A quantitative, selective and fast ultra-high performance liquid chromatography tandem mass spectrometry method for the simultaneous analysis of 33 basic drugs in hair (amphetamines, cocaine, opiates, opioids and metabolites).

Forensic testing for drugs of abuse in hair has become a useful diagnostic tool in determining chronic drug use as well as examining long-term drug history thorough segmental analysis. However, sensitive and specific analytical methods are needed. A simple, rapid and highly sensitive and specific method for the extraction and quantification of 33 opioids, opiates, cocaine, and amphetamines is presented. The method was fully validated according to international guidelines. Twenty milligrams of hair sample was pulverized and then incubated in the same disposable tube with methanol (under sonication at 45°C) during 4h. After centrifugation the supernatant was evaporated up to about 100 µL and a solid phase extraction (SPE) followed by separation and quantification using ultra performance liquid chromatography-tandem mass spectrometry (UHLC-MS/MS) were carried out. Chromatographic separation was achieved using a BEH phenyl column eluted with 0.1% formic acid: methanol (0.1% formic acid). Selectivity of the method was achieved by a combination of retention time, and two precursor-product ion transitions. Good intra-assay and inter-assay precision (relative standard deviations (RSDs) were observed (<15%) for most of the compounds. The lower limit of quantification was fixed at the lowest calibrator in the linearity experiments and it ranged from 0.006 to 0.063 ng/mg. No instability was observed in processed samples. Extraction efficiency varied from 37 to 107% (except for EDDP with a recovery of 5%) and matrix effects ranged from 52 to 160%, and for most of the compounds it was compensated by the internal standard (IS). The method was subsequently applied to authentic hair samples obtained from forensic and toxicology cases and to proficiency test (obtaining z-scores lower than 1 for most of the compounds). The validation and actual sample analysis results show that this method is rugged, precise, accurate, and well-suited for routine hair analysis.

Quantitative analysis of 26 opioids, cocaine, and their metabolites in human blood by ultra performance liquid chromatography-tandem mass spectrometry.

A sensitive and selective ultra performance liquid chromatographic-tandem mass spectrometric method was developed and fully validated for the simultaneous determination of (in order of chromatographic elution) methylecgonine, pholcodine, morphine, hydromorphone, oxymorphone, norcodeine, codeine, dihydrocodeine, oxycodone, 6-Monoacetylmorphine (6-MAM), hydrocodone, ethylmorphine, norfentanyl, benzoylecgonine, tramadol, normeperidine, meperidine, cocaine, pentazocine, cocaethylene, fentanyl, norbuprenorphine, 2-ethylidine-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), buprenorphine, propoxyphene, and methadone in blood. The matrixes analyzed during the validation experiments were as follows: citrated blank plasma for calibrators, fluoride blank plasma for internal quality control (QC), lyophilized serum for external QC, fluoride plasma and whole blood for authentic samples, and lyophilized serum and whole blood for proficiency testing schemes. Samples were extracted with cation exchange solid-phase extraction cartridges. The target drugs were separated and quantified in a chromatographic run of 8.1 minutes using 0.1% formic acid in water and methanol (with 0.1% formic acid) as mobile phase. The limit of quantification ranged from 0.5 to 2.5 ng/mL depending on the compound and the therapeutic concentration. The intra- and interassay precision was less than 15% for all the compounds (except for pentazocine and EDDP, which was <20%) determined with 2 internal and 2 external QC samples, and the bias was within ±15% (except for methylecgonine, which was <20%). Extraction efficiency was greater than 70% for all the compounds except for EDDP. Matrix effects were evaluated with authentic blood samples (n = 10), and they ranged from 47 to 95%, but they were compensated for most analytes using deuterated analogs as internal standards. Prepared samples were stable for 62 hours in the autosampler. This method was successfully applied to authentic samples (n = 120), involving the use of heroin, cocaine, tramadol, and methadone, and to proficiency testing schemes.

Ultra-performance liquid chromatography-tandem mass spectrometry method for the analysis of amphetamines in plasma.

A fast and selective ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method for the determination of amphetamines (amphetamine, methamphetamine, methylenedioxymethamphetamine, methylenedioxyamphetamine, methylenedioxyethylamphetamine, ephedrine, and p-methoxyamphetamine) in plasma has been developed and validated. Sample preparation was performed by liquid-liquid extraction using ethyl acetate. For optimized chromatographic performance with repeatable retention times, narrow and symmetrical peaks, and focusing all analytes at the column inlet, a gradient start, with acid mobile phase consisting of 0.1% formic acid and methanol was chosen. Positive electrospray ionization MS-MS detection was performed with two multiple reaction monitoring transitions for each analyte. Deuteriumlabeled internal standards were used for five of the analytes. The limit of detection was in the range 0.25-1.25 ng/mL, and the limit of quantification was fixed at the lowest calibrator of 2.5 ng/mL for all of the compounds. The RSD values of the intra- and interassay precision and accuracy were lower than 11% at four concentration levels, including two external quality controls. No or only minor matrix effects were observed, and the extraction method presented recoveries higher than 93% for all the compounds. Total run time, including equilibration, was 12 min. The method is routinely used at the National Institute of Criminalistics and Criminology for quantitative determination of the main amphetamines in plasma from forensic and driving under the influence cases.

Methadone determination in puparia and its effect on the development of Lucilia sericata (Diptera, Calliphoridae).

This paper describes a sensitive UPLC-MS/MS method for quantification of methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in single empty puparial case of Lucilia sericata. Larvae were reared on substrates spiked with different concentrations of methadone (0-4 µg/g). Methadone was quantified in puparia reared on high concentrated substrates (0.8-4 µg/g). The major metabolite of methadone (EDDP) was not detected, confirming rapid elimination of metabolites by the larvae before pupation. The effects of methadone on the development of L. sericata were also investigated. No effect on sex ratio was detected. A significant difference was calculated for emerged adults but no trends could be observed. Concerning the developmental curve, a significant difference was observed between control and high methadone concentrations using the Kolmogorov-Smirnov test.

Entomotoxicology, experimental set-up and interpretation for forensic toxicologists.

Forensic entomotoxicology studies the usefulness of insects as alternative toxicological samples. Use of insects as alternative matrix for drug detection is well documented and recommended when conventional matrices such as blood, urine or internal organs are no longer available. However, several limitations of entomotoxicology have been highlighted, especially concerning interpretation of the drug concentrations in insects on human forensic cases. In addition, the lack of knowledge in pharmacokinetic of drugs in insects, large variability of experimental set-up and toxicological analysis compromise the utility of this science. This review focuses on the current knowledge of factors influencing drug detection in insects. Reasons for the current limitations, but also recommendations for future research are discussed and proposed in this paper.

High-throughput analysis of amphetamines in blood and urine with online solid-phase extraction-liquid chromatography-tandem mass spectrometry.

An automated online solid-phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS-MS) method for the analysis of amphetamines in blood and urine was developed and validated. Chromatographic separation was achieved on a Nucleodur Sphinx RP column with an LC gradient (a mixture of 10 mM ammonium formate buffer and acetonitrile), ensuring the elution of amphetamine, methamphetamine, MDMA, MDA, MDEA, PMA, and ephedrine within 11 min. The method was fully validated, according to international guidelines, using only 100 and 50 microL of blood and urine, respectively. The method showed an excellent intra- and interassay precision (relative standard deviation < 11.2% and bias < 13%) for two external quality control samples (QC) for both matrices and three and two 'in house' QCs for blood and urine, respectively. Responses were linear over the investigated range (r(2) > 0.99, 2.5-400 microg/L for blood and 25-1000 microg/L for urine). Limits of quantification were determined to be 2.5 and 25 microg/L for blood and urine, respectively. Limits of detection ranged from 0.05 to 0.5 microg/L for blood and 0.25 to 2.5 microg/L for urine, depending on the compound. Furthermore, the analytes and the processed samples were demonstrated to be stable (in the autosampler for at least 72 h and after three freeze/thaw cycles), and no disturbing matrix effects were observed for all compounds. Moreover, no carryover was observed after the analysis of high concentration samples (15,000 microg/L). The method was subsequently applied to authentic blood and urine samples obtained from forensic cases, which covered a broad range of concentrations. The validation results and actual sample analyses demonstrated that this method is rugged, precise, accurate, and well-suited for routine analysis as more than 72 samples are analyzed non-stop in 24 h with minimum sample handling. The combination of the high-throughput online SPE and the well-known sensitivity and selectivity assured by MS-MS resulted in the elimination of the bottleneck associated with the sample preparation requirements and provided increased sensitivity, accuracy, and precision.

On-line solid-phase extraction combined with liquid chromatography-tandem mass spectrometry for high throughput analysis of 11-nor-Delta9-tetrahydrocannabinol-9-carboxylic acid in urine.

A simple, rapid and highly sensitive method for the analysis of THC-COOH in urine, using automated on-line solid-phase extraction (SPE) combined with liquid chromatography (LC)-mass spectrometry (MS/MS), is developed and fully validated according to international guidelines. Chromatographic separation was achieved on an Atlantis dC(18) column with an isocratical gradient, ensuring the elution of THC-COOH within 4.1 min. The total process time was 6 min and 500 microL of sample was required. SPE using C(8) cartridges was highly effective, reproducible and led to significant decreases in the interferences present in the matrix. The method showed an excellent intra- and inter-assay precision (relative standard deviation (RSD) <7% and bias <13%) for four external quality control (QC) samples and three 'in house' QCs. Responses were linear over the investigated range (r(2)>0.99, 5-200 microg/L). Limits of quantification (LOQ) and detection (LOD) were determined to be 5 micro/L and 0.25 microg/L, respectively. Furthermore, the analyte and the processed samples were demonstrated to be stable. Moreover, no carryover was observed after the analysis of high concentrated urine samples (5000 microg/L THC-COOH)). The method was subsequently applied to authentic samples previously screened by a routine immunoassay method.

Detection of diazepam in urine, hair and preserved oral fluid samples with LC-MS-MS after single and repeated administration of Myolastan and Valium.

Sedative agents are used to facilitate sexual assault due to their ability to render the victim passive, submissive and unable to resist. The primary pharmacological effect of the benzodiazepine tetrazepam is muscle relaxation, whereas the benzodiazepine diazepam acts on the central nervous system (CNS) exerting mainly sedation effects. Therefore, contrary to tetrazepam, diazepam is an often-abused drug, which can potentially be used as a date-rape drug. In this study, we describe the detection of low amounts of diazepam in Myolastan (Sanofi-Synthelabo S.A., Brussels, Belgium) and Epsipam (Will-Pharma, Wavre, Belgium) 50 mg tablet preparations by LC-MS-MS, GC-FID and HPLC-DAD. Considering the important forensic implication of this finding, a study was conducted with volunteers receiving a single or repeated dosage of Myolastan. Urine, hair and preserved oral fluid samples were analysed using a previously described sensitive and specific LC-MS-MS detection method allowing for the simultaneous quantification of tetrazepam, diazepam, nordiazepam, oxazepam and temazepam. This study demonstrates that diazepam can be observed in urine samples even after a single dose of Myolastan. In addition, maintaining therapy for 1 week results in the detection of both diazepam and nordiazepam in urine samples and of diazepam in the first hair segment. Importantly, comparing urine and hair samples after a single intake of diazepam versus the single and 1 week administration of Myolastan shows that the possible metabolic conversion of tetrazepam to diazepam is a more plausible explanation for the detection of diazepam in biological samples after the intake of Myolastan. As such, these results reveal that the presence of diazepam and/or nordiazepam in biological samples from alleged drug-facilitated assault cases should be interpreted with care.