In this paper, we demonstrate a rapid analysis procedure for use with a small set of rapidly mutating Y chromosomal short tandem repeat (Y-STR) loci that combines both rapid polymerase chain reaction (PCR) and microfluidic separation elements. The procedure involves a high-speed polymerase and a rapid cycling protocol to permit PCR amplification in 16 min. The resultant amplified sample is next analysed using a short 1.8-cm microfluidic electrophoresis system that permits a four-locus Y-STR genotype to be produced in 80 s. The entire procedure takes less than 25 min from sample collection to result. This paper describes the rapid amplification protocol as well as studies of the reproducibility and sensitivity of the procedure and its optimisation. The amplification process utilises a small high-speed thermocycler, microfluidic device and compact laptop, making it portable and potentially useful for rapid, inexpensive on-site genotyping. The four loci used for the multiplex were selected due to their rapid mutation rates and should proved useful in preliminary screening of samples and suspects. Overall, this technique provides a method for rapid sample screening of suspect and crime scene samples in forensic casework.

•The analysis of 4 similar cannabinoids is performed by surface enhanced Raman spectroscopy.•The analysis is performed by mixing gold nanoparticles with the sample and a magnesium chloride aggregating agent.•Support liquid extraction is used for urine extraction permitting a rapid and specific determination.Synthetic cannabinoids (K2, spice) present problems in forensic investigations because standard presumptive methods, such as immunoassays, are insufficiently specific for the wide range of potential target compounds. This issue can lead to problems with low sensitivity and yield false negative results. A potential solution to this problem is surface enhanced Raman spectroscopy (SERS).In this study we demonstrate the analysis of a set of structurally similar synthetic cannabinoids using SERS. The procedure involves mixing the analyte with gold nanoparticles prepared in a solution containing alkali or alkaline earth salt solutions. The salts produce aggregation of the nanoparticles with a resultant spectral enhancement due to the formation of spectral hotspots with enhanced field effects within the aggregate. Among the salts tested, 0.0167 M MgCl2 produced the lowest limit of detection and best overall sensitivity. The method produces clearly distinguishable spectra for each synthetic cannabinoid with detection limits as low as 18 ng/mL. Spiked urine samples were also analyzed following a cleanup procedure involving support liquid extraction. When using a portable Raman system, a higher concentration of MgCl2 was needed to produce similar a LOD. The results demonstrate that this procedure has great potential as a method for presumptive screening of synthetic cannabinoids.

•Single- and dual-locus age-predictors for saliva were developed from CpG sites in KLF14 and SCGN using a multivariate linear regression analysis.•The single-locus model explained 85% of age variance with a MAD of 5.8 years and the dual-locus model explained 84% of age variance with a MAD of 6.2 years in the training set.•The single-locus model predicted age (±8 years) with accuracy of 78.9% for younger subjects (≤40 years) and a MAD of 5.1 years in the validation set.•The age predictor for blood was based on CpG sites in SCGN and KLF14 and accounted for 71% of age variance with a MAD of 6.6 years in the training set.Recent developments in the analysis of epigenetic DNA methylation patterns have demonstrated that certain genetic loci show a linear correlation with chronological age. It is the goal of this study to identify a new set of epigenetic methylation markers for the forensic estimation of human age. A total number of 27 CpG sites at three genetic loci, SCGN, DLX5 and KLF14, were examined to evaluate the correlation of their methylation status with age. These sites were evaluated using 72 blood samples and 91 saliva samples collected from volunteers with ages ranging from 5 to 73 years. DNA was bisulfite modified followed by PCR amplification and pyrosequencing to determine the level of DNA methylation at each CpG site. In this study, certain CpG sites in SCGN and KLF14 loci showed methylation levels that were correlated with chronological age, however, the tested CpG sites in DLX5 did not show a correlation with age.Using a 52-saliva sample training set, two age-predictor models were developed by means of a multivariate linear regression analysis for age prediction. The two models performed similarly with a single-locus model explaining 85% of the age variance at a mean absolute deviation of 5.8 years and a dual-locus model explaining 84% of the age variance with a mean absolute deviation of 6.2 years. In the validation set, the mean absolute deviation was measured to be 8.0 years and 7.1 years for the single- and dual-locus model, respectively. Another age predictor model was also developed using a 40-blood sample training set that accounted for 71% of the age variance. This model gave a mean absolute deviation of 6.6 years for the training set and 10.3 years for the validation set. The results indicate that specific CpGs in SCGN and KLF14 can be used as potential epigenetic markers to estimate age using saliva and blood specimens. These epigenetic markers could provide important information in cases where the determination of a suspect’s age is critical in developing investigative leads.

•Copper ion carry-over during sample purification affects the DNA typing process.•Aggregation, charge effects, and conformational changes affect amplification and/or electrophoretic mobility.•Solid phase sample purification decreases copper carryover to subsequent steps.Copper wires are a common component of improvised explosive devices (IEDs) and are increasingly found in forensic evidence. When processed for DNA, ions from the metal can be co-extracted with the genetic material, and potentially cause complications during the analysis procedure. It was determined in this project that copper ions do have adverse effects on DNA processing by either interacting directly with the DNA or affecting the electrophoretic mobility of the molecule. It was also found that these negative effects could be improved by utilizing a purification method capable of reducing the amount of copper carry-over into the sample.This diagram illustrates the effects of a copper containing sample on the DNA processing workflow. During the purification process, copper ions interact with the nucleic acid causing aggregation and conformational changes which can affect electrophoretic conductivity. The end result is usually an inhibited profile.Download high-res image (109KB)Download full-size image

•Separation and identification of four microcystin (MC) variants commonly found in aquatic environments.•Separation by capillary electrophoresis with UV absorbance and time-of-flight mass spectrometry.•Study of the effect of cyclodextrin and pH on the separation.•Analysis of environmental samples.This paper demonstrates a method for the rapid separation and identification of four microcystin (MC) variants commonly found in aquatic environments. The procedure utilizes capillary electrophoresis (CE) coupled to UV absorbance and time-of-flight mass spectrometric (TOF–MS) detectors. All four analytes were effectively separated within 6 min using phosphate buffer in 50-μm ID capillaries with an applied electric field of 400 V/cm. The separation of the individual compounds was optimized through the adjustment of buffer, pH, and β-cyclodextrin content. Ultimately it was determined that, at a sufficiently high pH, all 4 compounds could be separated without the need for added cyclodextrins. The results provided accurate molecular information, assisting in the determination of compound identity. The method was then applied to environmental samples using solid phase extraction for isolation and pre-concentration. The results were comparable to those obtained by LC/MS, but with a shorter run time and lower sample and eluent consumption.

Correction for ‘The development of paper microfluidic devices for presumptive drug detection’ by Giacomo Musile et al., Anal. Methods, 2015, DOI: 10.1039/c5ay01432h.

A paper microfluidic device has been developed for the presumptive testing of seized drugs in forensic casework. The procedure involves creating hydrophilic channels on chromatographic paper using wax printing and thermal lamination. The channels are connected to a single stem that draws an unknown analyte solution up into 6 different lanes. A different colorimetric reaction occurs within each lane, permitting the multiplexed detection of a variety of different compounds, including cocaine, opiates, ketamine, and various phenethyl amines. Furthermore, the linear orientation of the lanes permits series of reactants to be placed in each channel, enhancing stability and permitting sequential interaction with the analyte as the solvent front passes through each individual reagent. The resultant device was characterized for sensitivity and tested with a variety of common interferences and drug diluents. It should prove a useful device for screening seized drugs.

In this paper the development of microfluidic paper-based analytical devices (μPADs) is described for rapid, on-site detection of improvised explosives. Five lane μPADs were designed and printed using wax ink on chromatography paper to create hydrophobic channels. Each channel contains colorimetric reagents capable of reacting with one or more explosive compounds resulting in a specific colorimetric reaction. Two devices were prepared, each capable of performing five simultaneous analyses on a single μPAD. The first μPAD was developed to detect inorganic explosives such as black powder, flash powder, and ammonium nitrate. It detects nitrate, nitrite, chlorate, and perchlorate oxidizers, as well as ammonium. The second μPAD was developed to detect military explosives such as TNT and RDX along with other high explosives like urea nitrate. It also detects organic peroxides such as TATP and its precursor hydrogen peroxide. All experiments were performed by dissolving the explosives in deionized water or 50:50 acetone/H2O as transport solvents with a detection time of around 5 minutes. Detection limits ranged from 0.39–19.8 μg of explosive compound. These two customized μPAD devices permit the on-site forensic testing of unknown explosives, thereby supplying law enforcement and military personnel with a resource for fast, easy detection of military, commercial, and homemade explosive components at low cost.

Benzodiazepines are among the most prescribed compounds and are commonly present in many toxicological screens. They are also of concern forensically in cases of drug facilitated sexual assault. Currently these compounds are predominantly analyzed using immunoassay techniques; however more specific screening methods are needed. This paper demonstrates the applicability of surface enhanced Raman spectroscopy as a method for the analysis and detection of benzodiazepines. The procedure involves mixing urine extracts with gold nanoparticles and appropriate aggregating agents for trace detection of these compounds and their metabolites. In this paper we will discuss the optimization of various parameters of this technique as well as its application to screening urine samples. Eleven different benzodiazepines and metabolites were examined, including 1,2-triazolo-benzodiazepines and 1,4-benzodiazpines. Experiments were performed using four different chloride salts, MgCl2, CaCl2, KCl, and NaCl, as aggregating agents for the colloidal gold nanoparticles. Overall it was found that each aggregating agent produced different levels of signal enhancement for each drug. MgCl2 provided the lowest limit of detection at 2.5 ng mL−1, and linearity over a wide range of concentrations for a variety of drugs chosen. It was also determined that the optimum MgCl2 concentration was 1.67 M. This method has shown the applicability of SERS for the detection of trace quantities of benzodiazepines in aqueous solutions as well as the optimization of the technique over a wide range of compounds. This technique can be utilized in the detection of trace benzodiazepines in toxicological samples following extraction of the analyte.

•• Detection of urea nitrate and ammonium as ion pairs in non-aqueous solvents by ESI MS.• Specific adduct ions were detected for each explosive.• The procedure can be used to detect fertilizer based explosives on surfaces.In this study methods for the detection of trace levels of the improvised explosives urea nitrate and ammonium nitrate were developed using electrospray ionization with infusion. By using a non-aqueous solvent mixture containing 95% acetone with 5% 2-methoxyethanol we were able to preserve the urea and ammonium nitrate ion pairs and discriminate between these and other similar salts. Negative ion electrospray ionization was used for urea nitrate detection and positive ion electrospray ionization was used for ammonium nitrate. Two specific adduct ions were detected for each explosive with ammonium nitrate producing m/z 178 [2AN+NH4]+ and m/z 258 ions [3AN+NH4]+ while urea nitrate produced m/z 185 [UN+NO3]− and m/z 248 [UN+HNO3+NO3]− The specificity of the analysis was examined by mixing the different explosives with various salts and interferents. Adduct ions formed in the gas phase were found to be useful in distinguishing between ion pairs and mixed salts. Overall the method demonstrates the sensitive detection of both explosives, and more specifically the potential to determine intact urea nitrate.

This paper details a method for the separation and identification of fourteen organic compounds commonly found as constituents in commercial smokeless powders using a hexyl acrylate-based porous monolith. Capillary electrochromatography (CEC) coupled to UV and time of flight-mass spectrometry (TOF-MS) methods were both explored. The CEC–UV method provides an effective and efficient method for the detection of all components in the additive package of the powder. The TOF-MS procedure provides better sensitivity and selectivity allowing an additional confirmation of the presence of the subset of those compounds, which are detectable via positive and/or negative ion electrospray ionization mass spectrometry. Both methods were used for the analysis of smokeless powder components in a mixed standard as well as in the determination of the composition of the additive package of individual powders.Highlights► The analysis of smokeless powder additives using CEC–UV and CEC/MS. ► The technique utilizes in-capillary synthesized acrylic monoliths. ► Detection is by UV and time of flight mass spectrometry. ► The mass spectrometry utilizes positive and negative ESI modes and exact mass measurements.

In this paper we have adapted a technique, previously used to determine the presence of urea in aqueous samples of wine and urine, to detect trace levels of urea nitrate explosives. The procedure involves the reaction of the uronium ion (protonated urea) with a fluorophore, xanthydrol. By modification of the procedure to utilize non-aqueous reagents, in neutral conditions, it can be made specific to the presence of the urea nitrate ion pair. The procedure includes selective detection of derivatization products by UV and fluorescence following separation by High-Performance Liquid Chromatography (HPLC). Analytical method development included optimization of HPLC conditions (solvent, gradient), UV and fluorescence wavelengths, and derivatization parameters (xanthydrol amount, reaction times, temperature). The extraction of urea nitrate from surfaces was also investigated and optimized. For best quantification, it was shown that an internal standard was required; this resulted in a quantification limit around 0.17 mM (21 mg/L). The entire procedure could be performed in less than 30 min per sample and potential interferences such as ammonium, nitrate, and urea did not produce a response under standard conditions.

Recently there has been concern regarding the use of flunitrazepam and other low-dose benzodiazepines in drug-facilitated sexual assault. These compounds are placed in drinks of unsuspecting victims and produce a sedative effect with anterorgrade amnesia. Chip-based microfluidic systems can provide a quick and disposable procedure for the detection of flunitrazepam and other nitrated benzodiazepines used in these crimes. This paper describes the application of indirect quenching of cyanine dye (Cy5) for detection of nitrated benzodiazepines. The separation is performed on a microfluidic device with a separation channel 8 cm long and 50 μm wide and utilizes indirect fluorescence detection with 635 nm laser excitation. The optimization of the separation using micellar electrokinetic chromatography with organic modifiers is described. A borate buffer containing 2.6 μM Cy5 dye, 15 mM sodium dodecyl sulphate (SDS) and 20% methanol is used. Complete separation of four target drugs occurs in under 2 min with limits of detection in the low μg/ml range. Overall the method provides a rapid and simple analysis for the presence of nitrated benzodiazepines in beverages and other similar preparations.

A novel electrolyte has been developed for the simultaneous separation of cations and anions in low explosive residue by capillary electrophoresis. This electrolyte contains 15 mM α-hydroxyisobutyric acid (HIBA) as the buffer, 6 mM imidazole as the cation chromophore, 3 mM 1,3,6-naphthalenetrisulfonic acid (NTS) as the anion chromophore, 4 mM 18-crown-6 ether as a cation selectivity modifier, and 5% (v/v) acetonitrile as an organic modifier. The pH was adjusted to 6.5 using tetramethylammonium hydroxide (TMAOH), an electroosmotic flow modifier. The method was optimized by varying the concentrations of α-HIBA, imidazole, and 1,3,6-NTS at three different pH values. The results provided a simultaneous indirect photometric analysis of both anions and cations with detection limits ranging from 0.5 to 5 ppm for anions and from 10 to 15 ppm for cations with a total run time of under 7 min. The method was then applied to the analysis of Pyrodex® RS and black powder, as well as several smokeless powders. The results obtained were consistent with previously reported results for separate anion and cation analysis and provide a faster, more complete analysis of each sample in a single chromatographic run.

PCR inhibitors can originate from a variety of sources and can co-extract with the DNA template, resulting in reduced amplification and/or dropped alleles. Currently real time PCR is used to provide a check for the presence of PCR inhibition by monitoring the quality of amplification of an internal control. In this paper we examine the effect of internal control length and sequences on its sensitivity to PCR inhibition by varying concentrations of commonly encountered PCR inhibitors. Data from both amplification and melt curves were evaluated. The results show that while amplicon sequence has minor effects on amplification efficiency and melt curves, amplicon length has a more dramatic effect, regardless of inhibitor type. Given the increasing variety of STR typing kits and their documented differences in performance with respect to inhibition, the data obtained in this study can be used to assist designers of real time PCR kits to adjust their internal PCR controls (IPC) to permit a more targeted estimation of inhibition.

The goal of this paper was to examine and compare two different commercially available approaches to the determination of the relative quantities of autosomal and Y chromosomal DNA using real-time PCR. One, Quantifiler® Duo, utilizes a TaqMan® assay with single copy probes for both autosomal human and Y quantification. The other method, Plexor HY® utilizes a primer quenching assay with multi-copy probes for its quantification of autosomal human and Y chromosomal DNA. To test these approaches we have utilized the NIST Human DNA Quantitation Standard Reference Material 2372, a set of three different NIST human DNA quantification standards, to examine the precision, accuracy and sensitivity of the real-time PCR assays. We also examined data from both systems utilizing casework samples. The results show that both systems produced linear estimates for DNA quantity over a broad range of input DNA. However we did observe some apparent copy number effects when comparing the three different NIST standards which we attributed to issues with sequence variations in the different standards. Overall, the single copy approach provided better accuracy while the multi-copy approach produced better sensitivity. Thus the choice of which system to use should depend upon the goals of the user.

•A developmental validation study was performed for epigenetic body fluid identification.•The procedure involves bisulfite modification, PCR and pyrosequencing.•The lowest concentration consistently detected varied from 0.1 to 10 ng.•The effect of degradation, inhibition and mixtures were determined.Determining the type and origin of body fluids in a forensic investigation can provide important assistance in reconstructing crime scenes. A set of epigenetic markers, ZC3H12D, BCAS4 and cg06379435, have been developed to produce unique and specific patterns of DNA methylation that can be used to identify semen, saliva, and blood, respectively. To ensure the efficacy of these markers, developmental validation studies were performed to determine the conditions and limitations of this new tool for forensic analysis. DNA was extracted from human samples and bisulfite modified using commercial bisulfite modification kits. Specific primers were used to amplify the region of interest and the methylation profile of the CpG sites were determined by pyrosequencing. The percent methylation values at each CpG site were determined in multiple samples and averaged for each tissue type. The versatility of these new markers is presented by showing the results of validation studies on sensitivity, human specificity, stability and mixture resolution. When testing the markers using different organisms, we did obtain positive results for certain non-human primate samples, however, all other tested species were negative. The lowest concentration consistently detected varied from 0.1 to 10 ng, depending on the locus, indicating the importance of primer design and sequence in the assay. The method also proved to be effective when inhibitors were present in the samples or when samples were degraded by heat. Simulated case- samples were also tested. In the case of mixtures of different cell types, the overall methylation values varied in a consistent and predictable manner when multiple cell types were present in the same sample. Overall, the validation studies demonstrate the robustness and effectiveness of this new tool for body fluid identification.

A paper microfluidic device has been developed for the presumptive testing of seized drugs in forensic casework. The procedure involves creating hydrophilic channels on chromatographic paper using wax printing and thermal lamination. The channels are connected to a single stem that draws an unknown analyte solution up into 6 different lanes. A different colorimetric reaction occurs within each lane, permitting the multiplexed detection of a variety of different compounds, including cocaine, opiates, ketamine, and various phenethyl amines. Furthermore, the linear orientation of the lanes permits series of reactants to be placed in each channel, enhancing stability and permitting sequential interaction with the analyte as the solvent front passes through each individual reagent. The resultant device was characterized for sensitivity and tested with a variety of common interferences and drug diluents. It should prove a useful device for screening seized drugs.

Correction for ‘The development of paper microfluidic devices for presumptive drug detection’ by Giacomo Musile et al., Anal. Methods, 2015, DOI: 10.1039/c5ay01432h.