Mutation rates at 14 STR loci in the population from Pernambuco, Northeast Brazil

Definition about mutation rates of short tandem repeats (STRs) loci used in forensic analysis are useful for the correct interpretation of resulting genetic profiles and the definition of criterions for exclusion in paternity testing. Germline mutation of 14 STR loci was studied for 54,105 parent–child allelic transfers from 2575 paternity testing cases carried out during 2000–2007 from the Pernambuco State, Northeast Brazil. The parenthood in each of these cases was highly validated (probability > 99.99%). We identified 43 mutations at 12 loci. Locus-specific mutation rate estimates varied between 2 × 10⁻⁴ and 2 × 10⁻³, and the overall mutation rate estimate was 8 × 10⁻⁴. Mutation events in the male germline were more frequent than in the female germline. The majority of the mutations could be explained by losses or gains of one repeat unit and there was no evidence for selection between insertion or deletion changes. Our data were compared with those of Portuguese and North-American populations for CSF1PO, D18S51, D21S11, D7S820, TH01, TPOX and demonstrated, despite the great difference in the size of the sample, that mutation rates of STR loci in a mixed population do not differ from that encountered in different populations.     

Typing of 49 autosomal SNPs by SNaPshot in the Slovenian population

A total of 157 unrelated individuals residing in Slovenia were typed for 49 of the autosomal single nucleotide polymorphisms (SNPs) in the SNPforID 52plex with the SNaPshot^® assay. We obtained full SNP profiles in all but one individual and perfect concordance was obtained in duplicated analyses. Allele frequencies are presented for the 49 SNPs. No deviation from HWE was observed for any SNP. F_IS and F_ST were estimated. A principal coordinate analysis performed on six populations (Slovenian, Danish, Somali, Greenland, Turkish and Chinese) showed that the Slovenian population grouped with the Danish population. The mean power of discrimination for the Slovenian population was 1.1 × 10⁻¹⁹, and the mean exclusion probability for trios was 99.96%.     

Population genetics of 15 autosomal STR loci in the population of Pomorze Zachodnie (NW Poland)

Allele frequency data and forensic efficiency parameters for 15 STR loci: D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, FGA were estimated from a sample of 600 unrelated individuals from the Pomorze Zachodnie (NW Poland). The combined MP and PE for all 15 loci are 3.9 × 10⁻¹⁸ and 0.9999988, respectively. Pairwise comparisons between Northwestern Poland and other Polish populations were performed.     

Voltammetric analysis of luminescent markers in gunshot residues

Currently, SEM-EDS is used to detect gunshot residue (GSR) from the presence of Ba, Pb, and Sb in the sample. However, the development of new nontoxic ammunition (NTA) has prevented conventional metals from being found. In this work, we aim to determine the presence of an inorganic luminescent chemical marker based on rare earth in gunshot residues using the technique of squarewave voltammetry (SWV). After firing, the luminescent complex [(Eu₂Zr)(btc)₃(Hbtc)_0.5.6H₂O], which is used as a chemical marker, can be detected under a UV lamp. An aqueous solution with 0.1 mol L⁻¹ KCl as supporting electrolyte can be easily collected on carbon paste electrode surfaces for SWV analysis A = 100 mV, f = 10 Hz, and step potential of 5 mV are required. The luminescent marker incorporated into the carbon paste electrode showed two anodic peak currents in the region of 0.4 V (vs Ag/AgCl) and at 0.75 V (vs Ag/AgCl) and also a cathodic one in 0.4 V (vs Ag/AgCl). SEM-EDS was able to analyze the same voltammetric results for conventional and nontoxic ammunition containing the luminescent marker. Therefore, voltammetry and SEM-EDS are valid for detecting the new residue marker in GSR. Despite this, the electrochemical method is still more advantageous because of its low cost and lack of expensive equipment and supplies in forensic laboratories.     

Determination of MDMA, MDA, MDEA and MBDB in oral fluid using high performance liquid chromatography with native fluorescence detection

This paper describes the analytical methodology for the determination of MDMA, MDA, MDEA and MBDB in oral fluid. After a liquid–liquid extraction, the analysis was carried out by high performance liquid chromatography (HPLC), with fluorescence detection. The detector wavelength was fixed at 285 nm for excitation and 320 nm for emission. The mobile phase, a mixture of phosphate buffer (pH = 5) and acetonitrile (75:25), and the column, Kromasil 100 C8 5 μm 250 mm × 4.6 mm, allowed good separation of the compounds in an isocratic mode in only 10 min. The method was validated and showed good limits of detection (2 ng/mL) and quantitation (10 ng/mL) for all the amphetamine derivatives. No interfering substances were detected. A stability study of these compounds in oral fluid stored at three different temperatures (−18, 4 and 20 °C) over 10 weeks was conducted, showing a time-dependent degradation of the four compounds.     

Development of a Pencil Drawn Paper-based Analytical Device to Detect Lysergic Acid Diethylamide (LSD)*†

The need for agile and proper identification of drugs of abuse has encouraged the scientific community to improve and to develop new methodologies. The drug lysergic acid diethylamide (LSD) is still widely used due to its hallucinogenic effects. The use of voltammetric methods to analyze narcotics has increased in recent years, and the possibility of miniaturizing the electrochemical equipment allows these methods to be applied outside the laboratory; for example, in crime scenes. In addition to portability, the search for affordable and sustainable materials for use in electroanalytical research has grown in recent decades. In this context, employing paper substrate, graphite pencil, and silver paint to construct paper-based electrodes is a great alternative. Here, a paper-based device comprising three electrodes was drawn on 300 g/m² watercolor paper with 8B pencils, and its efficiency was compared to the efficiency of a commercially available screen-printed carbon electrode. Square wave voltammetry was used for LSD analysis in aqueous medium containing 0.05 mol/L LiClO₄. The limits of detection and quantification were 0.38 and 1.27 μmol/L, respectively. Both electrodes exhibited a similar voltammetric response, which was also confirmed during analysis of a seized LSD sample, with recovery of less than 10%. The seized samples were previously analyzed by GCMS technique, employing the full scan spectra against the software spectral library. The electrode selectivity was also tested against 3,4-methylenedioxymethamphetamine (MDMA) and methamphetamine. It was possible to differentiate these compounds from LSD, indicating that the developed paper-based device has potential application in forensic chemistry analyses.     

Post-injection hybridization of complementary DNA strands on capillary electrophoresis platforms: A novel solution for dsDNA artifacts

Several laboratories have reported the occurrence of a split or n − 1 peak at the vWA locus in PowerPlex^® 16 and PowerPlex^® ES amplification products separated on 4- and 16-capillary electrophoresis instruments. The root cause of this artifact is post-PCR reannealing of the unlabeled, unincorporated vWA primer to the 3′-end of the tetramethylrhodamine (TMR)-labeled strand of the vWA amplicon. This reannealing occurs in the capillary post-electrokinetic injection. The split peak is eliminated by incorporation into the loading cocktail of a sacrificial hybridization sequence (SHS) oligonucleotide that is complementary to the vWA primer. The SHS preferentially anneals to the primer instead of the TMR-labeled strand of the vWA amplicon. In addition, the n − 10/n − 18 artifact that may be seen at the vWA locus was determined to be due to double-stranded amplicon formed post-electrokinetic injection into the capillary. This was also eliminated by adding in two Complementary Oligo Targets (COT1 and COT2) in addition to the SHS oligonucleotide into the loading cocktail. These three oligonucleotides are complementary to the 33 bases at the 5′-end of the unlabeled vWA amplicon strand and the 60 bases at its 3′-end and therefore compete for hybridization to the TMR-labeled amplicon strand. Incorporation of these three oligonucleotides in the Internal Lane Standard 600 (ILS600) eliminate both the split peak and n − 10/n − 18 artifact in PowerPlex^® 16 and PowerPlex^® ES amplification products without affecting sizing of alleles at the vWA locus or any locus in the PowerPlex^® 16, PowerPlex^® Y, PowerPlex^® ES, AmpFlSTR^® Profiler Plus^® ID, AmpFlSTR^® Cofiler^®, and AmpFlSTR^® SGM Plus^® kits.     

Confirmation by LC–MS of drugs in oral fluid obtained from roadside testing

The aim of this study was to assess the effectiveness of two current on-site oral fluid (OF) drug detection devices (OraLab and Dräger), as part of the Spanish participation in the Roadside Testing Assessment Project (ROSITA Project). The study was done in collaboration with the Spanish Traffic Police, in Galicia (NW Spain), during 2004 and 2005. A total of 468 drivers selected at the police controls agreed to participate through informed consent. In addition, saliva samples were collected and sent to the laboratory to confirm the on-site results. For this purpose, two different analytical liquid chromatography–mass spectrometry (LC–MS) methods were used to detect 11 drugs or metabolites in a 300 μL sample. Simultaneous analysis of morphine, 6-acetylmorphine, amphetamine, methamphetamine, MDA, MDMA, MDEA, MBDB, cocaine and benzoylecgonine was carried out using 100 μL of oral fluid, after an automated solid phase extraction. A different LC–MS method was performed to detect Δ⁹-THC in 200 μL of oral fluid using liquid–liquid extraction with hexane at pH 6. Both methods were fully validated, including linearity (1–250 ng/mL, 2–250 ng/mL) recovery (>50%), within-day and between-day precision (CV < 15%), accuracy (mean relative error < 15%), limit of detection (0.5 and 1 ng/mL), quantitation (1 and 2 ng/mL) and matrix effect. All of the positive cases and a random selection of 30% of the negatives were analyzed for confirmation analysis. Good results (sensitivity, specificity, accuracy, positive predictive value and negative predictive value > 90%) were obtained for cocaine and opiates by OraLab, and for cocaine by Dräger. However, the results for the other compounds could be improved for both detection devices. Differences in the ease of use and in the interpretation mode (visual or instrumental) were observed.     

Simultaneous analysis of six amphetamines and analogues in hair, blood and urine by LC-ESI-MS/MS: Application to the determination of MDMA after low Ecstasy intake

A rapid and sensitive method using LC-MS/MS triple stage quadrupole for the determination of traces of amphetamine (AP), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA, “ecstasy”), 3,4-methylenedioxyethamphetamine (MDEA), and N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine (MBDB) in hair, blood and urine has been developed and validated. Chromatography was carried out on an Uptisphere ODB C₁₈ 5 μm, 2.1 mm × 150 mm column (Interchim, France) with a gradient of acetonitrile and formate 2 mM pH 3.0 buffer. Urine and blood were extracted with Toxitube A^® (Varian, France). Segmented scalp hair was treated by incubation 15 min at 80 °C in NaOH 1 M before liquid–liquid extraction with hexane/ethyl acetate (2/1, v/v). The limits of quantification (LOQ) in blood and urine were at 0.1 ng/mL for all analytes. In hair, LOQ was <5 pg/mg for MA, MDMA, MDEA and MBDB, at 14.7 pg/mg for AP and 15.7 pg/mg for MDA. Calibration curves were linear in the range 0.1–50 ng/mL in blood and urine; in the range 5–500 pg/mg for MA, MDMA, MDEA and MBDB, and 20–500 pg/mg for AP and MDA. Inter-day precisions were <13% for all analytes in all matrices. Accuracy was <20% in blood and urine at 1 and 50 ng/mL and <10% in hair at 20 and 250 pg/mg. This method was applied to the determination of MDMA in a forensic case of single administration of ecstasy to a 16-year-old female without her knowledge during a party. She suffered from hyperactivity, sweating and agitation. A first sample of urine was collected a few hours after (T + 12 h) and tested positive to amphetamines by immunoassay by a clinical laboratory. Blood and urine were sampled for forensic purposes at day 8 (D + 8) and scalp hair at day 60 (D + 60). No MDMA was detected in blood, but urine and hair were tested positive, respectively at 0.42 ng/mL and at 22 pg/mg in hair only in the segment corresponding to the period of the offence, while no MDA was detectable. This method allows the detection of MDMA up to 8 days in urine after single intake.     

Evaluation of the Cozart RapiScan drug test system for opiates and cocaine in oral fluid

The purpose of this study was to evaluate the efficiency of the Cozart^® RapiScan (CRS) drug test system for detecting opiates and cocaine in oral fluid. Oral fluid samples were collected using the Cozart^® RapiScan collection system from 358 donors who were receiving treatment for their addiction and were monitored for drug misuse. A further 103 oral fluid samples were collected from volunteer donors who were not drug users. The samples were analyzed in the laboratory using the two-panel Cozart^® RapiScan cartridge for opiates and cocaine and confirmed using gas chromatography–mass spectrometry (GC–MS). The samples were stored frozen at −20 °C until analysis by GC–MS. The overall accuracy of the CRS for both opiates and cocaine was 100%. Samples spiked at 50% above and below the cut-off consistently gave negative and positive results respectively. A total of 88 samples were positive for various opiates and 111 samples were positive for cocaine and/or its metabolites. The CRS for opiates and cocaine in oral fluid, using a cut-off of 30 ng/mL morphine or benzoylecgonine equivalents in neat oral fluid, had overall efficiencies of 98% and 99%, respectively, versus GC–MS. A series of potential adulterants of oral fluid were evaluated and shown not to alter the outcome of the test result.     

Analysis of Ecstasy Tablets Using Capillary Electrophoresis with Capacitively Coupled Contactless Conductivity Detection

A method for the identification of 3,4‐methylenedioxymethamphetamine (MDMA) and meta‐chlorophenylpiperazine (mCPP) was developed employing capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C⁴D). Sample extraction, separation, and detection of “Ecstasy” tablets were performed in <10 min without sample derivatization. The separation electrolyte was 20 mm TAPS/Lithium, pH 8.7. Average minimal detectable amounts for MDMA and mCPP were 0.04 mg/tablet, several orders of magnitude lower than the minimum amount encountered in a tablet. Seven different Ecstasy tablets seized in Rio de Janeiro, Brazil, were analyzed by CE‐C⁴D and compared against routine gas chromatography‐mass spectrometry (GC‐MS). The CE method demonstrated sufficient selectivity to discriminate the two target drugs, MDMA and mCPP, from the other drugs present in seizures, namely amphepramone, fenproporex, caffeine, lidocaine, and cocaine. Separation was performed in <90 sec. The advantages of using C⁴D instead of traditional CE‐UV methods for in‐field analysis are also discussed.     

Validated method for the simultaneous determination of Δ-THC and Δ-THC-COOH in oral fluid, urine and whole blood using solid-phase extraction and liquid chromatography–mass spectrometry with electrospray ionization

A fully validated, sensitive and specific method for the extraction and quantification of Δ⁹-tetrahydrocannabinol (THC) and 11-nor-9-carboxy-Δ⁹-THC (THC-COOH) and for the detection of 11-hydroxy-Δ⁹-THC (11-OH THC) in oral fluid, urine and whole blood is presented. Solid-phase extraction and liquid chromatography–mass spectrometry (LC–MS) technique were used, with electrospray ionization. Three ions were monitored for THC and THC-COOH and two for 11-OH THC. The compounds were quantified by selected ion recording of m/z 315.31, 329.18 and 343.16 for THC, 11-OH THC and THC-COOH, respectively, and m/z 318.27 and 346.26 for the deuterated internal standards, THC-d₃ and THC-COOH-d₃, respectively. The method proved to be precise for THC and THC-COOH both in terms of intra-day and inter-day analysis, with intra-day coefficients of variation (CV) less than 6.3, 6.6 and 6.5% for THC in saliva, urine and blood, respectively, and 6.8 and 7.7% for THC-COOH in urine and blood, respectively. Day-to-day CVs were less than 3.5, 4.9 and 11.3% for THC in saliva, urine and blood, respectively, and 6.2 and 6.4% for THC-COOH in urine and blood, respectively. Limits of detection (LOD) were 2 ng/mL for THC in oral fluid and 0.5 ng/mL for THC and THC-COOH and 20 ng/mL for 11-OH THC, in urine and blood. Calibration curves showed a linear relationship for THC and THC-COOH in all samples (r² > 0.999) within the range investigated.The procedure presented here has high specificity, selectivity and sensitivity. It can be regarded as an alternative method to GC–MS for the confirmation of positive immunoassay test results, and can be used as a suitable analytical tool for the quantification of THC and THC-COOH in oral fluid, urine and/or blood samples.     

The Karimojong from Uganda: Genetic characterization using an X-STR decaplex system

The Karimojong, an African group from the Karamoja region of Northeast Uganda, were genetically analysed using a decaplex system for X chromosome short tandem repeats (X-STRs). A total of 255 individuals (117 males and 138 females) were genotyped for the following loci: DXS8378, DXS9898, DXS7133, GATA31E08, GATA172D05, DXS7423, DXS6809, DXS7132, DXS9902 and DXS6789. Allele frequencies and parameters for forensic evaluation were calculated for each STR. No association was found between any pairs of loci studied. DXS6789 was the most polymorphic marker in this sample, followed by DXS6809, with gene diversities of 84.79% and 83.94%, respectively. The less discriminating locus observed was DXS7133, with a gene diversity of 39.79%. High overall values of power of discrimination were obtained for female (1 in 1.8 × 10¹⁰) and male samples (1 in 1.6 × 10⁶), as well as high power of exclusion in father/mother/daughter trios (99.9997%), in father daughter duos (99.9862%) and in half sisters with same father (99.0331%). These results confirm the potential of this 10-plex in parentage testing and in human identification.     

Evaluation of the composition of street cocaine seized in two regions of Brazil

This work evaluates cocaine purity and the concentration ranges of adulterants and inorganic constituents for 31 street cocaine samples seized in two different regions of Brazil from July 2008 to May 2010. Cocaine and adulterants, such as caffeine, lidocaine and benzocaine, were quantified by Gas chromatography–mass spectrometry (GC–MS), and the inorganic constituents were determined by Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES) and ion chromatography (IC). The cocaine concentrations in the samples seized in the Amazonas state (AM samples) ranged from 154 to 978 mg g^? 1, and these samples did not contain any of the adulterants studied. The cocaine concentrations in the samples seized in the Minas Gerais state (MG samples) ranged from 63.9 to 753 mg g^? 1. Caffeine was the main adulterant found in 76% of the MG samples, ranging in concentration from 5.5 to 645.3 mg g^? 1. Lidocaine was found in 66.7% of the MG samples, with concentrations ranging from 16.3 to 576.7 mg g^? 1. Benzocaine was found in only one MG sample, at a concentration of 84.8 mg g^? 1. Fourteen elements were identified by ICP-OES, and a wide variation was observed in the concentrations of Ca, Mg, Na, P, Al, Fe, Mn and Zn. Pearson Product–moment Correlations between the analytes allowed the constituents to be associated with the chemicals used in the manufacturing of cocaine and with some common diluents. The study of the purity of cocaine and the presence and concentration of adulterants and inorganic constituents is important because the latter can have deleterious effects on health.     

Y-chromosome STR haplotypes in males from Greenland

A total of 272 males from Greenland were typed for 11 Y-chromosome STRs DYS19, DYS385a/b, DYS389-I, DYS389-II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438 and DYS439 with the PowerPlex^® Y System (Promega). A total of 146 different haplotypes were observed and the haplotype diversity was 0.9887. The number of haplotypes seen once was 108 and the most common haplotype was observed in 12 males. A significant F_ST value was observed (F_ST = 0.012, P < 0.00001) when comparing the population of 15 locations in Greenland assigned to 7 groups. The significance could mainly be attributed to the subpopulation of males from Tasiilaq (East of Greenland). The R_ST value was not statistically significant (R_ST = 0.016, P = 0.15).     

Determination of Nitrite in Whole Blood by High‐Performance Liquid Chromatography with Electrochemical Detection and a Case of Nitrite Poisoning

Although nitrite is widely used in meat processing, it is a major toxicity hazard to children and is responsible for the blue‐baby syndrome. A simple and effective method to determine nitrite in whole blood has been devised using ion chromatography with suppressed conductivity detection. The blood sample was deproteinized by adding acetonitrile and purified with mini‐cartridges to remove hydrophobic compounds, chloride ions, and metal ions. An aliquot of the filtrate was injected onto the ion chromatography. The retention time for nitrite was 13.8 min and the detection limit of nitrite in whole blood was 0.4 μmol/L. The calibration curve was linear (r² = 0.9999) over the concentration working range. The blood nitrite concentration of a victim who attempted suicide by ingesting sodium nitrite powder was determined using the present method. The basal levels for nitrite in human blood was determined with 7.1 ± 0.9 μmol/L (n = 12).     

Acidic and neutral drugs screen in blood with quantitation using microbore high-performance liquid chromatography–diode array detection and capillary gas chromatography–flame ionization detection

Blood previously acidified with aqueous saturated ammonium chloride solution was extracted with ethyl acetate. The dried extract was subjected to acetonitrile–hexane partition. The acetonitrile portion was analysed for the presence of acidic and neutral drugs by HPLC–DAD (200 mm×2.1 mm I.D. microbore ODS-Hypersil column) and GC–FID (25 m narrow-bore×0.25 mm I.D. HP-5 column with 0.33 μm film thickness). The protocol was found to be suitable for both clinical toxicology (including emergency toxicology) and postmortem toxicology. At least 66 drugs of interest were unequivocally identified by RRTs (HPLC) and UV spectra (DAD) match while another 12 were unequivocally identified by double RRTs match (HPLC and GC). Quantitation was facilitated by incorporating calibration blood standards in each assay batch. The five drugs most commonly encountered in clinical blood specimens (1150 cases) were: paracetamol (47.4% of the cases); chlormezanone (6.6%), theophylline (1.74%), naproxen (1.65%) and mefenamic acid (1.56%). The following drugs were detected in toxicologically significant quantities in postmortem blood specimens (245 cases): phenobarbitone (1.22% of the cases), naproxen (0.82%), chlormezanone (0.82%), theophylline (0.82%), carbamazepine (0.41%) and paracetamol (0.41%).     

Monitoring of Levamisole Concentration in Serum of Traffic Participants after Cocaine Consumption

This study highlights the problem of levamisole‐adulterated cocaine in context of active traffic participation. For the purposes of levamisole concentration monitoring in human serum, an analytical method based on LC‐MS/MS and solid‐phase extraction was applied. A Luna 5 μm C18 (2) 100 A, 150 mm × 2 mm column and a mobile phase consisting of A (H₂O/methanol = 95/5, v/v) and B (H₂O/methanol = 3/97, v/v), both with 10 mM ammonium acetate and with 0.1% acetic acid (pH = 3.2), were used. The validation experiments demonstrated that the method applied was appropriate for levamisole quantification in human serum. For 23% of levamisole‐positive samples, the concentrations exceeded 20 ng/mL. Therefore, the interaction of this drug with cocaine has to be considered as important for active traffic participation. As a consequence, monitoring of levamisole concentration in human serum is recommended, as long as it is used as cocaine adulterant.     

Determination of γ-hydroxybutyrate (GHB) and its precursors in blood and urine samples: A salting-out approach

γ-Hydroxybutyrate (GHB) is an increasingly popular drug of abuse that causes stimulation, euphoria, anxiolysis or hypnosis, depending on the dose used. Low doses of the drug are used recreationally, and also implicated in drug-facilitated sexual assaults. Because of the unusually steep dose–response curves, accidental GHB overdosing, leading to coma, seizures or death can occur. Being a controlled substance, GHB is often substituted with its non-scheduled precursors γ-butyrolactone (GBL) and 1,4-butanediol (BD), which are rapidly metabolized into GHB in the body. Here we describe an assay for GHB, GBL and BD in blood and/or urine samples. GHB and BD were extracted from diluted 200 μL aliquots of samples with t-butylmethylether (plus internal standard benzyl alcohol) in test tubes preloaded with NaCl. After acidification and centrifugation the solvent phase was transferred to a test tube preloaded with Na₂SO₄, incubated for 30 min, centrifuged again, and evaporated in vacuum. The residue was mixed with N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA) in acetonitrile, and injected into a GC–MS. When analyzing GBL, the salting-out step was omitted, and analysis was performed with a GC–FID apparatus. As revealed by the validation data this procedure is suitable for quantitative determination of GHB and its precursors in blood and/or urine samples.