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).     

Determination of Herbicides Paraquat,Glyphosate, and Aminomethylphosphonic Acid in Marijuana Samples by Capillary Electrophoresis

In this work, two methods were developed to determine herbicides paraquat, glyphosate, and aminomethylphosphonic acid (AMPA) in marijuana samples by capillary electrophoresis. For paraquat analysis, sample was extracted with aqueous acetic acid solution and analyzed by capillary zone electrophoresis with direct UV detection. The running electrolyte was 50 mmol/L phosphate buffer (pH 2.50). For glyphosate and AMPA, indirect UV/VIS detection was used, as these substances do not present chromophoric groups. Samples were extracted with 5 mmol/L hydrochloric acid. The running electrolyte was 10 mmol/L gallic acid, 6 mmol/L TRIS, and 0.1 mmol/L CTAB (pH = 4.7). The methods presented good linearity, precision, accuracy, and recovery. Paraquat was detected in 12 samples (n = 130), ranging from 0.01 to 25.1 mg/g. Three samples were positive for glyphosate (0.15–0.75 mg/g), and one sample presented AMPA as well. Experimental studies are suggested to evaluate the risks of these concentrations to marijuana user.     

Rapid Separation of Organophosphate Pesticides using Micellar Electrokinetic Chromatography and Short‐end Injection,

Organophosphate (OP) pesticides are highly toxic substances and are frequently represented as poisons. In order to qualify and quantify the selected OP pesticides (methyl paraoxon, ethyl paraoxon, methyl parathion, fenitrothion, and ethyl parathion), micellar electrokinetic chromatography and short‐end injection were investigated. This is the first time that this combination has been used to separate OP pesticides. A capillary with 8.5 cm effective length was used, and the analytes were separated within 2.1 min. Separation conditions including buffer (type, pH, and concentration), sodium dodecyl sulfate concentration, and separation voltage were optimized. The limit of detection (LOD) was estimated in the range of 10–20 μM. The OP pesticides spiked in artificial saliva and drinking water gave superior peak profiles, and good average recoveries 95.6% and 62.3%, respectively. Overall, a rapid method with excellent resolution and efficiency was developed and successfully applied in the analysis of potential sample matrixes.     

Evaluation of Total Nitrite Pattern Visualization as an Improved Method for Gunshot Residue Detection and its Application to Casework Samples

Visualization of nitrite residues is essential in gunshot distance determination. Current protocols for the detection of nitrites include, among other tests, the Modified Griess Test (MGT). This method is limited as nitrite residues are unstable in the environment and limited to partially burned gunpowder. Previous research demonstrated the ability of alkaline hydrolysis to convert nitrates to nitrites, allowing visualization of unburned gunpowder particles using the MGT. This is referred to as Total Nitrite Pattern Visualization (TNV). TNV techniques were modified and a study conducted to streamline the procedure outlined in the literature to maximize the efficacy of the TNV in casework, while reducing the required time from 1 h to 5 min, and enhancing effectiveness on blood‐soiled samples. The TNV method was found to provide significant improvement in the ability to detect significant nitrite residues, without sacrificing efficiency, that would allow for the determination of the muzzle‐to‐target distance.     

Analysis of nitrite in adulterated urine samples by capillary electrophoresis

A simple method for analyzing nitrite in urine has been developed to confirm and quantify the amount of nitrite in potentially adulterated urine samples. The method involved separation of nitrite by capillary electrophoresis and direct UV detection at 214 nm. Separation was performed using a bare fused silica capillary and a 25 mM phosphate run buffer at a pH of 7.5. Sample preparation consisted of diluting the urine samples 1:20 with run buffer and internal standard, and centrifuging for 5 min at 2500 rpm. The sample was hydrodynamically injected, then separated using -25 kV with the column maintained at 35 degrees C. The method had upper and lower limits of linearity of 1500 and 80 microg/mL nitrite, respectively, and a limit of detection of 20 microg/mL. The method was evaluated using the National Committee for Clinical Laboratory Standards (NCCLS) protocol (Document EP10-A2), and validated using controls, standards, and authentic urine samples. Ten anions, ClO-, CrO4(-2), NO3-, HCO3-, I-, CH3COO-, F-, SO4-, S2O8(-2), and Cl-, were tested for potential interference with the assay. Interferences with quantitation were noted for only CrO4(-2) and S2O8(-2). High concentrations of Cl- interfered with the chromatography. The method had acceptable accuracy, precision, and specificity.     

毛细管电泳高频电导法检测饮料中γ-羟基丁酸

目的建立饮料中γ-羟基丁酸（GHB）的毛细管电泳高频电导法检测分析方法。方法样品经稀释后直接进样,采用反向分离模式,以0.5mmol/LNa3PO4＋1.5mmol/LNa2HPO4＋0.1mmol/LCTAB为电泳分离缓冲介质,分离电压为16kV,进样时间为20s进行电泳分离及高频电导检测。结果该实验条件下,6.5min内可实现样品快速分析。GHB在10.0～150μg/mL范围内线性关系良好,r=0.990,检出限为3.0μg/mL（S/N=3）。选择的3种饮料样本不同添加浓度日间和日内RSD均小于6%,回收率均在95%以上。结论采用本文方法,样品处理简单,方法快速、灵敏,操作方便,可作为饮料中GHB的一种快速筛选法在相关检测中选用。     

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.     

Radiodense bullet wipe around osseous entrance gunshot wounds

“Bullet wipe” is the material deposited by a bullet on any surface with which it comes into contact after it is fired and may contain debris from the gun barrel, including particles of primer and metal fragments from previously fired bullets. X‐ray analysis is a non‐destructive method by which traces of metallic elements can be visually detected. The analysis of osseous defects for radiodense bullet wipe (RBW) assists in determining the presence or absence of perforating gunshot wounds, especially in fragmented, skeletonized remains. The aim of our current study was to determine the frequency of RBW around entrance firearms injuries that perforated bone. We prospectively analyzed entrance gunshot wounds for RBW over a three‐year period using digital X‐ray analysis (n = 59). We retrospectively reviewed the corresponding autopsy reports to determine the frequency of RBW by biologic sex, reported ancestry, age‐at‐death, location of wound, manner of death, range of fire, bullet caliber, and presence of bullet jacket. Data were analyzed by Fisher's exact test or Chi‐square test with significance levels accepted at p < 0.05. RBW was present in 66% (n = 39) of examined cases. Decedent characteristics did not significantly alter RBW distribution, including biologic sex (p = 0.75), reported ancestry (p = 0.49), and age‐at‐death (p = 0.43). Additionally, the location of the osseous entrance gunshot wound, manner of death, range of fire, and cartridge caliber did not affect RBW detection. All cases involving non‐jacketed rounds (n = 5) showed RBW (p = 0.30). To our knowledge, this study is the first to report the frequency of RBW detection from osseous entrance gunshot wounds.     

Optimization of Headspace Solid‐Phase Microextraction Technique for Extraction of Volatile Smokeless Powder Compounds in Forensic Applications

Smokeless powders are low explosives and are potentially found in cases involving firearms and improvised explosive devices. Apart from inorganic compound analysis, forensic determination of organic components of these materials appears as a promising alternative, especially the chromatographic techniques. This work describes the optimization of a solid‐phase microextraction technique using an 85 μm polyacrylate fiber followed by gas chromatography‐flame ionization detection for smokeless powder. A multivariate experimental design was performed to optimize extraction‐influencing parameters. A 2⁴ factorial first‐order design revealed that sample temperature and extraction time were the major influencing parameters. Doehlert matrix design has subsequently selected 66°C and 21 min as the compromised conditions for the two predetermined parameters. This extraction technique has successfully detected the headspace compounds of smokeless powders from different ammunition types and allowed for their differentiation. The novel technique allows more rapid sample preparation for chromatographic detection of smokeless powders.     

Blowfly Larval Tissues as a Secondary Detector for Determining Paraquat-Related Death in Rabbit Carcass

Paraquat poisoning is commonly associated with suicide or homicide in Malaysia. In a case involving advanced body decomposition, pathological analysis regarding the cause of death may become difficult or almost impossible. Insects serve as common alternative matrix for poison detection in forensic analysis. Paraquat detection via secondary bioaccumulation in fly larvae tissue has never been reported. In this study, tissues from blowfly larvae collected from a rabbit carcass with paraquat poisoning were analyzed for secondary bioaccumulation. Larvae samples were collected and analyzed using liquid–liquid extraction. The detection was performed via reduction of quaternary ammonium presence in paraquat and analyzed using gas chromatography–mass spectrometry (GC-MS) with selected ion monitoring mode (SIM mode). GC-MS showed the elution of reduced paraquat was at retention time 12.8 min. Blowfly larvae tissue has proven useful as a secondary detector in paraquat-related deaths.     

Study of the Behaviors of Gunshot Residues from Spent Cartridges by Headspace Solid‐Phase Microextraction–Gas Chromatographic Techniques

Gunshot residues, produced after shooting activity, have acquired their importance in analysis due to the notoriety of firearms‐related crimes. In this study, solid‐phase microextraction was performed to extract the headspace composition of spent cartridges using 85‐μm polyacrylate fiber at 66°C for 21 min. Organic compounds, that is, naphthalene, 2,6‐dinitrotoluene, 2,4‐dinitrotoluene, diphenylamine, and dibutyl phthalate were detected and analyzed by gas chromatography‐flame ionization detection technique. Evaluation of chromatograms for diphenylamine, dibutyl phthalate, and naphthalene indicates the period after a gunshot was discharged, whether it was 1 days, 2–4 days, <5 days, 10 days, 20 days, or more than 30 days ago. This study revealed the potential effects of environmental factors such as occasional wind blow and direct sunlight on the estimation of time after spent cartridges were discharged. In conclusion, we proposed reliable alternative in analyzing the headspace composition of spent cartridges in a simulated crime scene.     

The Effect of Skin Debris on Gunshot Residue Sampling and Detection

These experiments were designed to determine whether skin debris (desquamated epithelial cells and apparent skin oils) affects gunshot residue (GSR) particle detection on the sticky tape lift samples prepared for scanning electron microscopy (SEM). A dabbing experiment showed that GSR particles accumulate not only on the adhesive surface of the sampler, but also on the epithelial cell surfaces. Samplers were loaded with target GSR followed by dabbing 30 times on the back of a hand. Backscatter electron images were taken at 20 kV and for some at 30 kV of the same areas. The samplers were then treated with a sodium/calcium hypochlorite solution (bleach) to remove skin debris and again imaged in the SEM. Comparison of these images shows more GSR particles will likely be revealed at 30 kV than 20 kV and more particles revealed by the bleach treatment in an automated SEM system.     

离子色谱法检测全血中的亚硝酸盐及其代谢产物

目的建立全血中亚硝酸盐的离子色谱分析方法。方法将0.5mL全血和1mL水混合,用乙腈沉淀血液中的蛋白质后依次过C18柱、Ag柱和Na柱,用于去除其中的有机物和氯离子后进行离子色谱检测,并对蛋白沉淀溶剂、前处理柱等最佳实验条件进行考察。结果采用本文方法对空白添加全血进行检测,在色谱图上亚硝酸根离子及其氧化产物硝酸根离子的保留时间分别为10.02min、19.21min。选择乙腈作为蛋白沉淀剂,C18柱去除有机物,Ag柱去除氯离子,Na柱去除银离子。全血中亚硝酸根离子的检出限为0.05μg/mL,亚硝酸根和硝酸根的总回收率为95.9%~117.3%。结论本文方法简便,回收率好,灵敏度高,适用于中毒者血液中亚硝酸盐的检测。     

A Study of Background Levels of Antimony,Barium, and Lead on Vehicle Surface Samples by Graphite Furnace Atomic Absorption

Law enforcement agencies routinely sample for gunshot residue (GSR) by bulk techniques and often submit swabs taken from other surfaces besides the hands of the suspect shooter. This study aims to establish the prevalence of antimony, barium, and lead on normally handled automobile surfaces by graphite furnace atomic absorption analysis. No positives were determined on 50 sampled automobile surfaces above cutoff (positive) from background levels. Transfers of GSR particles from shooter hands to automobile surfaces were found to potentially allow for positive GSR determinations, but such transfers seem to be dependent on the shooting conditions and length of GSR exposure. We determined that our bulk analysis method yields an overall 87.94 ± 5.52% extraction efficiency from cotton swabs, while the LOQ determinations strengthening the fact that bulk analysis methods are valid and valuable tools for GSR investigations.     

The Survival of Gunshot Residues in Cremated Bone: An Inductively Coupled Plasma Optical Emission Spectrometry Study

Gunshot residue (GSR) has been sought and demonstrated on many types of material and with many techniques. Inductively coupled plasma optical emission spectrometry (ICP‐OES) could be a useful method on difficult substrates, but a systematic study on burnt material has never been performed. Hence, this study aims at evaluating the usefulness and reliability of this method on burnt samples. Sixteen adult bovine ribs (eight with soft tissues, eight totally skeletonized) were shot using two kinds of projectile (both 9 mm full metal‐jacketed or unjacketed). Then, every sample was led to complete calcination in an electric oven. The area of the gunshot entrance wound was swabbed and analyzed by ICP‐OES; the results were also correlated with a previously published parallel study by scanning electron microscopy (SEM) equipped with an SEM–energy dispersive X‐ray analyzer. ICP‐OES proved to be very sensitive and reliable even on degraded material and can be an appropriate nondestructive method for detecting residues on difficult and delicate substrates such as burnt bone.     

Detection of Gunshot Primer Residue on Bone in an Experimental Setting—An Unexpected Finding*

Abstract: Pork ribs with intact muscle tissue were used in an experimental attempt to identify bullet wipe on bone at distances from 1 to 6 feet with 0.45 caliber, full metal jacket ammunition. This resulted in the unexpected finding of primer‐derived gunshot residue (GSR) deep within the wound tract. Of significance is the fact that the GSR was deposited on the bone, under the periosteum, after the bullet passed through a Ziploc^® bag and c. 1 inch of muscle tissue. It is also important to note that the GSR persisted on the bone after the periosteum was forcibly removed. The presence of primer‐derived GSR on bone provides the potential to differentiate gunshot trauma from blunt trauma when the bone presents an atypical gunshot wound. In this study, the presence of gunshot primer residue at a distance of 6 feet demonstrates the potential for establishing maximum gun‐to‐target distance for remote shootings.     

LC/MS分析血浆中丁丙诺啡

目的建立血浆中丁丙诺啡液相色谱/质谱（LC/MS）分析方法。方法在含有丁丙诺啡的血浆中,加入内标奋乃静,加pH10.8缓冲溶液,用401有机担体作吸附剂、三氯甲烷作洗脱剂固相萃取,N2挥干,用50μL流动相定容后进行LC/MS分析。色谱条件：Thermo Hypersil-HyPURITY C18（150×2.1mm,5μm）,柱温：40℃,流动相：10mmol/LNH4AC（pH3.4）∶甲醇∶乙腈=36∶52∶12,流速：0.22mL/min。结果方法的线性范围为0.05～5.0μg/L（r=0.9998）,定量限0.05μg/L,检出限0.01μg/L（S/N=3）;3个浓度的质量控制样品（0.1μg/L,0.5μg/L,2.0μg/L）平均回收率分别为86.40%,92.72%,92.57%,RSD分别为4.51%,3.34%,2.09%。结论该方法操作简便、灵敏度高,可用于涉毒案件血浆中丁丙诺啡的分析。     

Detection of Gunshot Residue on Dark‐Colored Clothing Prior to Chemical Analysis

Detection of gunshot residue (GSR) is an arduous task for investigators. It is often accomplished with chemical tests, which can reveal elements and ions indicating the presence of GSR, but are likely to cause physical alteration to the pattern. In this study, the Spex Forensics Mini‐CrimeScope MCS 400, along with 16 accompanying wavelength filters, was applied to various GSR patterns and target types. Three dark shirt materials, four ammunition calibers, and eight ammunition manufacturers, along with the primer residue of the different manufacturer ammunitions were tested. Results indicate the alternate light source wavelength of 445 nm to be the optimal setting. In addition, target material plays a large role in the preservation of GSR patterns as particles burn. Furthermore, it can be extrapolated that residue, observed from a full round and firing distance of six inches, is mostly composed of unburnt gunpowder residue, not primer residue.     

Use of dynamically coated capillaries for the routine analysis of methamphetamine, amphetamine, MDA, MDMA, MDEA, and cocaine using capillary electrophoresis

A rapid, accurate, precise, reproducible, economical, and environmentally gentle method using capillary electrophoresis (CE) is presented for the routine analysis of methamphetamine, amphetamine, MDA, MDMA, MDEA, and cocaine in seized drugs. The methodology uses a 32 cm by 50 microm capillary (length to detector 23.5 cm) with a commercially available buffer kit and diode array UV detection. Dynamic coating of the capillary surface is accomplished by flushing with base for 1 min, a proprietary polycation for 1 min, and then a proprietary polyanion for 2 min. This approach provides a relatively high and stable electroosmotic flow (EOF), even at low pHs. The background electrolyte (BGE) contains 75 mM phosphate buffer (pH 2.5) with the same polyanion as above. Using this methodology, amphetamine, methamphetamine, MDA, MDMA, MDEA, and an internal standard (n-butylamphetamine) are baseline resolved in less than 5 min. The run-to-run migration time %RSDs and peak area %RSDs are typically <0.3% and <2.1%, respectively. The day-to-day and capillary-to-capillary migration time %RSDs are <1.5% and <2.1%, respectively. The %RSDs of the relative migration times compared with the internal standard on a day-to-day and capillary-to-capillary basis are <0.2% and <0.06%, respectively. The linear dynamic range using peak areas range from 0.003 to 0.10 mg/mL. The correlation coefficients are >0.9998, with all calibration curves passing at or near the origin. Similar data are obtained for cocaine and its internal standard henyltoloxamine. None of the compounds usually encountered in illicit samples interfere with the target compound (e.g., methamphetamine and cocaine) or the internal standard. Quantitative results for synthetic mixtures and seized exhibits are in good agreement with actual values, and also with results obtained from other techniques. The relatively high EOF for the dynamically coated capillary system allows for the screening of basic, acidic, and neutral adulterants in drug seizures; identification is facilitated by the use of automated UV library searches.     

The Erratic Behavior of Lesions in Burnt Bone

This study analyses depressed fractures (by blunt force trauma) and circular full‐thickness injuries (drill injuries and gunshot wounds) in charred bones. Fifty bovine ribs (total 104 lesions) were divided into three groups. The first group consisted in 20 depressed hammer‐produced fractures; in the second one, 60 round drill‐holes were produced (30 circular, 30 semicircular); in the third group, 12 fleshed and 12 skeletonized ribs were hit by 9‐mm bullets. Each specimen was carbonized in an electric oven up to 800°C. Morphological and metric analyses were performed before and after: morphological features were preserved, but depressed fractures showed an increase in their dimensions (p‐value < 0.05); the drilled holes shrunk (p‐value < 0.01); the charring cycle increased the number of fractures in samples with gunshot wounds differently in fleshed and defleshed ribs. This study showed the complex behavior of charred bone, for what concerns the interpretation of trauma and how caution should be applied.