Bioanalytical procedures and recent developments in the determination of opiates/opioids in human biological samples

The use and abuse of illegal drugs affects all modern societies, and therefore the assessment of drug exposure is an important task that needs to be accomplished. For this reason, the reliable determination of these drugs and their metabolites in biological specimens is an issue of utmost relevance for both clinical and forensic toxicology laboratories in their fields of expertise, including in utero drug exposure, driving under the influence of drugs and drug use in workplace scenarios. Most of the confirmatory analyses for abused drugs in biological samples are performed by gas chromatographic–mass spectrometric methods, but use of the more recent and sensitive liquid chromatography–(tandem) mass spectrometry technology is increasing dramatically. This article reviews recently published articles that describe procedures for the detection of opiates in the most commonly used human biological matrices, blood and urine, and also in unconventional ones, e.g. oral fluid, hair, and meconium. Special attention will be paid to sample preparation and chromatographic analysis.     

Current role of LC–MS(/MS) in doping control

Liquid chromatography–(tandem) mass spectrometry (LC–MS/MS) has revolutionized the detection assays used in doping control analysis over the last decade. New methods have enabled the determination of drugs that were formerly difficult to detect or undetectable at preceding sample concentrations, and complex and/or time-consuming procedures based on alternative chromatographic–mass spectrometric or immunochemical principles have been replaced by faster, more comprehensive and robust assays. A critical overview of the contributions of LC–MS(/MS) to sports drug testing is provided, including recent developments regarding low and high molecular weight drugs.     

Environmental analysis of fluorinated alkyl substances by liquid chromatography–(tandem) mass spectrometry: a review

Fluorinated alkyl substances (FASs) are widely distributed contaminants that have been found in many environmental, human and biological samples throughout the world. Perfluorochemicals are used in many industry and consumer products, such as polymers and surfactants, because they have unique and useful properties (they are stable, chemically inert and generally unreactive). However, these compounds have also been found to be toxic, persistent and bioaccumulative. In recent years various analytical methods have been developed for the analysis of FASs in environmental samples. Most of these methods are based on liquid chromatography coupled to mass spectrometry (LC–MS) or tandem mass spectrometry (LC–MS/MS), since this is considered to be the technique of choice. This article reviews the various LC–(tandem)MS methods described so far for the analysis of FASs in water, sediment, sludge and biota samples. It discusses the main experimental conditions used for sample pretreatment and for analysis as well as the most relevant problems encountered and the limits of detection achieved.     

Determination of adrenolytic drugs by SPME–LC–MS

Five adrenolytic drugs have been analyzed by liquid chromatography–mass spectrometry (LC–MS). Samples were prepared by solid-phase microextraction (SPME) using polypyrrole fibers coated on stainless steel support as an adsorbent for the drugs. Adsorption efficiencies were 95% and were close for all the drugs investigated. Relative standard deviations (RSD), calculated for samples prepared in standard solutions, were in the range 2.5–13%, however RSD values for the drugs in human plasma were 2.5–4.5%. Using LC–MS the limit of detection (LOD) and the limit of quantification (LOQ) were in the ranges 0.11–0.18 and 0.39–0.54 ng mL⁻¹, respectively, for the five drugs.     

Rapid drug detection in oral samples by porous silicon assisted laser desorption/ionization mass spectrometry

The demand for analysis of oral fluid for illicit drugs has arisen with the increased adoption of roadside testing, particularly in countries where changes in legislation allow random roadside testing of drivers for the presence of a palette of illicit drugs such as methamphetamine (MA), 3,4‐methylenedioxymethamphetamine (MDMA) and Δ⁹‐tetrahydrocannabinol (THC). Oral samples are currently tested for such drugs at the roadside using an immunoassay‐based commercial test kit. Positive roadside tests are sent for confirmatory laboratory analysis, traditionally by means of gas chromatography/mass spectrometry (GC/MS). We present here an alternative rapid analysis technique, porous silicon assisted laser desorption/ionization time‐of‐flight mass spectrometry (pSi LDI‐MS), for the high‐throughput analysis of oral fluids. This technique alleviates the need for sample derivatization, requires only sub‐microliter sample volumes and allows fast analysis (of the order of seconds). In this study, the application of the technique is demonstrated with real samples from actual roadside testing. The analysis of oral samples resulted in detection of MA and MDMA with no extraction and analysis of THC after ethyl acetate extraction. We propose that, subject to miniaturization of a suitable mass spectrometer, this technique is well suited to underpin the deployment of oral fluid testing in the clinic, workplace and on the roadside. Copyright © 2009 John Wiley & Sons, Ltd.     

Combination of Aqueous and Non-Aqueous Capillary Electrophoresis with Electrospray Mass Spectrometry for the Determination of Drug Residues in Water

 The work presented in this paper deals with the combination of capillary electrophoresis (CE) with electrospray mass spectrometry (MS) for the determination of drug residues in water. CE/MS methods have been developed based on either aqueous or non-aqueous ammonium acetate solutions as the carrier electrolyte for the separation of selected drugs. The different separation conditions were compared in terms of selectivity and detection limits; both aqueous and non-aqueous CE proved to be suitable for the present analytical task, exhibiting detection limits between 3 and 93 μg/dm³ (injected standard concentration) corresponding to concentrations between 5 and 19 ng/dm³ in the sample. A combination of liquid-liquid extraction and solid-phase extraction was investigated for sample pretreatment, yielding enrichment factors of 10000. The applicability of CE/MS was demonstrated for the analysis of several river water samples.     

Roadside Drug Testing Approaches

The purpose of this review is to present an overview of roadside drug testing, driving enforcement, and drunk/drug driving detection around the world. Drunk and drug driving is a severe problem, not only in the UAE, but also around the world. This has important implications for road safety as drunk or drug driving may increase the chances of a driver’s involvement in a road crash when compared to a drug-free driver. Recently, due to increases in drug-impaired drivers’ crash involvement, many mobile roadside drug testing devices have been introduced to the market. These devices use oral fluid, urine or blood matrices. These are on-the-spot tests, which are easy to use and are applied by law enforcement agencies and the public. Law enforcement agencies most commonly use oral fluid to detect the presence of illicit drugs in drivers. This review discusses all the available devices in the market used by the authorities. It also describes the type of drugs widely abused by drivers along with behavioral testing methods. The different types of matrices used for roadside drug testing are also evaluated. Sample collection, storage, and pre-treatment methods are discussed, followed by the confirmatory analysis of positive samples. This article will significantly help law enforcement agencies compare and evaluate all the reliable roadside testing devices and new emerging confirmatory devices available to them in the market. This will help them make an informed decision on which device to adapt to their individual needs.     

Slurry sampling electrothermal vaporization inductively coupled plasma mass spectrometry for the determination of Cr, Cd and Pb in plastics

Ultrasonic slurry sampling electrothermal vaporization dynamic reaction cell inductively coupled plasma mass spectrometry (USS–ETV–DRC–ICP–MS) for the determination of Cr, Cd and Pb in several plastic samples, using NH₄NO₃ as the modifier, is described. The influences of the instrumental operating conditions and the slurry preparation technique on the ion signals are investigated. A reduction in the intensity of the background at signals corresponding to chromium masses (arising from matrix elements) was achieved by using NH₃ as the reaction cell gas in the DRC. The method was applied to determine Cr, Cd and Pb in two polystyrene (PS) samples and a polyvinyl chloride (PVC) sample using two different calibration methods, namely standard addition and isotope dilution. The results were in good agreement with those for digested samples analyzed by ultrasonic nebulization DRC–ICP–MS. The precision between sample replicates was better than 17% with the USS–ETV–DRC–ICP–MS method. The method detection limits, estimated from standard addition curves, were about 6–9, 1–2 and 8–11 ng g⁻¹ for Cr, Cd and Pb, respectively, in the original plastic samples.     

Combination of Aqueous and Non-Aqueous Capillary Electrophoresis with Electrospray Mass Spectrometry for the Determination of Drug Residues in Water

Summary.  The work presented in this paper deals with the combination of capillary electrophoresis (CE) with electrospray mass spectrometry (MS) for the determination of drug residues in water. CE/MS methods have been developed based on either aqueous or non-aqueous ammonium acetate solutions as the carrier electrolyte for the separation of selected drugs. The different separation conditions were compared in terms of selectivity and detection limits; both aqueous and non-aqueous CE proved to be suitable for the present analytical task, exhibiting detection limits between 3 and 93 μg/dm³ (injected standard concentration) corresponding to concentrations between 5 and 19 ng/dm³ in the sample. A combination of liquid-liquid extraction and solid-phase extraction was investigated for sample pretreatment, yielding enrichment factors of 10000. The applicability of CE/MS was demonstrated for the analysis of several river water samples. Received August 25, 2000. Accepted October 17, 2000     

Optimisation of supercritical fluid extraction of polynuclear aromatic hydrocarbons from spiked soil and marine sediment standard reference material

 For the determination of 16 PAHs in soils and sediment samples by GC/FID and GC/MS, the dynamic off-line supercritical fluid extraction with both pure and modified carbon dioxide has been evaluated. The optimisation of extraction parameters was performed for four individual groups of PAHs according to their number of aromatic rings (2–3 rings, 4 rings, 5 rings and 6 rings) by varying pressure (200–510 bar), temperature (50–150 °C), extraction fluid volume (10–50 ml), and the methanol modifier concentration (0–10%). Using a five level spherical factorial experimental design the number of experiments required for optimisation was 45. In spiked soil samples extraction efficiencies of 80–100% were achieved for the individual groups of PAHs. At the optimal set of conditions 10–30% lower recoveries of PAHs were obtained for the standard reference material NIST SRM 1941a (marine sediment). The largest differences between extraction recoveries of native and spiked PAHs occurred at high molecular weight PAHs. Using SFE efficiency data for the standard reference material, cluster analysis proved that dividing the 16 PAHs into four groups according to their number of aromatic rings was appropriate and correct. Received: 2 February 1996/Revised: 26 November 1996/Accepted: 30 November 1996     

Determination of basic drugs of abuse in human serum by online extraction and LC–MS/MS

A new quantitation method for the determination of drugs of abuse (opiates, amphetamine and derivatives, cocaine, methadone and metabolites) in serum by using online extraction coupled to liquid chromatography (LC)–mass spectrometry (MS)/MS has been developed. The online extraction is carried out using two extraction columns simultaneously and one analytical column. One extraction column is loaded, while the other one is eluted by a gradient. The elution gradient also separates the analytes in the analytical column. For the sample preparation, serum is spiked with a mixture of deuterated analogues of the drugs. After protein precipitation with methanol/zinc sulphate, centrifugation, evaporation and reconstitution, the sample is injected into the LC system. The quantitation is based on the analysis of two multiple reaction monitoring transitions per drug. The recovery of the protein precipitation step is over 80% for all analytes. Intra- and interday precision, as relative standard deviation, is lower than 6%, and in the case of accuracy, RE is lower than 15%. Only the most polar analytes showed matrix effects. The limits of quantitation for the analysed compounds vary between 0.5 and 2.8 ng/mL. The developed method was used to quantify basic drugs in samples “from driving under the influence of drugs” cases. The results were compared with those obtained by using solid-phase extraction–GC–MS.     

On-line solid-phase extraction-gas chromatography-ion trap tandem mass spectrometric detection for the nanogram per liter analysis of trace pollutants in aqueous samples

Summary On-line solid-phase extraction-gas chromatographyion-trap tandem mass spectrometry (SPE-GC-MS/MS) has been used for the trace-level determination of polar and apolar pesticides. The SPE-GC interface, an Autoloop 2000, was operated at an injection temperature of 90°C which permitted the determination of thermolabile pesticides such as carbofuran and carbaryl. Rectilinear calibration curves were obtained for the analytes tested over a range of 0.1–500 ng L⁻¹, using a sample volume of 10–100 mL for enrichment on an SPE cartridge packed with styrene-divinylbenzene copolymer. The detection limits for the pesticides were in the 0.01–4 ng L⁻¹ range. For a number of pesticides acceptable tandem mass spectra were obtained at levels as low as 0.1 ng L⁻¹ level in real-life water samples. As a demonstration of the applicability of this technique for inorganic anions, bromide and nitrite were converted into 4-bromoacetanilide and 2-phenylphenol, respectively. The reaction products were pooled and subjected to simultaneous analysis by the present method using full-scan mass spectrometric detection. The detection limits were 0.3 and 2 ng L⁻¹, respectively.     

Simultaneous determination of Δ9-tetrahydrocannabinol and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol in oral fluid using isotope dilution liquid chromatography tandem mass spectrometry

The detection and confirmation of cannabinoids in oral fluid are important in forensic toxicology. Currently, the presence of Δ⁹-tetrahydrocannabinol (THC) is used for the detection of cannabis in oral fluid. A low concentration of 11-nor-9-carboxy-Δ⁹-tetrahydrocannabinol (THC-COOH) is found in oral fluid, which suggested a convenient and low-sensitivity confirmation assay can be used in a routine forensic laboratory. In this study, a highly sensitive isotope dilution liquid chromatography–tandem mass spectrometry method following dansylation was successfully developed for simultaneous determination of THC and THC-COOH in oral fluid. The dansylated derivatives dramatically demonstrated and enhanced the sensitivity of THC and THC-COOH. To avoid signal influenced by the matrix, a 5-min liquid chromatography gradient program was evaluated and optimized, which reduced the sample diffusion and caused sharp peaks (less than 12 s) and thus helped to achieve detection at a low level. The sensitivity, accuracy, and precision were also evaluated, and high quantitative accuracy and precision were obtained. The limit of quantitation of this approach was 25 pg/mL for THC and 10 pg/mL for THC-COOH in oral fluid. Finally, the method was successfully applied to eight suspected cannabis users. Among them, in six oral fluid samples THC-COOH was determined at a concentration from 13.1 to 47.2 pg/mL.     

Broad-spectrum drug screening of meconium by liquid chromatography with tandem mass spectrometry and time-of-flight mass spectrometry

Analysis of the major drugs of abuse in meconium has been established in clinical practice for detecting fetal exposure to illicit drugs, particularly for the ready availability of the sample and ease of collection from diapers, compared with neonatal hair and urine. Very little is known about the occurrence and detection possibilities of therapeutic and licit drugs in meconium. Meconium specimens (n = 209) were collected in delivery hospitals, from infants of mothers who were suspected to be drug abusers. A targeted analysis method by liquid chromatography–triple quadrupole mass spectrometry (LC-MS/MS) was developed for abused drugs: amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, morphine, codeine, 6-monoacetylmorphine, oxycodone, methadone, tramadol, buprenorphine, and norbuprenorphine. A separate LC-MS/MS method was developed for 11-nor-∆⁹-tetrahydrocannabinol-9-carboxylic acid. A screening method based on LC coupled to time-of-flight MS was applied to a broad spectrum of drugs. As a result, a total of 77 different compounds were found. The main drug findings in meconium were as follows: local anesthetics 82.5% (n = 172), nicotine or its metabolites 61.5% (n = 129), opioids 48.5% (n = 101), stimulants 21.0% (n = 44), hypnotics and sedatives 19.0% (n = 40), antidepressants 18.0% (n = 38), antipsychotics 5.5% (n = 11), and cannabis 3.0% (n = 5). By revealing drugs and metabolites beyond the ordinary scope, the present procedure helps the pediatrician in cases where maternal denial is strong but the infant seems to suffer from typical drug-withdrawal symptoms. Intrapartum drug administration cannot be differentiated from gestational drug use by meconium analysis, which affects the interpretation of oxycodone, tramadol, fentanyl, pethidine, and ephedrine findings.     

Wipe sampling procedure coupled to LC–MS/MS analysis for the simultaneous determination of 10 cytotoxic drugs on different surfaces

A simple wipe sampling procedure was developed for the surface contamination determination of ten cytotoxic drugs: cytarabine, gemcitabine, methotrexate, etoposide phosphate, cyclophosphamide, ifosfamide, irinotecan, doxorubicin, epirubicin and vincristine. Wiping was performed using Whatman filter paper on different surfaces such as stainless steel, polypropylene, polystyrol, glass, latex gloves, computer mouse and coated paperboard. Wiping and desorption procedures were investigated: The same solution containing 20% acetonitrile and 0.1% formic acid in water gave the best results. After ultrasonic desorption and then centrifugation, samples were analysed by a validated liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) in selected reaction monitoring mode. The whole analytical strategy from wipe sampling to LC–MS/MS analysis was evaluated to determine quantitative performance. The lowest limit of quantification of 10 ng per wiping sample (i.e. 0.1 ng cm⁻²) was determined for the ten investigated cytotoxic drugs. Relative standard deviation for intermediate precision was always inferior to 20%. As recovery was dependent on the tested surface for each drug, a correction factor was determined and applied for real samples. The method was then successfully applied at the cytotoxic production unit of the Geneva University Hospitals pharmacy.     

Comparison of extraction efficiencies and LC–MS–MS matrix effects using LLE and SPE methods for 19 antipsychotics in human blood

Antipsychotic drugs are frequently associated with sudden death investigations. Detection of these drugs is necessary to establish their use and possible contribution to the death. LC–MS(MS) methods are common; however accurate and precise quantification is assured by using validated methods. This study compared extraction efficiency and matrix effects using common liquid–liquid and solid-phase extraction procedures in both ante-mortem and post-mortem specimen using LC–MS–MS. Extraction efficiencies and matrix effects were determined in five different blank blood specimens of each blood type. The samples were extracted using a number of different liquid–liquid extraction methods and compared with a standard mixed-mode solid-phase extraction method. Matrix effects were determined using a post-extraction addition approach—the blank blood specimens were extracted as described above and the extracts were reconstituted in mobile phase containing a known amount of analytes. The extraction comparison of ante-mortem and post-mortem blood showed considerable differences, in particular the extraction efficiency was quite different between ante-mortem and post-mortem blood. Quantitative methods used for determination of antipsychotic drugs in post-mortem blood should establish that there are no differences in extraction efficiency and matrix effects, particularly if using ante-mortem blood as calibrator.     

Application of microextraction by packed sorbent to isolation of psychotropic drugs from human serum

A method of microextraction by packed sorbent (MEPS) followed by liquid chromatography with diode array detection has been developed and optimized for the extraction of six tricyclic antidepressants (amitriptyline, nortriptyline, imipramine, desipramine, doxepin, nordoxepin) from human serum. The optimal parameters of MEPS extraction (type of sorbent, volume of sample, composition, and volume of washing and elution solutions) for these drugs in spiked samples were defined. The developed MEPS procedure was validated and then successfully applied to the analysis of serum reference material. The limit of detection (0.02–0.05 μg/mL), intraday (2.7–8.8%) and interday (4.4–11.6%) precision (RSD), and the accuracy of the assay (94.5–108.8%) at three concentration levels—0.2, 0.5, and 0.8 μg/mL—were estimated. The accuracy of the method was evaluated by the analysis of certified reference material. Moreover, the validated procedure was compared with the solid-phase extraction technique. Finally, microextraction by packed sorbent was assessed as a suitable tool in forensic and clinical methods for serum sample preparations.     

Testing for Drugs in Hair – a Review of Chromatographic Procedures

This article reviews the analysis of drugs and drug metabolites in hair by chromatographic procedures, including the pretreatment steps, and the extraction methods. The general tendency in the last years, to highly sophisticated techniques (GC–MS–NCI, HPLC–MS, GC–MS–MS) illustrates the constant fight for sensitivity.     

Therapeutic drug monitoring by LC–MS–MS with special focus on anti-infective drugs

Liquid chromatography coupled to mass spectrometry nowadays plays an important role in the field of therapeutic drug monitoring (TDM), especially of new compounds for which no immunoassays are available. This paper reviews LC–MS(–MS) methods published recently for anti-infective drugs: antiretroviral drugs, other antiviral drugs, antibacterial drugs, antihelmintic drugs, antimalarial drugs, and other antiprotozoal drugs. An overview of the different methods is given, with special focus on selection of the internal standard and validation procedures.     

LC/ESI-MS/MS method for quantification of 28 synthetic cannabinoids in neat oral fluid and its application to preliminary studies on their detection windows

Serum and urine samples are commonly used for the analysis of synthetic cannabinoids in biofluids; however, their utilization as analytical matrices for drug abstinence control features some substantial drawbacks. While for blood collection invasive sampling is inevitable, the urinary analysis of synthetic cannabinoids is limited by the lack of available reference standards of the respective major metabolites. Moreover, the long detectability of synthetic cannabinoids in both matrices hampers the identification of a recent synthetic cannabinoid use. This article describes the development, validation and application of an LC/ESI-MS/MS method for the quantification of 28 synthetic cannabinoids in neat oral fluid (OF) samples. OF samples were prepared by protein precipitation using ice-cold acetonitrile. Chromatographic separation was achieved by gradient elution on a Luna Phenyl Hexyl column (50?×?2 mm, 5 μm), while detection was carried out on a QTrap 4000 instrument in positive ionization mode. The limits of detection ranged from 0.02 to 0.40 ng/mL, whereas the lower limits of quantification ranged from 0.2 to 4.0 ng/mL. The method was applied to authentic samples collected during two preliminary studies in order to obtain insights into the general detectability and detection windows of synthetic cannabinoids in this matrix. The results indicate that synthetic cannabinoids are transferred from the blood stream into OF and vice versa only at a very low rate. Therefore, positive OF samples are due to contamination of the oral cavity during smoking. As these drug-contaminations could be detected up to approximately 2 days, neat oral fluid appears to be well suited for detection of a recent synthetic cannabinoid use.