples taken from three different areas. There was also low variability (average 1.8% RSD) in repeat samples taken from the same location for 11 other non-users. Endogenous GHB concentrations from the LOD/LOQ to 5.60 ng/mg were determined for the 22 donors using the synthetic hair as a calibrator. These results demonstrate the successful application of a synthetic hair matrix in the analysis of GHB in human hair.

Evaluation of Four Fingerprint Development Methods for Touch Chemistry Using Matrix-Assisted Laser Desorption Ionization/Time-of-Flight Mass Spectrometry(.).

Four preparation techniques for MALDI/TOF mass spectrometry were compared to determine the ability to gather intelligence for investigations through the chemical analysis of latent fingerprints, defined as "touch chemistry." Compatible fingerprint development processes used for identification along with new techniques are necessary to evaluate touch chemistry. Ten volunteers deposited fingerprints from solvent residues containing drugs and explosives onto microscope slides. The developers included (A) fingerprint powder, (B) MALDI matrix, (C) fingerprint powder and lifting, and (D) cyanoacrylate fuming with fingerprint powder. Qualitative identification was based on ion images and spectra. The highest average detection rates (88%) were found using methods A and B. Methods C (52%) or D (18%) had limited success. Results demonstrate the importance of imaging coupled to extracted mass spectral data in detecting analytes in deposited fingerprints. Overall, the results suggest continued development of touch chemistry applications could prove useful for gathering intelligence and forensically relevant information.

An improved method for the analysis of GHB in human hair by liquid chromatography tandem mass spectrometry.

The abuse of gamma-hydroxybutyric acid (GHB) and its suspicion in cases of suspected drug-facilitated sexual assault is of keen interest to forensic toxicology laboratories. This paper reports an extraction, separation and detection procedure for GHB in hair utilizing a combination of liquid-liquid extraction and solid-phase extraction using ethyl acetate and Oasis Max(®) cartridge, respectively, after the hair sample was digested. Analysis was by LC-MS-MS using a gradient separation on an Acclaim(®) Trinity(TM) P1 column performing three multiple-reaction monitoring (MRM) transitions each for GHB and its internal standard. The procedure was validated over a range from 0.4 to 50 ng/mg with estimated limit of detection (LOD) of 0.33 and an administratively set limit of quantitation (LOQ) of 1.2 ng/mg. Twenty hair specimens collected from individuals with no known exposure to GHB were analyzed for matrix interferences and to establish initial background levels of GHB. A wide range of endogenous GHB levels were observed in these samples (from less than the LOQ to 4.4 ng/mg). The results suggest the need for additional studies to better establish the full range of endogenous GHB levels in hair and that extreme caution is required in interpreting GHB findings in hair samples.

Chemical analysis of pharmaceuticals and explosives in fingermarks using matrix-assisted laser desorption ionization/time-of-flight mass spectrometry.

Chemical analysis of latent fingermarks, "touch chemistry," has the potential of providing intelligence or forensically relevant information. Matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI/TOF MS) was used as an analytical platform for obtaining mass spectra and chemical images of target drugs and explosives in fingermark residues following conventional fingerprint development methods and MALDI matrix processing. There were two main purposes of this research: (1) develop effective laboratory methods for detecting drugs and explosives in fingermark residues and (2) determine the feasibility of detecting drugs and explosives after casual contact with pills, powders, and residues. Further, synthetic latent print reference pads were evaluated as mimics of natural fingermark residue to determine if the pads could be used for method development and quality control. The results suggest that artificial amino acid and sebaceous oil residue pads are not suitable to adequately simulate natural fingermark chemistry for MALDI/TOF MS analysis. However, the pads were useful for designing experiments and setting instrumental parameters. Based on the natural fingermark residue experiments, handling whole or broken pills did not transfer sufficient quantities of drugs to allow for definitive detection. Transferring drugs or explosives in the form of powders and residues was successful for preparing analytes for detection after contact with fingers and deposition of fingermark residue. One downfall to handling powders was that the analyte particles were easily spread beyond the original fingermark during development. Analyte particles were confined in the original fingermark when using transfer residues. The MALDI/TOF MS was able to detect procaine, pseudoephedrine, TNT, and RDX from contact residue under laboratory conditions with the integration of conventional fingerprint development methods and MALDI matrix. MALDI/TOF MS is a nondestructive technique which provides chemical information in both the mass spectra and chemical images.

The role of variations in growth rate and sample collection on interpreting results of segmental analyses of hair.

Segmental analysis of hair for drugs, metabolites, and poisons has been widely reported in the scientific literature over the past two decades. Two fundamental assumptions in interpreting results of such analyses are (1) an average linear growth rate of head hair of 1cm/month and (2) that sample collections occur with the hair being cut directly next to the scalp. The purpose of this study was to evaluate the variability associated with growth rate of human head hair, as well as the ability to uniformly collect hair next to the scalp. The results were used to determine how these factors affect the interpretation of results generated in segmental analysis of hair. A thorough literature review was conducted to assess the range of linear growth of human head hair from the vertex posterior and occipital regions. The results were compiled to establish the average (1.06cm/month), as well as the range of possible growth rates of head hair. The range was remarkable and suggests that conclusions based on the 1-cm/month growth rate could be significantly skewed. A separate study was undertaken to evaluate collection of hair next to the scalp. Fourteen individuals were provided oral instructions, as well as a written standard collection procedure for head hair. The experience levels among the collectors varied from novice to expert. Each individual collected hair from dolls with short- and long-hair. Immediately following each collection, the sampling area was evaluated to determine how close to the scalp the cuts were made, as well as the variability in the lengths of hair remaining at the sampled area. From our collection study, we determined that 0.8±0.1cm of hair was left on the scalp after cutting. When taking into account the amount of hair left on the scalp after collecting, the use of a growth rate of 1.06cm/month, and the assumption that it takes two weeks for newly formed hair in the follicle to reach the scalp, we find that the first 1-cm segment of hair typically corresponds to hair formed 1.3±0.2 to 2.2±0.4 months (95% confidence) earlier. The impact of these findings as it relates to the corresponding time for each additional segment is demonstrated. As a result, we recommend that hair collection be delayed 8 weeks after a suspected ingestion to ensure that the sample fully represents the exposure period. The results of this study suggest that the variability in the growth rate of human head hair, as well as the inconsistent collection of hair, significantly affect the interpretation of results from segmental analysis of hair.

A semi-automated solid-phase extraction liquid chromatography/tandem mass spectrometry method for the analysis of tetrahydrocannabinol and metabolites in whole blood.

Marijuana is one of the most commonly abused illicit substances in the USA, making cannabinoids important to detect in clinical and forensic toxicology laboratories. Historically, cannabinoids in biological fluids have been derivatized and analyzed by gas chromatography/mass spectrometry (GC/MS). There has been a gradual shift in many laboratories towards liquid chromatography/mass spectrometry (LC/MS) for this analysis due to its improved sensitivity and reduced sample preparation compared with GC/MS procedures. This paper reports a validated method for the analysis of Delta(9)-tetrahydrocannabinol (THC) and its two main metabolites, 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THC-COOH) and 11-hydroxy-Delta(9)-tetrahydrocannabinol (THC-OH), in whole blood samples. The method has also been validated for cannabinol (CBD) and cannabidiol (CDN), two cannabinoids that were shown not to interfere with the method. This method has been successfully applied to samples both from living people and from deceased individuals obtained during autopsy. This method utilizes online solid-phase extraction (SPE) with LC/MS. Pretreatment of samples involves protein precipitation, sample concentration, ultracentrifugation, and reconstitution. The online SPE procedure was developed using Hysphere C8-EC sorbent. A chromatographic gradient with an Xterra MS C(18) column was used for the separation. Four multiple-reaction monitoring (MRM) transitions were monitored for each analyte and internal standard. Linearity generally fell between 2 and 200 ng/mL. The limits of detection (LODs) ranged from 0.5 to 3 ng/mL and the limits of quantitation (LOQs) ranged from 2 to 8 ng/mL. The bias and imprecision were determined using a simple analysis of variance (ANOVA: single factor). The results demonstrate bias as <7%, and imprecision as <9%, for all components at each quantity control level.

Rapid analysis of cocaine and metabolites in urine using a completely automated solid-phase extraction-high-performance liquid chromatography-tandem mass spectrometry method.

A rapid, simple, and completely automated method for the analysis of cocaine and its metabolites in urine has been developed. The method utilizes online solid-phase extraction (SPE) with liquid chromatographic separation and tandem mass spectrometric detection (MS-MS). An efficient online SPE procedure was developed using Hyspheretrade mark MM anion sorbent. A gradient chromatography method with a Gemini C6-Phenyl (50 x 3.00-mm i.d., 5 microm) column was used for the separation of all compounds. Detection was by positive ion mode electrospray ionization MS-MS. Multiple reaction monitoring (MRM) was used to enhance the selectivity and sensitivity of the method. Two MRM transitions were monitored for each analyte and one transition for each internal standard. Linearity was analyte-dependent but generally ranged from 7 to 1000 ng/mL. The limits of detection for the method ranged from 3 to 23 ng/mL and the limits of quantitation ranged from 7 to 69 ng/mL. Quality control (QC) samples were analyzed for each analyte in triplicate at 50, 200, and 800 ng/mL. The bias and precision were determined using a simple analysis of variance (ANOVA: single factor). The results demonstrated bias as < 5% and % precision as < 9% for all components at each QC level.

An automated SPE/LC/MS/MS method for the analysis of cocaine and metabolites in whole blood.

As laboratories are called upon to develop novel, fast, and sensitive methods, here we present a completely automated method for the analysis of cocaine and its metabolites (benzoylecgonine, ecgonine methyl ester, ecgonine and cocaethylene) from whole blood. This method utilizes an online solid-phase extraction (SPE) with high performance liquid chromatographic separation and tandem mass spectrometric detection. Pretreatment of samples involve only protein precipitation and ultracentrifugation. An efficient online solid-phase extraction (SPE) procedure was developed using Hysphere MM anion sorbent. A gradient chromatography method with a Gemini C6-Phenyl (50mmx3.00mm i.d., 5microm) column was used for the complete separation of all components. Analysis was by positive ion mode electrospray ionization tandem mass spectrometry, using multiple reaction monitoring (MRM) to enhance the selectivity and sensitivity of the method. For the analysis, two MRM transitions are monitored for each analyte and one transition is monitored for each internal standard. With a 30-microL sample injection, linearity was analyte dependent but generally fell between 8 and 500ng/mL. The limits of detection (LODs) for the method ranged from 3 to 16ng/mL and the limits of quantitation (LOQs) ranged from 8 to 47ng/mL. The bias and precision were determined using a simple analysis of variance (ANOVA: single factor). The results demonstrate bias as <7%, and %precision as <9% for all components at each QC level.

Guidance for improved detection of drugs used to facilitate crimes.

Reports of drug-facilitated crimes have significantly increased since the mid-1990s. When individuals report that they were robbed or assaulted while incapacitated by a drug, toxicologic testing may be needed to help substantiate the alleged victim's claims. Most often, these cases have involved strong central nervous system depressant drugs, which have the capability of preventing individuals from consenting to the action of the perpetrator or fighting off their attackers. For all intents and purposes, the drug acts as the offender's weapon, so many jurisdictions require analytical proof of its presence for criminal charges of the drug-facilitated crime to be filed. This article provides information on the manner in which drug-facilitated crimes occur, the drugs that are used to commit these crimes, and recommendations to improve the detection of these drugs through toxicologic analyses.

Ethanol analysis from biological samples by dual rail robotic autosampler.

Detection, identification, and quantitation of ethanol and other low molecular weight volatile compounds in liquid matrices by headspace gas chromatography-flame ionization detection (HS-GC-FID) and headspace gas chromatography-mass spectrometry (HS-GC-MS) are becoming commonly used practices in forensic laboratories. Although it is one of the most frequently utilized procedures, sample preparation is usually done manually. Implementing the use of a dual-rail, programmable autosampler can minimize many of the manual steps in sample preparation. The autosampler is configured so that one rail is used for sample preparation and the other rail is used as a traditional autosampler for sample introduction into the gas chromatograph inlet. The sample preparation rail draws up and sequentially adds a saturated sodium chloride solution and internal standard (0.08%, w/v acetonitrile) to a headspace vial containing a biological sample, a calibrator, or a control. Then, the analytical rail moves the sample to the agitator for incubation, followed by sampling of the headspace for analysis. Using DB-624 capillary columns, the method was validated on a GC-FID and confirmed with a GC-MS. The analytes (ethanol, acetonitrile) and possible interferences (acetaldehyde, methanol, pentane, diethyl ether, acetone, isopropanol, methylene chloride, n-propanol, and isovaleraldehyde) were baseline resolved for both the GC-FID and GC-MS methods. This method demonstrated acceptable linearity from 0 to 1500 mg/dL. The lower limit of quantitation (LOQ) was determined to be 17 mg/dL and the limit of detection was 5 mg/dL.

Further evidence of in vitro production of gamma-hydroxybutyrate (GHB) in urine samples.

This study was designed to supplement previous studies that documented in vitro production of gamma-hydroxybutyrate (GHB) in urine samples. Urine samples were provided by subjects who reported that they had never used GHB (n=31). The specimens were stored under standard conditions of refrigeration (5 degrees C) without any preservatives added. All specimens were repeatedly analyzed for the presence of endogenous GHB over a 6-month period using a previously reported headspace GC-MS method. Significant elevations in GHB were observed in many of the urine samples as storage time increased. As a result, the in vitro production of GHB may increase the apparent GHB concentrations in urine during storage. This potential for an artificial increase in GHB concentration must be appreciated when establishing the threshold between endogenous and exogenous concentrations of GHB.