UPLC-Orbitrap® Screening for over 35 Drugs of Abuse and Metabolites in Biological Fluids in Under 10 min.

We present a UPLC®-High Resolution Mass Spectrometric method to simultaneously screen for 19 benzodiazepines, 12 opiates, cocaine and three metabolites, and 3 "Z-drug" hypnotic sedatives in both blood and urine specimens. Sample processing consists of a high-speed, high-temperature enzymatic hydrolysis for urine samples followed by a rapid supported liquid extraction (SLE). The combination of ultrahigh-resolution chromatography with high resolution mass spectrometry allows all analytes to be uniquely detected with a 10 min analytical run. Limits of detection for all target analytes are 3 ng/mL or better, with only 300 µL of specimen used for analysis. The combination of low sample volume with fast processing and analysis makes this method a suitable replacement for immunoassay screening of the targeted drug classes, while providing far superior specificity and better limits of detection than can routinely be obtained by immunoassay.

Rapid screening for drugs of abuse in biological fluids by ultra high performance liquid chromatography/Orbitrap mass spectrometry.

We present a UPLC(®)-High Resolution Mass Spectrometric method to simultaneously screen for nineteen benzodiazepines, twelve opiates, cocaine and three metabolites, and three "Z-drug" hypnotic sedatives in both blood and urine specimens. Sample processing consists of a high-speed, high-temperature enzymatic hydrolysis for urine samples followed by a rapid supported liquid extraction (SLE). The combination of ultra-high resolution chromatography with high resolution mass spectrometry allows all 38 analytes to be uniquely detected with a ten minute analytical run. Limits of detection for all target analytes are 3ng/mL or better, with only 0.3mL of specimen used for analysis. The combination of low sample volume with fast processing and analysis makes this method a suitable replacement for immunoassay screening of the targeted drug classes, while providing far superior specificity and better limits of detection than can routinely be obtained by immunoassay.

An HPLC-HR-MS-MS method for identification of anticoagulant rodenticides in blood.

This paper presents a fully validated method for the qualitative identification of bromadiolone, brodifacoum, coumachlor, coumatetralyl, difenacoum and warfarin in whole blood specimens. Samples are protein precipitated with acetonitrile, processed via solid-phase extraction and analyzed by high-performance liquid chromatography with high resolution tandem mass spectrometric detection. Limits of detection were 10 ng/mL or better for all analytes.

Benzodiazepines and metabolites from biological fluids by liquid chromatography electrospray tandem mass spectrometry.

Solid-phase extraction and liquid chromatography-tandem mass spectrometry are invaluable techniques for the determination of benzodiazepines and metabolites in biological matrices. The reason for using tandem mass spectrometry is to increase limits of detection without the need for chemical derivatization. Here we describe a technique for the detection of 26 benzodiazepines and metabolites at a detection limit of approximately 1-2 ng/mL in blood and 1-5 ng/mL in urine when screened using a data-dependent scan method.

A gas chromatography-thermal conductivity detection method for helium detection in postmortem blood and tissue specimens.

In cases of death by inert gas asphyxiation, it can be difficult to obtain toxicological evidence supporting assignment of a cause of death. Because of its low mass and high diffusivity, and its common use as a carrier gas, helium presents a particular challenge in this respect. We describe a rapid and simple gas chromatography-thermal conductivity detection method to qualitatively screen a variety of postmortem biological specimens for the presence of helium. Application of this method is demonstrated with three case examples, encompassing an array of different biological matrices.

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.

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.

A comprehensive study on the variations in urinary concentrations of endogenous gamma-hydroxybutyrate (GHB).

This study was designed to supplement previous attempts to establish an accurate range of normal endogenous gamma-hydroxybutyrate (GHB) concentrations in random antemortem urine samples. Furthermore, its purpose was to ascertain the effect of gender, race, age, medications, and select medical conditions on endogenous concentrations of GHB in urine and the proposed endogenous urinary GHB cutoff of 10 microg/mL. Urine samples (n = 207) were provided by subjects who reported that they had never used GHB. As part of the collection process, subjects also completed a short survey to collect information about gender, race, age, orally ingested medications, and select medical conditions. All specimens were analyzed in duplicate for the presence of endogenous GHB using a previously reported headspace gas chromatography-mass spectrometry method. The data were analyzed for tendencies among different population groups. GHB concentrations ranged from 0.00 to 2.70 microg/mL in all specimens, with a median concentration of 0.24 microg/mL. Males (n = 130) had an average endogenous GHB concentration of 0.27 microg/mL (0.00-2.70 microg/mL), whereas females (n = 77) averaged 0.29 microg/mL (0.00-0.98 microg/mL). Select medical conditions and participants' race, age ranges, and medications that were used within 48 h prior to collection were also evaluated. We believe this to be the most comprehensive study on endogenous GHB concentrations in urine to date. The results of this study will aid the interpretation of low GHB concentrations measured in urine samples, particularly in investigations of drug-facilitated crimes.