Rapid screening procedures for a variety of complex forensic samples using laser diode thermal desorption (LDTD) coupled to different mass spectrometers.

RATIONALE: The applications shared in this paper demonstrate the wide variety of samples that can be analyzed when Laser Diode Thermal Desorption (LDTD) is interfaced with a high-resolution mass spectrometer and show the speed at which high quality data can be generated from complex matrices. METHODS: Samples are solvent extracted and spotted in a 96-well plate. In the case of biological fluids, hydrolysis followed by solid-phase extraction is required. The solvent in the 96-well plate is evaporated followed by mass spectrometric (MS) analysis with atmospheric pressure chemical ionization. Where applicable, the instrument is operated in data-dependent mode, with a full-scan mass spectrum followed by MS/MS spectra of the top 10 ions with a total runtime of 0.4 min. RESULTS: Four applications (MAAQ and Tear Gas, twelve rodenticides, seven explosives, and 40 drugs of abuse) are reported in this paper. MAAQ, tear gas, and rodenticides were identified by full-scan, followed by MS/MS experiments at levels of 125 µg/L, 125 µg/L, and 500 µg/L, respectively. Explosives were all identified at 102 µg/L by full-scan experiments. The drugs of abuse were identified by multiple reaction monitoring (MRM) experiments at defined cutoff levels from 2 to 1000 µg/L. CONCLUSIONS: Interfacing LDTD with a mass spectrometer allows for rapid screening of a wide range of samples, with either minimal or complex sample preparation. Using a high-resolution mass spectrometer with the combination to perform full-scan and MS/MS experiments adds a high level of specificity.

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 analysis of forensic-related samples using two ambient ionization techniques coupled to high-resolution mass spectrometers.

RATIONALE: This paper highlights the versatility of interfacing two ambient ionization techniques, Laser Diode Thermal Desorption (LDTD) and Atmospheric Solids Analysis Probe (ASAP), to high-resolution mass spectrometers and demonstrate the method's capability to rapidly generate high-quality data from multiple sample types with minimal, if any, sample preparation. METHODS: For ASAP-MS analysis of solid and liquid samples, the material was transferred to a capillary surface before being introduced into the mass spectrometer. For LDTD-MS analysis, samples were solvent extracted, spotted in a 96-well plate, and the solvent was evaporated before being introduced into the mass spectrometer. All analyses were performed using Atmospheric Pressure Chemical Ionization in positive mode. RESULTS: Seven consumer "Spice" packets were combined and analyzed by both ASAP and LDTD, which identified 11 synthetic cannabinoids/cathinones by full MS and MS/MS experiments. To further show the usefulness of these techniques, black tar heroin was analyzed, which resulted in the identification of heroin and its impurities (monoacetylmorphine, papaverine, and noscapine). These experiments were performed on the LTQ-Orbitrap to demonstrate the ability to perform both parallel and serial MS and MSn experiments. CONCLUSIONS: Interfacing LDTD and ASAP to high-resolution mass spectrometers allows for expeditious analysis of a wide range of samples, with minimal or no sample preparation. Both allow for rapid full scan, MS/MS, and/or MSn experiments from a single sample introduction. Published in 2017. This article is a U.S. Government work and is in the public domain in the USA.

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.

Rapid analysis of forensic samples using an atmospheric solid analysis probe interfaced to a linear ion trap mass spectrometer.

RATIONALE: This paper highlights the simplicity of interfacing an Atmospheric Solid Analysis Probe (ASAP) to a Linear Ion Trap Mass Spectrometer and shows that this technique can be used for the rapid generation of high-quality data from a range of sample types with minimal or no sample preparation. METHODS: For a solid sample or surface deposit, the process entails rubbing a capillary melting tube a few times on the sample to transfer material to the capillary surface and then introducing it into the source of the mass spectrometer. Similarly, for a liquid sample, a capillary tube is dipped into the sample to just coat the surface or a few microliters may be applied to the tip of a capillary before being analyzed by Atmospheric Pressure Chemical Ionization in both positive and negative mode. RESULTS: A rodenticide containing brodifacoum, black tar heroin and its impurities (morphine, codeine, noscapine, papaverine, and monoacetylmorphine), crack cocaine and 1-methylaminoanthraquinone dyestuff were successfully analyzed directly without any sample preparation. All compounds were detected using full scan mass spectrometry (MS), followed by confirmation by MS/MS. Preliminary results suggest that this technique could be used for quantitation. CONCLUSIONS: Interfacing the ASAP to an ion trap mass spectrometer allows the ability to perform full scan, MS(n) experiments, and rapid positive/negative switching from a single sample introduction. Because of these features, this instrument is very useful for rapid, routine analysis and for confirmation with the use of in-house MS/MS libraries.

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.

A fast method for screening and/or quantitation of tetrahydrocannabinol and metabolites in urine by automated SPE/LC/MS/MS.

Marijuana is one of the most commonly used illicit substances. The high usage of this substance results in it being commonly encountered in clinical samples throughout the USA and Europe. Due to its wide availability and use, marijuana is also commonly encountered in forensic toxicology laboratories. The proposed method utilized an automated solid phase extraction (SPE) coupled to liquid chromatography/mass spectrometry (LC/MS). The automated SPE procedure was developed using Hysphere C8-EC sorbent, and the high performance liquid chromatography (HPLC) separation was performed using an Xterra MS C(18) column with a total runtime of 10 min. The standard curves linearity generally fell between 6 and 500 ng/mL. The limits of detection ranged from 2 to 4 ng/mL, and the limits of quantitation ranged from 8 to 12 ng/mL. The bias and imprecision were determined using a simple analysis of variance (single factor). The results demonstrate bias as <11% and percent imprecision as <12% for all components at four quality control levels. This method has been in use for over 2 years and has been applied to numerous forensic samples. When compared to other published methods, it exceeds others in its simplicity and speed of analysis. This method takes advantage of robotics and automation for a total analysis time of 10 min, including sample preparation, separation, and detection.

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.

Screening of cocaine and its metabolites in human urine samples by direct analysis in real-time source coupled to time-of-flight mass spectrometry after online preconcentration utilizing microextraction by packed sorbent.

Microextraction by packed sorbent (MEPS) has been evaluated for fast screening of drugs of abuse with mass spectrometric detection. In this study, C8 (octyl-silica, useful for nonpolar to moderately polar compounds), ENV(+) (hydroxylated polystyrene-divinylbenzene copolymer, for extraction of aliphatic and aromatic polar compounds), Oasis MCX (sulfonic-poly(divinylbenzene-co-N-polyvinyl-pyrrolidone) copolymer), and Clean Screen DAU (mixed mode, ion exchanger for acidic and basic compounds) were used as sorbents for the MEPS. The focus was on fast extraction and preconcentration of the drugs with rapid analysis using a time-of-flight (TOF) mass spectrometer as the detector with direct analysis in a real-time (DART) source. The combination of an analysis time of less than 1 min and accurate mass of the first monoisotopic peak of the analyte and the relative abundances of the peaks in the isotopic clusters provided reliable information for identification. Furthermore, the study sought to demonstrate that it is possible to quantify the analyte of interest using a DART source when an internal standard is used. Of all the sorbents used in the study, Clean Screen DAU performed best for extraction of the analytes from urine. Using Clean Screen DAU to extract spiked samples containing the drugs, linearity was demonstrated for ecgonine methyl ester, benzoylecgonine, cocaine, and cocaethylene with average ranges of: 65-910, 75-1100, 95-1200, and 75-1100 ng/mL (n = 5), respectively. The limits of detection (LOD) for ecgonine methyl ester, benzoylecgonine, cocaine, and cocaethylene were 22.9 ng/mL, 23.7 ng/mL, 4.0 ng/mL, and 9.8 ng/mL respectively, using a signal-to-noise ratio of 3:1.

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.

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.

Analysis of trace amount of bank dye and lachrymators from exploding bank devices by solid-phase microextraction and gas chromatography-mass spectrometry.

Solid-phase microextraction (SPME) is a fast, solvent-free alternative to conventional sample preparation techniques. This technique involves exposing a fused silica fiber that has been coated with a stationary phase to an aqueous solution or its headspace to selectively extract compounds from their matrix. The fiber is then removed, and the analytes are thermally desorbed in the injector of a gas chromatograph. By sampling from the headspace above sample matrices, SPME can be used to extract target analytes from very complex matrices. In this study, SPME in the headspace is used in developing a method for the dye 1-methylaminoanthraquinone (MAAQ) and two lachrymators: orthochlorobenzalmalononitrile (CS) (tear gas) and 2-chloroacetophenone (CN) (tear gas). The focus is to develop a robust method to minimize sample preparation and to reduce matrix interferences encountered by other extraction techniques. In developing the method, several fibers are studied for their affinity for the compounds of interest. Although this method is developed for qualitative analysis, the extraction time and temperature profile are thoroughly investigated to provide the optimal conditions. The use of a salt solution is evaluated to increase the partitioning of MAAQ into the headspace. Using this method, qualitative extraction is achieved for the analysis of CN, CS, and MAAQ from its matrices. CN and CS are extracted in less than 5 min, though MAAQ needed more than 15 min to achieve a reasonable response. If more sensitivity is required, the use of a salt solution increases the response of MAAQ by 90-fold.

Analysis of ethyl carbamate in wines using solid-phase extraction and multidimensional gas chromatography/mass spectrometry.

The method describes a rapid and accurate procedure for the analysis of ethyl carbamate in wines. The separation of the ethyl carbamate (EC), the target analyte, from alcohol and the sample matrix is a challenge to many analytical chemists. After alcohol removal from the sample, EC was extracted and concentrated by solid-phase extraction. For analysis of EC, large-volume injection on a programmable temperature vaporization (PTV) inlet was used followed by multidimensional gas chromatography/mass spectrometry (MDGC/MS) using electron-impact ionization (EI). For quantitation, the ratio of ions produced during EI at m/z 62 (EC) and 64 (isotopically labeled EC) was monitored. The use of solid-phase extraction and MDGC/MS removes the majority of the matrix interference encountered in other methods. A linear dynamic range was established from 0.387 to 1160 ng/mL, with a limit of detection at 0.1 ng/mL and limit of quantitation at 1 ng/mL.