A case of fatal overdose involving both hydromorphone and kratom

Kratom is a plant originating in Southeast Asia that has been used for its dose-dependent stimulant and opioid effects. The main active compound in kratom is mitragynine, an alkaloid with affinity for the mu-opioid receptor. Toxicity and fatalities related to kratom use have increased substantially in recent years. In this case report, we describe a 44-year-old man who was found deceased in bed. The only significant finding at autopsy was abdominal distension with >4 L of ascites. Toxicology testing was performed on femoral blood which showed 79 ng/mL of hydromorphone, 560 ng/mL of mitragynine, and 240 ng/mL of olanzapine. In addition, creatinine and urea in vitreous humor were significantly elevated, consistent with renal impairment. Death was attributed to hydromorphone toxicity with mitragynine being a contributing factor.     

Identification of Opioids and Related Substances using Handheld Raman Spectrometers

This study describes the performance of handheld Raman devices for detecting one hundred opioids and related substances including fentanyl and several analogs. Using a single “parent” device, signatures (spectra) with excellent signal-to-noise ratios were generated using <5 mg of most compounds. The signatures were added to a method (library), which was electronically transferred to three “daughter” devices. The devices were able to discriminate different salt forms and isomers. On average, the daughter devices yielded a true-positive rate of 97.3% for generating an alarm for opioids and were 93.3% effective for correctly identifying the opioid. The devices yielded true-negative, false-positive and false-negative rates of 100%, 0%, and 2.7%, respectively, where false negatives were due to weak signal and fluorescence. These data demonstrate that the parent-daughter electronic transfer method was successful and effective, which permits the ability to develop methods in the laboratory that can be seamlessly pushed out to field devices.     

Infrared Spectra of U.S. Automobile Original Finishes (Post – 1989). VIII: In Situ Identification of Bismuth Vanadate Using Extended Range FT‐IR Spectroscopy,Raman Spectroscopy,and X‐Ray Fluorescence Spectrometry

Chrome Yellow (PbCrO₄·xPbSO₄) was a common pigment in U.S. automobile OEM finishes for more than three decades, but in the early 1990s its use was discontinued. One of its main replacements was Bismuth Vanadate (BiVO₄·nBi₂MoO₆, n = 0–2), which was commercially introduced in 1985, as this inorganic pigment also produces a very bright hue and has excellent outdoor durability. This paper describes the in situ identification of Bismuth Vanadate in automotive finishes using FT‐IR and dispersive Raman spectroscopy and XRF spectrometry. Some differentiation of commercial formulations of this pigment is possible based on far‐infrared absorptions, Raman data, and elemental analysis. The spectral differences arise from the presence or absence of molybdenum, the use of two crystal polymorphs of BiVO₄, and differences in pigment stabilizers. Bismuth Vanadate is usually not used alone, and it is typically found with Isoindoline Yellow, hydrous ferric oxide, rutile, Isoindolinone Yellow 3R, or various combinations of these.     

Clarifying the complex chemistry of cobalt(II) thiocyanate-based tests for cocaine using single-crystal X-ray diffraction and spectroscopic techniques

Cobalt(II) thiocyanate-based tests are routinely used to screen cocaine products, with the formation of a blue species interpreted as a positive response. An array of other organic bases has been identified as false positives – including well-documented cocaine product adulterant lidocaine and its salt. False positives prompt continued test development, though improvements are hindered by unresolved product structures and reaction pathways. Toward greater clarity, cobalt(II) thiocyanate reactions with cocaine hydrochloride, along with lidocaine and its salt, were investigated using multiple analytical techniques. Reactions involving cocaine hydrochloride yielded glassy, amorphous blue material while reactions of lidocaine hydrochloride monohydrate produced larger, needle-like crystals whose structure was determined via single-crystal X-ray diffraction to be an ion pair (Hlidocaine⁺)₂([Co(SCN)₄]²⁻)·H₂O. While the blue precipitate isolated from reactions involving cocaine hydrochloride was unsuitable for crystallographic structure determination, comparative ultraviolet–visible, attenuated total reflectance infrared, and Raman spectroscopic analysis – along with elemental analysis – supports that this solid is comprised of a comparable ion pair (Hcocaine⁺)₂[Co(SCN)₄]²⁻. Pink crystals isolated from lidocaine reaction vessels were identified as coordination compounds cis-[CoL₂(SCN)₂] and trans-[CoL₂(SCN)₂] where L = lidocaine, while pink crystals from both cocaine hydrochloride and lidocaine hydrochloride monohydrate reaction vessels were the coordination polymer trans-[Co(H₂O)₂(SCN)₂]·H₂O. The results presented herein enable reaction optimization to favor a desired product, whether ion pair or coordination species.     

Analysis of Δ-tetrahydrocannabinol in oral fluid samples using solid-phase extraction and high-performance liquid chromatography–electrospray ionization mass spectrometry

An analytical method using solid-phase extraction (SPE) and high-performance liquid chromatography–mass spectrometry (LC–MS) has been developed and validated for the confirmation of Δ⁹-tetrahydrocannabinol (THC) in oral fluid samples. Oral fluid was extracted using Bond Elut LRC-Certify solid-phase extraction columns (10 cm³, 300 mg) and elution performed with n-hexane/ethyl acetate. Quantitation made use of the selected ion-recording mode (SIR) using the most abundant characteristic ion [THC + H⁺], m/z 315.31 and the fragment ion, m/z 193.13 for confirmation, and m/z 318.00 for the protonated internal standard, [d₃-THC + H⁺]. The method proved to be precise for THC, in terms of both intra-day and inter-day analyses, with coefficients of variation less than 10%, and the calculated extraction efficiencies for THC ranged from 76 to 83%. Calibration standards spiked with THC between 2 and 100 ng/mL showed a linear relationship (r² = 0.999). The method presented was applied to the oral fluid samples taken from the volunteers during the largest music event in Portugal, named Rock in Rio-Lisboa. Oral fluid was collected from 40 persons by expectoration and with Salivette^®. In 55% of the samples obtained by expectorating, THC was detected with concentration ranges from 1033 to 6552 ng/mL and in 45% of cases THC was detected at concentrations between 51 and 937 ng/mL. However, using Salivette^® collection, 26 of the 40 cases had an undetectable THC.     

Discrimination Between Infant and Adult Bloodstains Using Micro‐Raman Spectroscopy: A Preliminary Study

In the present study, we used micro‐Raman spectroscopy with high‐resolution analysis to discriminate between bloodstains from infants and bloodstains from adults. Raman peaks were detected at 674, 754, 976, 1002, 1105, 1127, 1176, 1248, 1340, 1368, 1390, 1560, and 1611 cm^?1; these peaks were derived from hemoglobin, albumin, and glucose. However, a peak was obtained at 1105 cm^?1, which was assigned to histidine; this peak was observed only for bloodstains from adults. Human adult hemoglobin (HbA) is composed of an α₂β₂ tetramer structure, whereas human fetal hemoglobin (HbF) is composed of an α₂γ₂. Therefore, the lack of a Raman peak at 1105 cm^?1 in bloodstains from infants indicates the possibility of two histidine substitutions (His116Ile and His143Ser) in the γ chain of HbF. This study discriminates between bloodstains from infants and bloodstains from adults using micro‐Raman spectroscopy, with beneficial implications in forensic science.     

Validation of an LC–MS Method for the Detection and Quantification of BZP and TFMPP and their Hydroxylated Metabolites in Human Plasma and its Application to the Pharmacokinetic Study of TFMPP in Humans*

Abstract: An LC–MS method was developed for benzylpiperazine (BZP) and trifluoromethylphenylpiperazine (TFMPP), constituents of “party pills” or “legal herbal highs,” and their metabolites in human blood plasma. Compounds were resolved using a mixture of ammonium formate (pH 4.5, 0.01 M) and acetonitrile (flow rate of 1.0 mL/min) with a C18 column. Calibration curves were linear from 1 to 50 ng/mL (R² > 0.99); the lower limit of quantification (LLOQ) was 5 ng/mL; the accuracy was >90%; the intra‐ and interday relative standard deviations (R.S.D) were <5% and <10%, respectively. Human plasma concentrations of TFMPP were measured in blood samples taken from healthy adults (n = 6) over 24 h following a 60‐mg oral dose of TFMPP: these peaked at 24.10 ng/mL (±1.8 ng/mL) (C_max) after 90 min (T_max). Plasma concentrations of 1‐(3‐trifluoromethyl‐4‐hydroxyphenyl) piperazine peaked at 20.2 ng/mL (±4.6 ng/mL) after 90 min. TFMPP had two disposition phases (t_½ = 2.04 h (±0.19 h) and 5.95 h (±1.63 h). Apparent clearance (Cl/F) was 384 L/h (±45 L/h).     

Evaluation of Suspected Counterfeit Pharmaceutical Tablets Declared to Contain Controlled Substances Using Handheld Raman Spectrometers

This study describes the performance of handheld Raman devices for determining whether suspect pharmaceutical tablets declared to contain controlled substances were consistent with authentic (CWA) or not consistent with authentic (NCWA) tablets using a simple, rapid, field-friendly method capable of being used by nonexperts. Twenty-five authentic products and 84 known NCWA tablets were examined using three “parent” devices for a total of 327 analyses. On average, the parent devices yielded a true pass rate of 100%, a true fail rate of 98.4%, a false pass rate of 1.6%, and a false fail rate of 0%. The methods/libraries were then transferred to 13 identical “daughter” devices, which were used to examine 10 suspect finished dosage forms in duplicate (six known NCWA tablets and four authentic tablets) for a total of 260 measurements. On average, the daughter devices had a true pass rate of 100%, a true fail rate of 95.5%, a false pass rate of 4.5%, and a false fail rate of 0.0%. These data demonstrate that the parent–daughter electronic transfer method was successful, which permits the ability to develop methods in the laboratory that can be seamlessly pushed out to field devices. The methods can then be used to (i) prioritize samples for additional testing using other more time-consuming laboratory-based techniques needed to detect and quantify active ingredients and (ii) help support the interdiction of dangerous tablets at ports of entry, thereby preventing them from reaching the supply chain.     

Differentiation of Commercially Available Kratom by Purported Country of Origin using Inductively Coupled Plasma–Mass Spectrometry,

Kratom is a plant material exhibiting both analgesic and stimulant effects and is also forensically relevant since it is abused as a “legal high.” It is regulated in several countries but not scheduled in the United States at the federal level. This study used inductively coupled plasma–mass spectrometry (ICP–MS) to measure the concentrations of 13 elements in 19 kratom samples obtained from an online distributor selling kratom, from Borneo, Malaysia, Indonesia, Thailand, and Vietnam, for the purpose of using the elements to discriminate among purported country of origin, “suborigin,” and strain. Analysis of variance revealed statistical differences in concentrations of elements from each group, while discriminant function analysis (using leave-one-out classification) successfully classified kratom samples by their purported country of origin (100%), “suborigin,” (100%), and strain (86%). Our method illustrates the possibility of utilizing ICP–MS for determination of commercially available kratom samples by purported origin, “subororign,” or by product line.     

In Situ Identification of Semen Stains on Common Substrates via Raman Spectroscopy,,

The spectroscopic identification of body fluids in situ is a major objective in forensic science. This approach offers the confirmatory, nondestructive, rapid, and on‐scene identification of various body fluids. Although Raman spectroscopy has shown tremendous promise toward this goal in prior proof‐of‐concept experiments, a significant challenge which still remains is substrate interference. Here, an approach for detecting semen stains in situ on various substrates using Raman spectroscopy is explored. Simulated semen evidence was prepared on skin, glass, and various fabrics. Raman data were accumulated from stains without any pretreatment using a common confocal mapping spectrometer using 785 nm laser excitation. The results demonstrate that the spectroscopic interferences encountered by substrates can be reduced and eliminated using a combination of existing subtraction techniques and chemometric models. Heterogeneous substrates proved most challenging, however, automatic subtraction treatment, and location of fluid hotspots was able to elucidate a clear spectroscopic signature of semen in every instance.     

Application of the Optimized Summed Scored Attributes Method to Sex Estimation in Asian Crania

The optimized summed scored attributes (OSSA) method was recently introduced and validated for nonmetric ancestry estimation between American Black and White individuals. The method proceeds by scoring, dichotomizing, and subsequently summing ordinal morphoscopic trait scores to maximize between‐group differences. This study tests the applicability of the OSSA method for sex estimation using five cranial traits given the methodological similarities between classifying sex and ancestry. A large sample of documented crania from Japan and Thailand (n = 744 males, 320 females) are used to develop a heuristically selected OSSA sectioning point of ≤1 separating males and females. This sectioning point is validated using a holdout sample of Japanese, Thai, and Filipino (n = 178 males, 82 females) individuals. The results indicate a general correct classification rate of 82% using all five traits, and 81% when excluding the mental eminence. Designating an OSSA score of 2 as indeterminate is recommended.     

Quantitative analysis of mitragynine, codeine, caffeine, chlorpheniramine and phenylephrine in a kratom (Mitragyna speciosa Korth.) cocktail using high-performance liquid chromatography

A simple HPLC technique for determining mitragynine, codeine, caffeine, chlorpheniramine and phenylephrine in 'kratom cocktail' was developed. The analytical method for mitragynine, codeine and caffeine used an Eclipse XDB-C8 column. A Lichrospher CN column was using for analysing chlorpheniramine and phenylephrine. The correlation coefficient of each standard was between 0.9957 and 0.9993. The precision of the methods were between 0.700 and 7.108% RSD. The concentration of mitragynine, codeine, caffeine, chlorpheniramine and phenylephrine in 'kratom cocktail' was 90.021, 234.174, 73.986, 7.053 and 1.486 mg/L, respectively.     

Pharmacokinetics of ethanol in patients with renal failure before and after hemodialysis

We studied the pharmacokinetics of ethanol in seven patients suffering from terminal renal failure before and after they underwent hemodialysis. Ethanol (0.40 g/kg) was administered in the morning after an overnight fast by a constant rate intravenous (IV) infusion over 45 min. After removing a mean fluid volume of 2.46±0.48 liters (±SD), span 1.76–3.43 liters by hemodialysis, the same subjects received a second IV infusion of ethanol after they had eaten lunch. At exactly timed intervals of 0, 45, 90, 105, 120, 135, 150, 165, and 180 min from the start of the infusion, two blood-samples were drawn and the plasma portion of one of them was obtained by centrifugation. The concentration of ethanol in blood and plasma was determined by headspace gas chromatography and the water-content of whole blood was determined from the change in weight after desiccation. Plasma always contained a higher concentration of ethanol than whole blood and the mean plasma/whole blood ratio in patients with renal failure was 1.07:1 (span 1.05–1.10). The rate of ethanol disappearance from blood (β-slope) was faster (0.185±0.013 versus 0.157±0.022 g/l/h), the C₀ value was higher (0.79±0.08 versus 0.73±0.10 g/l) and the apparent volume of distribution (V_d) of ethanol was lower (0.507±0.049 versus 0.558±0.078 l/kg) after hemodialysis. The water content of whole blood was significantly higher (P<0.001) before dialysis (88.6±1.97 g/100 ml) compared with after dialysis (87.4±2.01 g/100 ml). The higher V_d for ethanol and lower C₀ as well as higher blood-water content are to be expected for a over hydrated condition before hemodialysis. The swifter rate of ethanol elimination from blood (β-slope) after hemodialysis should be interpreted with caution because eating a meal before the second infusion of ethanol is a confounding factor. Nevertheless, the rate of elimination of ethanol from blood in patients with renal failure agreed reasonably well with values expected for healthy subjects, namely mean 0.15 g/l/h spanning from 0.10 to 0.20 g/l/h.     

Evaluation and classification of fentanyl-related compounds using EC-SERS and machine learning

Multiple analytical techniques for the screening of fentanyl-related compounds exist. High discriminatory methods such as GC–MS and LC–MS are expensive, time-consuming, and less amenable to onsite analysis. Raman spectroscopy provides a rapid, inexpensive alternative. Raman variants such as electrochemical surface-enhanced Raman scattering (EC-SERS) can provide signal enhancements with 10¹⁰ magnitudes, allowing for the detection of low-concentration analytes, otherwise undetected using conventional Raman. Library search algorithms embedded in instruments utilizing SERS may suffer from accuracy when multicomponent mixtures involving fentanyl derivatives are analyzed. The complexing of machine learning techniques to Raman spectra demonstrates an improvement in the discrimination of drugs even when present in multicomponent mixtures of various ratios. Additionally, these algorithms are capable of identifying spectral features difficult to detect by manual comparisons. Therefore, the goal of this study was to evaluate fentanyl-related compounds and other drugs of abuse using EC-SERS and to process the acquired data using machine learning—convolutional neural networks (CNN). The CNN was created using Keras v 2.4.0 with Tensorflow v 2.9.1 backend. In-house binary mixtures and authentic adjudicated case samples were used to evaluate the created machine-learning models. The overall accuracy of the model was 98.4 ± 0.1% after 10-fold cross-validation. The correct identification for the in-house binary mixtures was 92%, while the authentic case samples were 85%. The high accuracies achieved in this study demonstrate the advantage of using machine learning to process spectral data when screening seized drug materials comprised of multiple components.     

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.     

A short note on the identification of mitragynine

The leaves of Mitragyna speciosa are used as a substitute for opium. Criminal penalties for possession of mitragynine in this country are assigned for possession of defined quantities of mitragynine and its identification as Mitragyna speciosa by thin-layer chromatography and gas chromatography.     

Counterfeit Adderall Containing Aceclofenac from Internet Pharmacies

A nontargeted approach based on liquid chromatography equipped with a quadrupole time‐of‐flight mass detector (LC‐MS Q‐TOF) joined to nuclear magnetic resonance (NMR) analysis allowed rapid identification and quantification of the anti‐inflammatory drug aceclofenac in illegal Adderall tablets. The largest chromatographic peak had m/z = 354.030 and m/z = 376.012 matching, respectively, the ionic structures (M + H)⁺ and (M + Na)⁺ of a molecule M. The accurate mass data generated the molecular formula C₁₆H₁₃Cl₂NO₄. A screening of the pharmaceutical active substances having that molecular formula together with the MS/MS fragmentation pattern suggested aceclofenac. Aceclofenac structure was unambiguously confirmed by ¹H and ¹³C NMR experiments. The aceclofenac content was 90 mg/tablet (RSD 2%) as detected by quantitative NMR. Information on the identity and content of illegal drugs is required for legal purposes; it supports in evaluating the effective impact on users safety, and it is useful for control laboratories using a targeted approach in their analytical activities.     

Transferrin (Tf) polymorphism: An analysis by isoelectric focusing

The polymorphism of the transferrin (Tf) system was studied in a total of 300 unrelated Japanese individuals from Miyagi prefecture, the northern part of Japan, using isoelectric focusing in thin-layer polyacrylamide gel. In our population samples three common phenotypes and nine variants were observed. The calculated allele frequencies were Tf^C₁ = 0.773, Tf^C₂ = 0.212, Tf^D_chi (Chinese) = 0.008, and the combined frequencies for the Tf^B_var (variants) = 0.007. Family data (n = 44) were in accordance with an autosomal codominant fashion of inheritance. The use of isoelectric focusing procedure among Japanese will raise the probability of excluding a man falsely accused of paternity to 15.8% as compared with 1.5% when the conventional electrophoretic methods are used.