Single hair analysis for gamma-hydroxybutyric acid-Method optimization, validation, and application.

As gamma-hydroxybutyric acid (GHB) underlies fast metabolization, its determination from hair may presumably offer a detection window superior to that of body fluids. Due to the wide range of endogenous concentration levels, the evidence of an exogenous ingestion is challenging. As already shown for other drugs, the temporal resolution obtained by applying single hair microanalysis provides further information. Therefore, a method for the extraction and quantification of GHB in 2-mm hair segments (seg) was optimized and validated (limit of detection [LOD]: 2.5 pg/seg, lower limit of quantification [LLOQ]: 5 pg/seg), and five single hairs were examined, each for three non-users and for three (alleged) users. A major challenge was the choice of appropriate extraction tubes without remains of GHB. In two samples from non-users, GHB could not or could only be detected in trace amounts. In the third sample, concentrations between the LOD and 31.1 pg/seg (mean: 9.5, median: 8.4; each pg/seg) were detected with decreasing values towards the tips. In two samples of persons with assumed GHB intake, maximum concentrations of 6.8 and 30.7 pg/seg were measured, but no significant concentration peaks indicating a single ingestion could be observed. The third sample showed concentrations of 7.6-55.2 pg/seg (mean: 28.8, median: 29.6; each pg/seg). In this case, the obtained profiles showing at least two reproducible concentration maxima between 20 and 40 mm point to an ingestion of GHB. The concentration profiles from single hairs were reproducible in each case, reflecting the concentration course of routine 1-cm segmental analysis. These are the first results published on GHB testing in segmented single hairs, and the results must be verified further.

Micro-segmentation of retinal image lesions in diabetic retinopathy using energy-based fuzzy C-Means clustering (EFM-FCM).

Diabetic retinopathy (DR) is a prevalent cause of global visual impairment, contributing to approximately 4.8% of blindness cases worldwide as reported by the World Health Organization (WHO). The condition is characterized by pathological abnormalities in the retinal layer, including microaneurysms, vitreous hemorrhages, and exudates. Microscopic analysis of retinal images is crucial in diagnosing and treating DR. This article proposes a novel method for early DR screening using segmentation and unsupervised learning techniques. The approach integrates a neural network energy-based model into the Fuzzy C-Means (FCM) algorithm to enhance convergence criteria, aiming to improve the accuracy and efficiency of automated DR screening tools. The evaluation of results includes the primary dataset from the Shiva Netralaya Centre, IDRiD, and DIARETDB1. The performance of the proposed method is compared against FCM, EFCM, FLICM, and M-FLICM techniques, utilizing metrics such as accuracy in noiseless and noisy conditions and average execution time. The results showcase auspicious performance on both primary and secondary datasets, achieving accuracy rates of 99.03% in noiseless conditions and 93.13% in noisy images, with an average execution time of 16.1 s. The proposed method holds significant potential in medical image analysis and could pave the way for future advancements in automated DR diagnosis and management. RESEARCH HIGHLIGHTS: A novel approach is proposed in the article, integrating a neural network energy-based model into the FCM algorithm to enhance the convergence criteria and the accuracy of automated DR screening tools. By leveraging the microscopic characteristics of retinal images, the proposed method significantly improves the accuracy of lesion segmentation, facilitating early detection and monitoring of DR. The evaluation of the method's performance includes primary datasets from reputable sources such as the Shiva Netralaya Centre, IDRiD, and DIARETDB1, demonstrating its effectiveness in comparison to other techniques (FCM, EFCM, FLICM, and M-FLICM) in terms of accuracy in both noiseless and noisy conditions. It achieves impressive accuracy rates of 99.03% in noiseless conditions and 93.13% in noisy images, with an average execution time of 16.1 s.

Analysis of Forty-Two Psychoactive Substances in a Single Hair by Micro-Segmental Technique.

OBJECTIVES: To establish an LC-MS/MS method based on single hair micro-segmental technique, and verify the detection of 42 psychoactive substances in 0.4 mm hair segments. METHODS: Each piece of single hair was cut into 0.4 mm segments and extracted by sonication and the segments were immersed in dithiothreitol-containing extraction medium. Mobile phase A was the aqueous solution containing 20 mmol/L ammonium acetate, 0.1% formic acid, and 5% acetonitrile. Mobile phase B was acetonitrile. An electrospray ionization source in positive ion mode was used for data acquisition in multiple reaction monitoring (MRM) mode. RESULTS: The 42 psychoactive substances in hair had a good linear relationship within their respective linear ranges (r>0.99), the limits of detection were 0.2-10 pg/mm, the limits of quantification were 0.5-20 pg/mm, the intra-day and inter-day precisions were 1.5%-12.7%, the intra-day and inter-day accuracies were 86.5%-109.2%, the recovery rates were 68.1%-98.2%, and the matrix effects were 71.3%-111.7%. The method was applied to hair samples collected from one volunteer at 28 d after a single dose of zolpidem, with zolpidem detected in 5 hairs was 1.08-1.60 cm near the root tip, and the concentration range was 0.62-20.5 pg/mm. CONCLUSIONS: The micro-segmental technique of single hair analysis can be applied to the investigation of drug-facilitated sexual assault cases.

Analysis of Forty-Two Psychoactive Substances in a Single Hair by Micro-Segmental Technique / 法医学杂志

OBJECTIVES@#To establish an LC-MS/MS method based on single hair micro-segmental technique, and verify the detection of 42 psychoactive substances in 0.4 mm hair segments.@*METHODS@#Each piece of single hair was cut into 0.4 mm segments and extracted by sonication and the segments were immersed in dithiothreitol-containing extraction medium. Mobile phase A was the aqueous solution containing 20 mmol/L ammonium acetate, 0.1% formic acid, and 5% acetonitrile. Mobile phase B was acetonitrile. An electrospray ionization source in positive ion mode was used for data acquisition in multiple reaction monitoring (MRM) mode.@*RESULTS@#The 42 psychoactive substances in hair had a good linear relationship within their respective linear ranges (r>0.99), the limits of detection were 0.2-10 pg/mm, the limits of quantification were 0.5-20 pg/mm, the intra-day and inter-day precisions were 1.5%-12.7%, the intra-day and inter-day accuracies were 86.5%-109.2%, the recovery rates were 68.1%-98.2%, and the matrix effects were 71.3%-111.7%. The method was applied to hair samples collected from one volunteer at 28 d after a single dose of zolpidem, with zolpidem detected in 5 hairs was 1.08-1.60 cm near the root tip, and the concentration range was 0.62-20.5 pg/mm.@*CONCLUSIONS@#The micro-segmental technique of single hair analysis can be applied to the investigation of drug-facilitated sexual assault cases.

Application of single hair analysis in a doping case involving amphetamine.

A previously published method for single hair analysis has been applied to a doping case for further clarification. Amphetamine could be detected in multiple micro segments resulting in two distinct concentration peaks in several hairs. The consumption of a contaminated food supplement as possible source for the amphetamine is discussed.

Measurement of three-dimensional distributions of drugs in nails using liquid chromatography/tandem mass spectrometry after micro-segmentation to elucidate drug uptake routes.

Ingested drugs can be taken up into nails and hairs from the bloodstream and these drugs can be stably retained over several months or more. Free edges of nails can often be used as a specimen to prove drug intake. However, the mechanism of drug uptake into the nails is unclear. Although it is presumed that there are horizontal uptake routes at the nail root and vertical uptake routes at the nail bed against the direction of growth, three-dimensional distribution analysis is required to verify this phenomenon. Herein, we first developed a method to measure the three-dimensional distribution of drugs in the nails using the combination of micro-segmentation of nails (0.2 × 1.5 × 0.06 mm size) and highly sensitive quantification of drugs by LC-MS/MS. Carbinoxamine was administered as a model compound to a subject in a single dose, and then the free edges of big toenails were collected every several weeks over a year. The three-dimensional distribution of carbinoxamine in each free edge was visualized and arranged along the collection period. Carbinoxamine was localized in the lower layer during the early collection period (up to 78 days after drug ingestion) and in the upper and middle layers later (134 days or later). The changes in the drug distribution in the nails along the collection period implied that two-way drug uptake takes place in the whole nail through both the nail bed and the nail root simultaneously. The developed analytical method could be useful to elucidate the mechanism of drug uptake into the nails.