In vitro metabolic studies and machine learning analysis of mass spectrometry data: A dual strategy for differentiating alpha-pyrrolidinohexiophenone (α-PHP) and alpha-pyrrolidinoisohexanophenone (α-PiHP) in urine analysis.

Synthetic cathinones are some of the most prevalent new psychoactive substances (NPSs) globally, with alpha-pyrrolidinoisohexanophenone (α-PiHP) being particularly noted for its widespread use in the United States, Europe, and Taiwan. However, the analysis of isomeric NPSs such as α-PiHP and alpha-pyrrolidinohexiophenone (α-PHP) is challenging owing to similarities in their retention times and mass spectra. This study proposes a dual strategy based on in vitro metabolic experiments and machine learning-based classification modelling for differentiating α-PHP and α-PiHP in urine samples: (1) in vitro metabolic experiments using pooled human liver microsomes and liquid chromatography tandem quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) were conducted to identify the key metabolites of α-PHP and α-PiHP from the high-resolution MS/MS spectra. After 5 h incubation, 71.4 % of α-PHP and 64.7 % of α-PiHP remained unmetabolised. Nine phase I metabolites were identified for each compound, including primary ß-ketone reduction (M1) metabolites. Comparing the metabolites and retention times confirmed the efficacy of in vitro metabolic experiments for differentiating NPS isomers. Subsequently, analysis of seven real urine samples revealed the presence for various metabolites, including M1, that could be used as suitable detection markers at low concentrations. The aliphatic hydroxylation (M2) metabolite peak counts and metabolite retention times were used to determine α-PiHP use. (2) Classification models for the parent compounds and M1 metabolites were developed using principal component analysis for feature extraction and logistic regression for classification. The training and test sets were devised from the spectra of standard samples or supernatants from in vitro metabolism experiments with different incubation times. Both models had classification accuracies of 100 % and accurately identified α-PiHP and its M1 metabolite in seven real urine samples. The proposed methodology effectively distinguished between such isomers and confirmed their presence at low concentrations. Overall, this study introduces a novel concept that addresses the complexities in analysing isomeric NPSs and suggests a path towards enhancing the accuracy and reliability of NPS detection.

Quantitative determination and metabolic profiling of synthetic cathinone eutylone in vitro and in urine samples by liquid chromatography tandem quadrupole time-of-flight mass spectrometry.

In Taiwan, synthetic cathinones are the most prevalent new psychoactive substances, and their use is growing continuously. Urine samples are currently analysed to determine drug abuse, but the metabolic profiles and metabolites of these compounds are not widely reported. Given that cases of eutylone abuse have been growing since 2020, this study established a method employing supported liquid extraction combined with liquid chromatography tandem quadrupole time-of-flight mass spectrometry to identify and quantify eutylone and its metabolites in urine samples. Method validation was performed, and eight authentic samples were analysed. Moreover, in vitro metabolism experiments were conducted, and metabolites were generated by incubating eutylone with human liver microsomes and cytosol. Metabolite characterisation was achieved by confirming the accurate mass and product ions in full MS/MS spectra. Five metabolites were identified in in vitro experiments; they resulted from eutylone N-dealkylation, ß-ketone reduction, demethylenation, aliphatic hydroxylation and sequential demethylenation and O-methylation. The metabolic profile was obtained evaluating the metabolites at different incubation times: Demethylenation occurred first, followed by N-dealkylation, ß-ketone reduction and aliphatic hydroxylation. Three additional metabolites were identified in authentic samples. Based on in vitro and in vivo evidence, we propose that the demethylenation and O-methylation metabolite, the ß-ketone reduction metabolite, and the ß-ketone reduction, demethylenation and O-methylation metabolite are the most appropriate biomarkers of eutylone consumption. Using these markers can help expand the eutylone detection window and provide information for toxicology research.

A novel approach to evaluate the extent and the effect of cross-contribution to the intensity of ions designating the analyte and the internal standard in quantitative GC-MS analysis.

In gas chromatography-mass spectrometry methods of analysis adopting the analyte's isotopic analog as the internal standard (IS), the cross-contribution (CC) phenomenon -- contribution of IS to the intensities of the ions designating the analyte, and vice versa -- has been demonstrated to affect the quantitation data. A novel approach based on the deviations of the empirically observed concentrations of a set of standards was developed to assess the accuracy of the empirically derived CC data. This approach demonstrated that normalization of ion intensities derived from the analyte and the IS generates reliable CC data. It further demonstrated that an ion-pair (designating the analyte and the IS) with approximately 5% or higher CC will result in a very limited linear calibration range.

GC-MS analysis of multiply derivatized opioids in urine.

Opiates such as hydrocodone, hydromorphone, oxycodone, noroxycodone, and oxymorphone reportedly may interfere with the analysis of morphine and codeine. The analysis of these compounds themselves also is an important issue. Thus, double derivatization approaches utilizing methoxyamine and hydroxylamine to first form oxime products with keto-opiates, followed by the derivatization with trimethylsilyl (TMS) or propionyl groups, have been developed for the simultaneous analysis of these compounds. However, these studies have not included all compounds of interest and resulted in inadequate chromatographic resolution or significant intensity cross-contribution between the ions designating the analyte and its deuterated internal standard for certain compounds. By exploring three-step derivatization approaches with the combination of various derivatization groups and orders, this study concluded that application of methoxyimino/propionyl/TMS groups, in the order listed, facilitated the simultaneous analysis of eight opiates (morphine, 6-acetylmorphine, hydromorphone, oxymorphone, codeine, hydrocodone, oxycodone and noroxycodone) in urine samples, achieving satisfactory limits of quantitation and detection. In addition, the adapted approach resulted in two usable products for morphine and codeine providing alternatives, should interferences render any of these products non-usable.

Gas chromatography-mass spectrometry analysis of ketamine and its metabolites--a comparative study on the utilization of different derivatization groups.

Method of chemical derivatization is the main difference among the GC-MS based methodologies reported for the analysis of ketamine and its major metabolites (norketamine and dehydronorketamine). These approaches included acylation and silylation resulting in the formation of acetyl, trifluoroacetyl, heptafluorobutyryl, and pentafluorobenzoyl (for acylation); and possibly trimethylsilyl and t-butyldimethylsilyl (for silylation) derivatives. This study evaluates the merits of these approaches based on the following criteria: reaction yields and ionization efficiency of the derivatization products; chromatographic characteristics; and cross-contributions to the intensities of ions designating the analyte and the internal standard. Pentafluorobenzoyl-derivatives were found to provide the best performance characteristics.

Famprofazone as the source of methamphetamine and amphetamine in urine specimen collected during sport competition.

During a sport competition event in Taiwan, one urine specimen was found positive for both methamphetamine (2688 ng/mL) and amphetamine (462 ng/mL). The specimen donor claimed that she had taken Gewolen (a nonprescription drug manufactured in Taiwan) for treating abdominal pain and the medication was presented. Laboratory investigation confirmed that Gewolen contains famprofazone, which is known to metabolize to methamphetamine and amphetamine and is included in the prohibited list of the World Anti-Doping Agency. Study on the excretion profiles of three volunteers ingesting 50 mg famprofazone produced the following patterns similar to that observed in the case specimen: (a) the ratio of methamphetamine to amphetamine was approximately 6 to 1; (b) d- and l-enantiomers of both methamphetamine and amphetamine were present, while the amount of l-methamphetamine was 3-4-fold greater than its counterpart. The data suggested that famprofazone (as the ingredient of Gewolen) was likely the source of the prohibited drugs found in the case specimen.

Comparison of Rehydration Techniques for Fingerprinting the Deceased after Mummification.

Postmortem decay causes fingertip decomposition, desiccation, shriveling, and rigidity, reducing the possibility of obtaining sufficiently clear fingerprints for identification. In this study, five rehydration solutions (ammonium hydroxide, sodium carbonate, potassium hydroxide, urea, and warm water) followed by three fingerprint recording methods (photograph, inking roll, and dusting tape) were investigated to process mummified fingertips from an unidentified cadaver. The results show that sodium carbonate treatment is the most effective for minutiae restoration, followed by ammonium hydroxide treatment. This study also demonstrates that even those fingertips that previously failed in urea solution, 1% potassium hydroxide solution, and warm water treatment could be further improved with sodium carbonate solution to obtain qualified minutiae for fingerprint matching. The optimal procedure is rehydrating the desiccated fingertips with sodium carbonate solution for 24 h followed by dusting the finger and transferring the print to adhesive tape.

Artifacts in the GC-MS profiling of underivatized methamphetamine hydrochloride.

When underivatized methamphetamine hydrochloride (MA.HCl) in methanol is subjected to the instant gas chromatographic-mass spectrometric (GC-MS) profiling with old inlet liners at temperatures above 200 degrees C, appreciable amounts of N,N-dimethylamphetamine (DMA) and amphetamine (AP) are produced. The presence of these two artifacts is attributed to the N-demethylation and N-methylation reactions of MA as well as methyl group exchange with methanol. These artifacts are only produced in old injection port liners and at elevated temperatures. The formation of artifacts is proportional to concentration of MA.HCl. It is suggested that special cautions and measures be undertaken to prevent artifacts.

Latent Fingerprint Development on Thermal Paper Using Traditional Ninhydrin and 1,2-indanedione.

Thermal paper poses a significant challenge to latent print development as it tends to change color when traditional fingerprint development formulations are applied to it. In this study, the optimal components of ninhydrin, 1,2-indanedione, 1,8-diazafluoren-9-one (DFO), and 5-methylthioninhydrin (5-MTN) for yielding clear fingerprints on thermal paper were determined by systematically adjusting the relative amounts of the reagents, polar solvents, and the nonpolar diluent petroleum ether, followed by validation on text-printed thermal paper. Specifically, 3.0% ethyl acetate as the polar solvent in petroleum ether was found to be the optimal combination; the optimal dilution ratios of ninhydrin, DFO, and 5-MTN original solutions with petroleum ether were 1 to 2, 11, and 7, respectively. The optimal concentration of 1,2-indanedione in petroleum ether was 0.125 g/L, with a string of 0.5% ethyl acetate in petroleum ether.

The Influence of Selected Fingerprint Enhancement Techniques on Forensic DNA Typing of Epithelial Cells Deposited on Porous Surfaces.

Fingerprints deposited at crime scene can be a source of DNA. Previous reports on the effects of fingerprint enhancement methods have focused mainly on fingermarks deposited in blood or saliva. Here, we evaluate the effects of fingerprint enhancement methods on fingerprints deposited on porous surfaces. We performed real-time quantification and STR typing, the results of which indicated that two methods (iodine fuming and 1,2-indanedione in ethyl acetate enhancement) had no effect on the quantity of DNA isolated and resultant STR alleles when compared to control samples. DNA quantities and allele numbers were lower for samples enhanced with silver nitrate and 1,2-indanedione in acetic acid when compared to control samples. Based on DNA quantity, quality, and observable stochastic effects, our data indicated that iodine fuming and 1,2-indanedione in ethyl acetate were the preferred options for the enhancement of fingerprints on porous surfaces.

Enantiomeric determination of amphetamines: exploring a novel one-step solid-phase microextraction-based approach.

The recent advances in fiber manufacturing technology, solid-phase microextraction (SPME) is now widely studied for its effectiveness for the pretreatment of various categories of samples. This study explores a novel SPME approach for enantiomeric analysis of amphetamines, in which absorption/derivatization are accomplished in one step. Specifically, (S)-(-)-N-(Trifluoroacetyl)-prolyl chloride was adopted as the chiral derivatizing reagent and added directly into the sample matrix. Analytical parameters, such as temperature, absorption/desorption duration, and the amount of derivatizing reagent, were studied to determine their effects on the yield of analytes. The derivatization products resulting from this study show excellent desorption characteristics on the polydimethylsiloxane-coated fiber adopted in this study. Optimal operational parameters (absorption: 70 degrees C for 10 min; injection: 250 degrees C for 5 min) cause minimal negative impact on the fiber, allowing repeated use of the fiber for more than 30 times. For quantitation, data were collected under selected ion monitoring (SIM) mode using m/z 237 and 251 to designate derivatized amphetamine and methamphetamine. This method was evaluated and proved to be effective in (a) quantitative determination of the enantiomeric pairs of amphetamine and methamphetamine--in terms of repeatability, linearity, and limits of detection and quantitation; and (b) generating case-specimen data comparable to those derived from a conventional Liquid-liquid extraction approach. Good linearity for the calibration curves were established in the range of 1000-50 ng/mL for both analytes. The limits of detection for these analytes were 30 ng/mL.

Simultaneous determination of amphetamine and methamphetamine enantiomers in urine by simultaneous liquid-liquid extraction and diastereomeric derivatization followed by gas chromatographic-isotope dilution mass spectrometry.

A simple, rapid, reliable, and economic analytical scheme starting with in situ liquid-liquid extraction and asymmetric (or diastereomeric) chemical derivatization (ChD) followed by gas chromatography (GC)-isotope dilution mass spectrometry (MS) is described for the simultaneous determination of D- and L-amphetamine (AP) and methamphetamine (MA) in urine which could have resulted from the administration of various forms of questioned amphetamines or amphetamines-generating drugs. By using L-N-trifluoroacetyl-1-prolyl chloride (L-TPC) as chiral derivatizing agent, resolutions of 2.2 and 2.0 were achieved for the separation of AP and MA enantiomeric pairs, respectively, on an ordinary HP-5MS capillary column. The GC-MS quantitation was carried out in the selected ion monitoring (SIM) mode using m/z 237 and 251 as the quantifier ions for the respective diastereomeric pairs of AP-L-TPC and MA-L-TPC. The calibration curves plotted for the two pairs of analytes stretch with good linearity down to 45 ng/mL, and the limits of detection and quantitation determined were as low as 40 and 45 ng/mL, respectively. Also, a comparative study using 10 real-case urine specimens previously screened as positive for MA administration showed mostly tolerable biases between the two sums (of concentration) of D- and L-MA obtained via an asymmetric L-TPC-ChD approach and via an ordinary pentafluoropropionylation (PFPA-ChD) approach, respectively, as well as between the two sums of D- and L-AP obtained thereupon, thus validating the proposed analytical scheme as a promising forensic protocol for the detailed analysis of enantiomeric amphetamines in urine.

Enantiomeric determination of ephedrines and norephedrines by chiral derivatization gas chromatography-mass spectrometry approaches.

Concerned with variations in abuse potential and control status among various isomers of ephedrines and norephedrines, this study was conducted to develop an effective method for the simultaneous analysis of eight ephedrine-related compounds along with structurally similar cathinones. Among various approaches studied, a 60-m HP-5MS (0.25 mm i.d., 0.25 microm film thickness) was successfully used to characterize the following compounds that were derivatized with (-)-alpha-methoxy-alpha-trifloromethylphenylacetic acid (MTPA): (+)-cathinone, (-)-cathinone, (+)-norephedrine, (-)-norephedrine, (+)-norpseudoephedrine, (+)-ephedrine, (-)-ephedrine, (-)-pseudoephedrine, (+)-pseudoephedrine. (-)-Cathine standard was not available, but should also be resolvable under this analytical procedure. This method was successfully applied to the analysis of selected cold remedies for characterizing the enantiomeric compositions of the compounds present in these samples.

Latent fingermark development using low-vacuum vaporization of ninhydrin.

The vacuum technique is a method of vaporizing a solid material to its gas phase, helping deposit reagents gently on target surfaces to develop latent fingermarks. However, this application is rarely reported in the literature. In this study, a homemade fume hood with a built-in vacuum control system and programmable heating system designed by the Taiwan Criminal Investigation Bureau is introduced. Factors that affect the instrument's performance in developing fingermarks are discussed, including the quantity of chemicals for vaporization, heating program arrangement, and paper of different materials. The results show that fingermarks are effectively developed by vaporizing solid ninhydrin. This would be an alternative application in selecting a solvent-free method for protecting the environment and reducing health hazards in the lab. In terms of the heating program, the result indicates that under a low-vacuum condition (50 mTorr), 80-90 °C is a suitable temperature range for ninhydrin vaporization, allowing ninhydrin to be vaporized without bumping and waste. In terms of the performance on different material papers, this instrument demonstrates its capacity by developing latent fingermarks on thermal paper without discoloration or damaging the original writing, and the same results are also observed on Taiwan and United States banknotes. However, a coherent result could be hardly obtained using the same vaporization setting because different banknotes have their own surface features and water absorption ability or other unique factors may influence the effect of ninhydrin deposition. This study provides a reliable application for developing latent fingermarks without using solvents, and it is also expected to contribute to environmental protection along with the trend of green chemistry technology.

Identification of tiny and thin smears of automotive paint following a traffic accident.

Three complementary methods in the order of stereomicroscopy, micro-FT-IR spectroscopy and solubility tests were used for the purpose of matching known and questioned paint samples in an auto accident case. Grayish green paint smears scattered on a silvery gray coated plastic bar were taken from a blue car and referred to as questioned samples. Green paint chips were collected as known samples from a green car. These were analyzed to determine whether the paint smears found in the blue car could have been the transfers from the green car. Although each of the three methods, when used alone, suffered from unequal bases for making comparison (i.e., layering whole paint vs. smeared paint), insufficient specificity of methodology and the interfering background coating beneath the smeared paint, the limitations were significantly relieved when three methods were used in combination. Based on the results presented in this report, the questioned grayish green paint smears collected from the blue car and the known green paint chips from the green car are of the same class of paint; that is, the possibility of the above stated paint transfers cannot be eliminated.

Distribution characteristics of methamphetamine and amphetamine in urine and hair specimens collected from alleged methamphetamine users in northern Taiwan.

This study was conducted to better understand the distribution characteristics of methamphetamine and amphetamine in urine and hair specimens collected from alleged methamphetamine users in the local population. It is anticipated that the data hereby obtained will be helpful to the interpretation of the time and pattern of drug use. Eight alleged methamphetamine-using arrestees from Keelung Police Department (north of Taipei, Taiwan) consented to contribute both urine and hair specimens. Each arrestee contributed seven urine specimens collected at 0, 12, 24, 48, 72, 96, and 120 h, respectively, after the arrest. Hair specimens were cut into 2-cm sections. The limits of detection and quantitation of the urine protocol were 40 and 50 ng/mL, respectively, for both amphetamine and methamphetamine, while the corresponding limits of detection and quantitation for the hair protocol were 0.8 and 1.0 ng/mg, respectively. The concentration variations of methamphetamine and amphetamine in the urine specimens exhibited three distinct patterns: (a) continuous decrease in the analytes' concentrations for specimens collected at hours 0-120; (b) increase in the analytes' concentrations in specimens collected at hours 0-12, followed by decrease; (c) increase in analytes' concentrations in specimens collected at later times. Together with the amphetamine/methamphetamine concentration ratios found in these urine specimens, the observed trends in the changes of the analytes' concentrations are helpful for the interpretation on the time of drug use. Unlike urine specimens, amphetamine/methamphetamine concentration ratios in various hair specimens and hair sections remain relatively constant.