RESUMEN

DNA adducts are widely recognized as biomarkers of exposure to environmental carcinogens and associated health effects in toxicological and epidemiological studies. This study presents a targeted and sensitive method for comprehensive DNA adductome analysis using ultra-high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS). The method was developed using calf thymus DNA, with careful optimization of mass spectrometric parameters, chromatographic separation conditions, and pretreatment methods. Ultimately, a targeted method was established for 41 DNA adducts, which showed good linearity (R2 ≥0.992), recovery (80.1-119.4 %), accuracy (81.3-117.8 %), and precision (relative standard deviation <14.2 %). The established method was employed to analyze DNA adducts in peripheral blood cells from pregnant women in Shanxi and Beijing. Up to 23 DNA adducts were successfully detected in samples of varying sizes. From 2 µg of maternal DNA samples, seven specific adducts were identified: 5-methyl-2'-deoxycytidine (5-MedC), 5-hydroxymethyl-2'-deoxycytidine (5-HmdC), N6-methyl-2'-deoxyadenosine (N6-MedA), 8-hydroxy-2'-deoxyguanosine (8-OHdG), 5-hydroxy-2'-deoxycytidine (5-OHdC), 1,N6-etheno-2'-deoxyadenosine (1,N6-εdA), and N2-methyl-2'-deoxyguanosine (N2-MedG). This study reveals that exposure to higher concentrations of ambient air pollutants may elevate the levels of DNA methylation and oxidative damage at different base sites, highlighting the application potential of DNA adducts as sensitive biomarkers of air pollution exposure.

Asunto(s)

RESUMEN

Crocus sativus L. (C. sativus) has its stigma as the main valuable part used. With extremely low production and high prices, stigma is considered a scarce resource. As a result, its petals, considered as by-products, are often discarded, leading to significant waste. We developed a UPLC-Q-Orbitrap HRMS method for qualitative analysis of stigmas and petals and a UHPLC-QQQ-MS/MS method for simultaneous quantification of 9 characteristic active compounds for the first time, and compared their biological activity in vitro. The results indicated that a total of 63 compounds were identified in the petals and stigmas. The content of flavonoids in the petals was significantly superior to that in the stigma, and the content of quercetin in the petals was 50 times higher than that in the stigma. The results of the in vitro evaluation of biological activity indicated that both the petals （•OH: IC50=39.70 mg/mL; DPPH: IC50=28.37 mg/mL; ABTS: IC50=0.9868 mg/mL）and stigma （•OH: IC50=34.41 mg/mL; DPPH: IC50=38.99 mg/mL; ABTS: IC50=3.194 mg/mL）demonstrated comparable antioxidant activities. However, the tyrosinase inhibitory activity in petals （IC50=21.17 mg/mL） was weaker than that in stigma（IC50=1.488 mg/mL）. This study provides a fast, reliable, and efficient analytical method that can be used for the quality assessment of petals as a natural resource and its related products in the food and pharmaceutical industries.

Asunto(s)

RESUMEN

This study investigated antibiotic utilization in artisanal dairies and residue occurrence throughout the raw milk cheese production chain using commercial testing (Charm KIS and Eclipse Farm3G) and UHPLC-QqQ-MS/MS and LC-QqQ-MS/MS. The cross-sectional survey results revealed gaps in the producers' knowledge of antibiotic use. Commercial testing detected antibiotic levels close to the LOD in 12.5 % of the samples, mainly in raw milk and whey, with 10.0 % testing positive, specifically in fresh and ripened cheeses, indicating that antibiotics are concentrated during cheese-making. Chromatographically, several antibiotics were identified in the faeces of healthy animals, with chlortetracycline (15.7 ± 34.5 µg/kg) and sulfamethazine (7.69 ± 16.5 µg/kg) predominating. However, only tylosin was identified in raw milk (3.28 ± 7.44 µg/kg) and whey (2.91 ± 6.55 µg/kg), and none were found in fresh or ripened cheeses. The discrepancy between commercial and analytical approaches is attributed to compounds or metabolites not covered chromatographically.

Asunto(s)

RESUMEN

ObjectiveTo establish an ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry（UHPLC-QqQ-MS） for determination of the active ingredients in Erdongtang， and to predict the targets and pathways of anti-insulin resistance action of this formula. MethodThe analysis was performed on an ACQUITY UPLC BEH C18 column（2.1 mm×100 mm， 1.7 μm） with the mobile phase of 0.1% formic acid aqueous solution（A）-acetonitrile（B） for gradient elution（0-3 min， 90%-87%A； 3-6 min， 87%-86%A； 6-9 min， 86%-83%A； 9-11 min， 83%-75%A； 11-18 min， 75%-70%A； 18-19 min， 70%-52%A； 19-22 min， 52%A； 22-25 min， 52%-5%A； 25-27 min， 5%-90%A； 27-30 min， 90%A）. The contents of active ingredients in Erdongtang was detected by electrospray ionization（ESI） and multiple reaction monitoring（MRM） mode under positive and negative ion modes. On this basis， network pharmacology was applied to predict the targets and pathways of Erdongtang exerting anti-insulin resistance effect. ResultThe 20 active ingredients in Erdongtang showed good linear relationships within a certain mass concentration range， and the precision， stability， repeatability and recovery rate were good. The results of determination showed that the ingredients with high content in 15 batches of samples were baicalein（1 259.39-1 635.78 mg·L-1）， baicalin（1 078.37-1 411.52 mg·L-1）， the ingredients with medium content were mangiferin（148.59-217.04 mg·L-1）， timosaponin BⅡ（245.10-604.89 mg·L-1）， quercetin-3-O-glucuronide（89.30-423.26 mg·L-1）， rutin（46.91-1 553.61 mg·L-1）， glycyrrhizic acid（55.97-391.47 mg·L-1）， neomangiferin（37.45-127.03 mg·L-1）， nuciferine（0.89-63.48 mg·L-1）， hyperoside（6.96-136.78 mg·L-1）， liquiritin（30.89-122.78 mg·L-1）， liquiritigenin（26.64-110.67 mg·L-1）， protodioscin（58.57-284.26 mg·L-1）， the ingredients with low content were wogonin（7.16-20.74 mg·L-1）， pseudoprotodioscin（5.49-22.96 mg·L-1）， ginsenoside Rb1（7.31-23.87 mg·L-1）， ginsenoside Rg1（10.78-28.33 mg·L-1）， ginsenoside Re（7.78-24.76 mg·L-1）， ophiopogonin D（2.08-4.29 mg·L-1）， methylophiopogonanone A（0.74-1.67 mg·L-1）. The results of network pharmacology indicated that the mechanism of anti-insulin resistance exerted by Erdongtang might be related to the phosphatidylinositol 3-kinase/protein kinase B（PI3K/Akt） signaling pathway. ConclusionThe established UHPLC-QqQ-MS has the advantages of simple sample processing， strong exclusivity and high sensitivity， and can simultaneously determine the contents of the main ingredients from seven herbs in Erdongtang， which can lay the foundation for the development of Erdongtang compound preparations. The results of the network pharmacology can provide a reference for the mechanism study of Erdongtang in the treatment of type 2 diabetes mellitus.

RESUMEN

This study comprehensively analyzed the active components of Sanhan Huashi Formula using qualitative and quantitative mass spectrometry techniques, laying the foundation for understanding its pharmacological substance basis. UHPLC-LTQ-Orbitrap-MS and GC-MS technologies were used to analyze and identify the volatile and non-volatile components in Sanhan Huashi Formula. UHPLC-QQQ-MS/MS technology was used to simultaneously determine the content of 27 major active components in the formula. The results showed that 308 major chemical components were identified in Sanhan Huashi Formula, among which 60 compounds were identified by comparing with reference standards, mainly including alkaloids, flavonoids, coumarins, triterpenoid saponins, amino acids, and nucleosides. GC-MS technology preliminarily identified 52 volatile compounds, with γ-eudesmol and ß-eudesmol as the main components. The quantitative results demonstrated good linearity(r>0.99) for the 27 active components, indicating the stability, simplicity, and reliability of the established method. Among them, amygdalin, nodakenin, arecoline, ephedrine, and pseudoephedrine had relatively high content and were presumably the main pharmacologically active substances. In conclusion, this study systematically and comprehensively characterized the major chemical components and patterns in Sanhan Huashi Formula, providing a basis for understanding its pharmacological mechanisms and clinical applications.

Asunto(s)

RESUMEN

The recovery of raw materials offers an opportunity for applying the principles of circular bioeconomy. The phenolic composition of three underused wine byproducts (skin, seed, and bunch stem) was analyzed through UHPLC-QqQ-MS/MS to evaluate the intercultivar variability comparing red and white grape cultivars from La Rioja (Spain) and the influence of the winemaking, comparing conventional fermentation and carbonic maceration. We observed that the red skin, especially from Graciano, is rich in anthocyanins, whereas the white skin contains mainly phenolic acids, flavonols, and flavan-3-ols, with Maturana Blanca being the richest variety. Seeds are rich in flavan-3-ols and lignans with Maturana Blanca and Viura, respectively, the richest cultivars. Stems contain high amounts of flavan-3-ols, lignans, and stilbenes, with the red cultivars of Garnacha and Tempranillo being the richest samples. Carbonic maceration has a negative effect on the phenolic amount compared to conventional fermentation. In synthesis, we observed that each type of byproduct from red or white grape cultivars has a particular phenolic composition that can result in obtaining different ingredients with particular phenolic composition for target applications.

Asunto(s)

RESUMEN

Prostaglandins have stimulative influence on the human uterus and therefore were introduced to medical treatment in reproductive healthcare as labor inductors or abortifacients. The UHPLC-ESI-QqQ-MS/MS method was developed for six prostaglandins: carboprost, cloprostenol, dinoprost (PGF2α), dinoprostone (PGE2), misoprostol and sulprostone (substances for pregnancy termination) in pharmaceutical samples and was applied for the toxicological examination of pills containing misoprostol (collected during gynecological examination). There were used two internal standards: misoprostol-d5 and PGF2α-d4. The quantification of analytes was performed in the MRM mode. The linearity of method was in the range from 0.1 to 10 µg/mL, with a coefficient of determination above 0.997 (R2) for each compound. The precision and accuracy values did not exceed ±5.0%. Analysis of the pills revealed the presence of two substances: misoprostol and diclofenac. Misoprostol and diclofenac dose per sample were as follows: 608.8 ng (sample 1), 708.4 ng (sample 2), 618.8 ng (sample 3) and 67.7 mg (sample 1), 65.3 mg (sample 2) 67.3 mg (sample 3), respectively. A simple, precise and reliable method can be applied for routine examinations in terms of clinical and forensic toxicology examinations as well as in quality control of drugs for pharmaceutical purposes (original drugs and counterfeit medications).

RESUMEN

INTRODUCTION: Areca nut is an economic crop and an important component in traditional Chinese medicine (TCM) and ethnomedicine. The crop is rich in alkaloids and flavonoids. Most previous studies have focused on the chemical components, especially alkaloids, in crops from certain areca nut-producing areas. OBJECTIVE: The purpose of this study was to compare the differences in areca nut seeds in two main cultivation areas, identify differential metabolites, and evaluate seed quality in different production areas. METHODS: A widely targeted metabolomics method based on ultrahigh-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QQQ-MS), combined with the TCM systems pharmacology (TCMSP) database and multivariate statistical analysis, was used in this study to maximise the differentiation between quality characteristics of areca nut seeds from China and Southeast Asian regions. RESULTS: Altogether, 1031 metabolites were identified in areca nut seeds; by querying the TCMSP database, 375 metabolites were identified as the main active ingredients. Moreover, the research showed that the metabolic profiles of areca nut seeds from China (ASCN) and Southeast Asia (ASSA) exhibit significant differences, and the difference is mainly reflected in 318 compounds. The relative content of 146 metabolites in ASCN was significantly higher than that in ASSA. Through Kyoto Encyclopedia of Genes and Genomes (KEGG) comparative analysis, areca nut seed metabolites in Chinese production areas were determined to have a wider metabolic pathway. CONCLUSION: The areca nut seeds from cultivation areas possess many metabolites that are beneficial for health, including alkaloids, amino acids, phenolic acids, and lipids. Thus, compared with ASSA, ASCN have a higher medicinal value. This study provides a direction for the subsequent development and utilisation of areca nut seeds.

Asunto(s)

RESUMEN

Glucosinolates (GSLs) and their degradation products in radish confer plant defense, promote human health, and generate pungent flavor. However, the intact GSLs in radish have not been investigated comprehensively yet. Here, an accurate qualitative and quantitative analyses of 15 intact GSLs from radish, including four major GSLs of glucoraphasatin (GRH), glucoerucin (GER), glucoraphenin (GRE), and 4-methoxyglucobrassicin (4MGBS), were conducted using UHPLC-HRMS/MS in combination with UHPLC-QqQ-MS/MS. Simultaneously, three isomers of hexyl GSL, 3-methylpentyl GSL, and 4-methylpentyl GSL were identified in radish. The highest content of GSLs was up to 232.46 µmol/g DW at the 42 DAG stage in the 'SQY' taproot, with an approximately 184.49-fold increase compared to the lowest content in another sample. That the GSLs content in the taproots of two radishes fluctuated in a similar pattern throughout the five vegetative growth stages according to the metabolic profiling, whereas the GSLs content in the '55' leaf steadily decreased over the same period. Additionally, the proposed biosynthetic pathways of radish-specific GSLs were elucidated in this study. Our findings will provide an abundance of qualitative and quantitative data on intact GSLs, as well as a method for detecting GSLs, thus providing direction for the scientific progress and practical utilization of GSLs in radish.

RESUMEN

Pulsatilla chinensis (P.chinensis) is a traditional Chinese medicine used for the treatment of intestinal amebiasis diseases, vaginal trichomoniasis and bacterial infections. Tritepenoid saponins were important components of P.chinensis. Therefore, we asssessmented expression profiling of triterpenoids in different fresh tissues of P.chinensis by ultra high performance liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) and ultra high performance liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-QQQ-MS). Firstly, we identified 132 triterpenoids, including 119 triterpenoid saponins, 13 triterpenoid acids and forty seven of them were first determined in Pulsatilla genus, including new aglycones and new ways of rhamnose linking to the aglycone. Secondly, we established the analytical method to analysis triterpenoids content of P.chinensis and comprehensively verified the analytical method by linearity, precision, repeatability, stability and recovery. At last, we quantified 119 triterpenoids simultaneously based on UHPLC-QQQ-MS. The results show that the types and contents of triterpenoids had obvious tissue distribution. New components like rhamnose directly linked to the aglycone mainely distributed in aboveground tissues. Additionally, We identified 15 chemical ingredients as differential components between the aboveground and underground tissues of P.chinensis. This study provides an efficient analysis strategy for the qualitative and quantitative analysis of triterpenoids in P.chinensis even in other traditional Chinese medicines. At the same time, it provides important informations to explain the biosynthetic pathway of triterpenoid saponins in P.chinensis.

Asunto(s)

RESUMEN

Dendrobium officinale Kimura et Migo (D. officinale), one of the nine everlasting types of grass, has gained increasing attention owing to its important roles in alternative medicines and drug discovery. Due to its natural resources being in danger of being extinct, imitation wild planting is becoming increasingly common. To assess the product's quality completely, an efficient ultrahigh performance liquid chromatography-triple quadrupole tandem mass spectrometry (UHPLC-QQQ-MS/MS) method was established to simultaneously quantify nine phenolic compounds in D. officinale samples. The extraction parameters, including solvent, solvent concentration, solid-liquid ratio, and extraction time, were systematically optimized with the single-factor test. The results demonstrated that extraction with a 1:200 solid-to-liquid ratio of 80% methanol for 1.5 h was the most efficient condition for the extraction of flavonoids. Satisfactory retention times and resolution of the nine analytes were acquired on the Thermo Scientific Hypersil GOLD column with multiple reaction monitoring in negative ion scanning mode. The method was validated to demonstrate its selectivity, linearity, precision, accuracy, and robustness. Thus, the verified UHPLC-QQQ-MS/MS method was successfully applied to the quantification of phenolic components present in D. officinale samples. The results indicated that the quantity and composition of phenolic components in D. officinale from various provenances were significantly different. This work provides a theoretical foundation for the cultivation and assessment of wild D. officinale quality.

RESUMEN

Polar plant growth regulators, used alone or doped in fertilizers, are most effective and widely utilized plant growth regulators (PGRs) in agriculture, which play important roles in mediating the yield and quality of crops and foodstuffs. The application scope has been extended to herbal medicines in the past 2 decades and relevant study is inadequate. The aim of this study is to establish a QuPPe-based extraction method containing low-temperature and d-SPE cleanup procedure followed by the detection on a selective multiresidue ultrahigh-performance liquid chromatography - triple quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS) in three herbal matrices. This simple, accurate, versatile and robust method was verified according to the validation criteria of the SANTE/12682/2019 guideline document. The analytical range was from 2.5 to 200 µg/L, and the average recoveries were in the range of 64.6-117.8% (n = 6). The optimized method was applied to 135 herbal medicines thereof. Result showed that the detection frequency of chlormequat was the highest in the investigated PGRs, with the positive rate of 15.6%. Improvement of the detection method for polar PGRs will enrich the coverage of PGRs, which is conducive to safeguard public health and ensure drug safety. Supplementary Information: The online version contains supplementary material available at 10.1007/s10337-023-04254-3.

RESUMEN

Yunnan pickled tea is produced from fresh tea-leaves through fixation, rolling, anaerobic fermentation and sun-drying. In this study, widely targeted metabolomics using UHPLC-QQQ-MS/MS and HPLC analysis were carried out to elaborate its quality formation during the whole process. Results confirmed the contribution of preliminary treatments and anaerobic fermentation to the quality formation. A total of 568 differential metabolites (VIP > 1.0, P < 0.05, FC > 1.50 or < 0.67) were screened through OPLS-DA. (-)-Epigallocatechin and (-)-epicatechin significantly (P < 0.05) increased from the hydrolyzation of ester catechins, such as (-)-epigallocatechin gallate and (-)-epicatechin gallate in anaerobic fermentation. Additionally, the anaerobic fermentation promoted vast accumulations of seven essential amino acids, four phenolic acids, three flavones and flavone glycosides, pelargonidin and pelargonidin glycosides, flavonoids and flavonoid glycosides (i.e. kaempferol, quercetin, taxifolin, apigenin, myricetin, luteolin and their glycosides) through relevant N-methylation, O-methylation, hydrolyzation, glycosylation and oxidation.

Asunto(s)

RESUMEN

This study aimed to develop and evaluate a method for simultaneously analyzing malondialdehyde (MDA) and two typical toxic α,ß-unsaturated aldehydes, 4-hydroxy-2-hexenal (HHE), and 4-hydroxy-2-nonenal (HNE), using ultra-high-performance liquid chromatography with triple quadrupole mass spectrometry (UHPLC-QqQ-MS/MS). The method possessed widely linear range (10-1000 ng/mL). The limit of detection (LOD) and limit of quantitation (LOQ) of MDA, HHE, and HNE were 2.0 and 5.0 ng/g, 2.0 and 5.0 ng/g, and 0.1 and 0.3 ng/g, respectively; the recovery rates all fall into 95.56-104.22 %. The method was sufficiently precise (<5%), and not affected by the analysis matrix. Application to 17 food products revealed total MDA, HHE, and HNE contents were 0.11-3.56, 0.05-3.32, and 0.09-3.70 µg/g, respectively. It will be useful in future research on the influence of food composition and main substrate structure on the generation and distribution of these three aldehydes and the implementation of corresponding control methods.

Asunto(s)

RESUMEN

Stilbenes, especially resveratrol and resveratrol dimers, can quench singlet oxygen (1O2) effectively. Studies found resorcinol, catechol, carbon-carbon double bonds in resveratrol dimers and resveratrol monomers all contributed to quenching 1O2. However, which structures play a key role in quenching of 1O2 by stilbenes had not yet been determined. To explore it, UHPLC-QQQ-MS2 and UHPLC-QTOF-MS2 were used to analyze and compare the 1O2 quenching activities of piceatannol, resveratrol, dihydroresveratrol, pterostilbene, trimethoxystilbene and oxyresveratrol in vitro. The results showed that all six compounds exhibited some capacity to quench 1O2. Catechol [i.e., C6H4(OH)2] had the strongest capacity to quench of 1O2 amongst the stilbenes tested followed by the presence of carbon-carbon double bonds. This offers insight a route for screening for stilbenes with higher activities that might have a role in development of novel food-related antioxidants and functional foods with potential health benefits.

Asunto(s)

RESUMEN

Synthetic cathinones comprise a large amount of substances present on the dark market, which creates an undeniably worldwide problem and still is posing a threat. A 22-year-old man was brought to the Emergency Room from a party, where he had ingested orally 20 g of mephedrone. The man exhibited a disorder of consciousness with no logical verbal contact and dilated pupils. Moreover, a metabolic acidosis was present. The patient died after an hour from an admission to the ER. Blood and vitreous humor collected during an autopsy were analyzed with the use of an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-QqQ-MS/MS) with the use of C18 column in multiple reaction monitoring (MRM) mode. Both biological specimens were prepared using liquid-liquid extraction (LLE) with the use of ethyl acetate and 0.5 M ammonium carbonate water solution (pH 9). The limit of quantification (LOQ) of the method was 0.5 ng/ml in both matrices; precision and accuracy values did not exceed ±15%. Recovery of the method was in the range of 86.1%-102.7%. Determined concentrations of 4-CMC were 8542 and 9874 ng/ml in blood and vitreous humor, respectively. Other substances present in both biological materials were: atropine, diazepam, lidocaine, and its metabolite norlidocaine, as well as methcathinone and ethyl alcohol. The concentration presented in here described case is the highest ever reported 4-CMC concentration. Important aspect is also receiving other NPS by recreational users than intended, which lead to accidental poisoning (in presented case user assumed 4-CMC was 4-MMC).

Asunto(s)

RESUMEN

A quantitative analysis method for six principal active constituents (acubin, geniposidic acid, chlorogenic acid, pinoresinol di-O-glucopyranoside, geniposide, and pinoresinol 4-O-glucopyranoside) of crude Eucommiae Cortex (EC) and its salt-processed product extracts was developed to investigate and compare their pharmacokinetic behaviors in adenine-induced renal fibrotic rats in vivo. UHPLC-QqQ-MS/MS technology was employed. Scan was conducted in negative ion mode and quantitative determination was carried out by MRM paired ion. The established method was fully validated by specificity, linearity, precision, accuracy, stability, recovery, and matrix effect, and the results of methodological investigation met the requirements of biological sample analysis. Then, a quick, sensitive, and accurate method was successfully established, which could simultaneously measure the contents of six active constituents of crude and salt-processed EC extracts in rat plasma. After a single administration to renal fibrotic rats of crude EC and its salt-processed product extracts, the plasma concentration of each constituent at different time points was measured, the pharmacokinetic parameters were calculated and the concentration time curves were structured. The experiment was approved by the experimental animal ethics committee from Nanjing University of Chinese Medicine (No. 202103A008). The results showed that compared to the crude Eucommiae Cortex group, the tmax of aucubin, pinoresinol di-O-glucopyranoside, geniposide, and pinoresinol 4-O-glucopyranoside in the salt-processed Eucommiae Cortex group rat plasma were significantly lower than those in the crude group (P < 0.05, P < 0.01); the Cmax and AUC0-48 h of chlorogenic acid, the Cmax, AUC0-48 h and AUC0-∞ of pinoresinol di-O-glucopyranoside, and the Cmax of geniposide and pinoresinol 4-O-glucopyranoside were significantly higher than those in the crude group (P < 0.05, P < 0.01). Our investigation found that compared to crude Eucommiae Cortex, a variety of active ingredients could play a role of quick effect with higher peak blood concentration and bioavailability after oral administration of salt-processed Eucommiae Cortex, which were consistent with the traditional Chinese medicine theory of "salt-processing enhancing drug into kidney meridian", providing an experimental basis for the selection of quality control indexes and the in-depth study of processing mechanisms and metabolic rules in vivo of Eucommiae Cortex and its salt-processed product.

RESUMEN

Benzodiazepines exhibit central nervous system depressive activity as well as sedative, hypnotic, and anticonvulsant properties, which enable to use them as medical treatment in anxiety, epilepsy, insomnia and alcohol withdrawal syndrome. However, from 2000s illegal benzodiazepine derivatives have started to emerge on illicit drug market as new psychoactive substances (NPSs) monitored in many countries. Analysis of both pharmaceutical drugs and NPSs from benzodiazepines group could be challenging, as usually very low concentrations need to be determined. Thus, an ultra-sensitive UHPLC-QqQ-MS/MS method was developed for simultaneous determination of 54 benzodiazepines (pharmaceutical drugs, NPS and their metabolites) and 3 z-drugs with one metabolite in biological fluid samples. The lower limit of quantification for most substances was 50 pg/mL, whereas for 17 substances as low as 10 pg/mL was achieved. Together with reduced sample volume to 100 µL it makes the developed method suitable for a sensitive multidrug toxicological analysis. Presented method was applied in routine toxicological practice as well as for the determination of benzodiazepines, z-drugs and their metabolites in 25 authentic biological fluids (blood, urine, vitreous humor and bile), both antemortem and postmortem. 19 different compounds, including benzodiazepines, their metabolites and z-drugs were determined. Antemortem blood concentrations were within 0.2-114.5 ng/mL, whereas concentrations in antemortem urine samples were 0.03-102.6 ng/mL. In postmortem specimens, concentrations ranged within 0.2-473.2 ng/mL, 0.5-94.1 ng/mL, 1.3-208.8 ng/mL and 41.5-42.0 ng/mL in blood, vitreous humor, urine and bile, respectively. The developed method is suitable for a forensic toxicology analysis, as well as clinical toxicology which is evidenced by the positive results of international proficiency tests.

Asunto(s)

RESUMEN

This study comprehensively analyzed the active components of Sanhan Huashi Formula using qualitative and quantitative mass spectrometry techniques, laying the foundation for understanding its pharmacological substance basis. UHPLC-LTQ-Orbitrap-MS and GC-MS technologies were used to analyze and identify the volatile and non-volatile components in Sanhan Huashi Formula. UHPLC-QQQ-MS/MS technology was used to simultaneously determine the content of 27 major active components in the formula. The results showed that 308 major chemical components were identified in Sanhan Huashi Formula, among which 60 compounds were identified by comparing with reference standards, mainly including alkaloids, flavonoids, coumarins, triterpenoid saponins, amino acids, and nucleosides. GC-MS technology preliminarily identified 52 volatile compounds, with γ-eudesmol and β-eudesmol as the main components. The quantitative results demonstrated good linearity(r>0.99) for the 27 active components, indicating the stability, simplicity, and reliability of the established method. Among them, amygdalin, nodakenin, arecoline, ephedrine, and pseudoephedrine had relatively high content and were presumably the main pharmacologically active substances. In conclusion, this study systematically and comprehensively characterized the major chemical components and patterns in Sanhan Huashi Formula, providing a basis for understanding its pharmacological mechanisms and clinical applications.

Asunto(s)

RESUMEN

The aim of the study was the development and validation of the UHPLC-QqQ-MS/MS method for the determination of mifepristone in human blood as well as the identification and quantification of its metabolites after self-induced pharmacological abortion. The metabolic pathway in humans was proposed after examination of an authentic casework. The fast and simple preanalytical procedure was successfully applied (pH9, tert-butyl-methyl ether). The validation parameters of the method were as follows: limit of quantification: 0.5 ng/mL; coefficients of determination: >0.999 (R2), intra- and inter-day accuracy and precision values did not exceed ± 13.2%. The recovery and matrix effect were in the range of 96.3−114.7% and from −3.0 to 14.7%, respectively. Toxicological analysis of the mother's blood (collected the day after the pregnancy termination) revealed the presence of five compounds: mifepristone (557.4 ng/mL), N-desmethyl-mifepristone (638.7 ng/mL), 22-OH-mifepristone (176.9 ng/mL), N,N-didesmethyl-mifepristone (144.5 ng/mL) and N-desmethyl-hydroxy-mifepristone (qualitatively). To our knowledge, the study presented in this paper is the first report on the concentrations of mifepristone and its metabolites in maternal blood samples after performing a self-induced abortion. The established UHPLC-QqQ-MS/MS method is suitable for forensic toxicological analysis as well as in terms of clinical toxicology in future investigations (examination of pharmacokinetics, bioavailability and metabolism of RU-486).

Asunto(s)