Fast detection of tryptamine in meat products with azide-functionalized covalent organic frameworks confined in molecularly imprinted polymers.

Tryptamine is a biogenic amine that affects organoleptic quality through the generation of off-odours in foods. Herein, imine-based covalent organic frameworks (COFs) were synthesized via Schiff base reactions and postmodified with click chemistry to generate azide-functionalized COFs with tunable azide units on the walls. The combination of molecular imprinting with COFs enabled the specific recognition of the targets. The resulting optosensing system (azide-functionalized COFs@MIPs) was used as a sample-to-answer analyser for detecting tryptamine (detection time within 10 min). A linear relationship was observed for the fluorescence response to tryptamine concentrations in the range of 3-120 µg L-1, with a limit of detection of 1.74 µg L-1. The recoveries for spiked samples were satisfactory, with relative standard deviations <9.90%. The optosensing system is a potential tool for the quantitative detection of tryptamine in meat products because of its lower cost, shorter processing time, and simpler processing steps compared to conventional chromatographic techniques.

Facile and green chemistry-compatible fluorescence spectroscopic applications of acid red 87 used to evaluate eletriptan, antimigraine, in its pharmaceutical and biological samples.

Eletriptan (ETR), a selective pharmaceutical agent agonist of the 5-hydroxytryptamine1 receptor subtype, are primarily used to treat acute migraine attacks. ETR is a triptan-class medication that works by narrowing cerebral blood vessels and reducing chemicals that produce headache pain, light and sound sensitivity, and nausea. Due to its effectiveness in reducing migraine symptoms, it is a worthwhile choice for those looking for quick and efficient treatment. A green, raid, one-pot and straightforward fluorescence spectrometric method was employed to evaluate ETR in tablets and biological samples. By introducing the ETR drug and the fluorescent ligand, Acid red 87, in an acidic buffer, a quenching of the ligand native fluorescent was promptly produced. The quenching action was simply attributed to the selective ion-pair complex generation between the cationic target and the selected ligand. An increase in ETR concentration was linearly proportional to the quenching response in the 50.0 - 500.0 ng/mL range. The optimal configurations for adjusting the system's variable parameters were determined by examining the ETR-Acid red 87 system's response. Additionally, the sensor that was developed met the standards set by the International Council for Harmonisation of Technical Requirements of Pharmaceuticals for Human Use. The sensitivity thresholds of the approach were 13.8 and 42.0 ng/mL for the detection and quantification parameters, respectively, LOD and LOQ. This approach proficiently evaluated the pharmaceutical and biological samples of ETR. Finally, the proposed approach not only simplifies the analysis process but also limits the badimpact on the environment, making it a promising technique for analytical applications.

Survey of the biogenic amines in craft beer from the Chinese market and the analysis of the formation regularity during beer fermentation.

This study evaluated the levels of eight biogenic amines in 59 craft beers of five styles and monitored the changes during beer fermentation, showing that putrescine and tryptamine were the most abundant at maximum values of 46.14 mg/L and 89.97 mg/L, respectively. This research indicated for the first time that dark beer, such as Stout/Porter, displayed the highest total biogenic amine content due to considerable tryptamine accumulation, with a maximum value of 116.95 mg/L. The total biogenic amine level increased gradually during the segmental saccharification and main fermentation stages, representing the two critical control points for their formation during beer fermentation. This study provides a theoretical basis and technical guidance for the safe and standardized production of craft beer and the formulation of biogenic amines limit standards, which is highly significant for protecting the health of consumers.

Effect of collagen casing on the quality characteristics of fermented sausage.

OBJECTIVE: Fermented sausage is popular all over the world for its rich nutrition and unique flavor. Sausage casing is one of the key factors affecting the quality of fermented sausage. However, there is little information involved in this field. METHODS: In this study, collagen casings were used as a wrapping material, and natural casings (pig casings) were used as a control. The effects of the two types of casings on biogenic amine content and other quality characteristics of fermented sausage were analyzed with increasing the storage time. RESULTS: The results showed that with storage time increasing, the hardness and gumminess of fermented sausage in collagen casing (CC) group were higher than those in pig casing (PC) group (P<0.05), while the elasticity in CC group was lower than that in PC group (P<0.05). In the processing and storage period, there was no significant difference in the type and content of flavor substances between the two groups. More importantly, the contents of tryptamine, putrescine, cadaverine, histamine, tyramine and phenyethylamine in fermented sausage of CC group were lower than those in PC group (P<0.05). CONCLUSION: In conclusion, this study revealed that CC could improve the quality characteristics of fermented sausage and reduce the content of biogenic amines in fermented sausage, which provides a theoretical basis for the choice of casings in industrial production in the future.

Determination of 8 biogenic amines in aquatic products and their derived products by high-performance liquid chromatography-tandem mass spectrometry without derivatization.

A method for the determination of 8 biogenic amines in aquatic products and their derived products was established by HPLC-MS/MS without derivatization. The samples were extracted by 5% perchloric acid solution. N-hexane was used to clean the extract. The analytes were separated by a column of ACQUITY UPLC HSS T3 (100 mm × 2.1 mm, 1.8 µm), and gradient eluted with a mixed solution of (0.5% formic acid) and acetonitrile. Good linearity was obtained with correlation coefficients (R2) >0.99. This method achieved higher sensitivity (from 0.1 mg/kg for tyramine, 2-phenylethylamine and tryptamine to 1.0 mg/kg for spermidine, spermine, cadaverin, histamine and putrescine). The average recoveries were demonstrated in the range of 70.9%-113.1%, with relative standard deviations (RSDs) from 0.33% to 10.81%. This method was suitable for the detection of BAs in aquatic products and their products.

Murexide-derived in vitro electrochemical sensor for the simultaneous determination of neurochemicals.

This work highlights the protocol employed for the simultaneous electroanalysis of tryptamine, serotonin and dopamine using a conducting poly-murexide-based electrode. To date, this is the first-of-its-kind report of simultaneous electrochemical determination of these three targets. Features of the developed electrode were identified by employing FE-SEM analysis. Under optimized conditions, the analytes underwent an irreversible electro-oxidation at the modified electrode surface, with a linear range of 0.5-40 µΜ, 0.4-40.4 µΜ and 0.5-40 µΜ for dopamine, serotonin and tryptamine, respectively. The electrolytic medium employed for the sensing was a phosphate-buffered solution with pH 7. The specificity of the developed electrode was also satisfactory in the presence of other biomolecules including L-phenylalanine, L-serine, glucose and ascorbic acid. Thus, the developed murexide-derived conducting-polymer-based electrode was used for the simultaneous sensing of the neurochemicals dopamine, serotonin and tryptamine. Electroanalysis was also demonstrated for these targets in human serum.

Transfer of triptans into human breast milk and estimation of infant drug exposure through breastfeeding.

Clinical data on the transfer of triptans into human breast milk remain scarce. In a lactation study including 19 breastfeeding women with migraine, we examined the excretion of six different triptans into milk. Following intake of a single dose, each participant collected seven breast milk samples at predefined intervals up to 24 hours after dose. Triptan concentrations in milk were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Infant drug exposure was estimated by calculating the relative infant dose (RID). Twenty-two breast milk sample sets were obtained for sumatriptan (n = 8), rizatriptan (n = 5), zolmitriptan (n = 4), eletriptan (n = 3), almotriptan (n = 1) and naratriptan (n = 1). Based on the average concentration in milk throughout the day, estimated mean RIDs (with range in parenthesis) were as follows: eletriptan 0.6% (0.3%-0.8%), sumatriptan 0.7% (0.2%-1.8%), rizatriptan 0.9% (0.3%-1.4%), almotriptan 1.8% (-), zolmitriptan 2.1% (0.7%-5.3%) and naratriptan 5.0% (-). Infant drug exposure through breastfeeding appears to be low and indicates that use of the triptans in this study is compatible with breastfeeding. Naratriptan may not be first choice in breastfeeding mothers initiating triptans during the neonatal period.

Determination of six underivatized biogenic amines by LC-MS/MS and study of biogenic amine production during trout (Salmo trutta) storage in ice.

Biogenic amines (BAs) are natural components of food produced mainly during metabolism in animals and plants. The determination of BAs is important because of their potential toxicity and their potential use as food spoilage indicators. In the present study, a method for the determination of six BAs (putrescine, cadaverine, histamine, ß-phenylethylamine, tyramine, and tryptamine) by Liquid Chromatography - Tandem Mass Spectrometry (LC-MS/MS) with Atmospheric Pressure Chemical Ionisation (APCI) source has been used on trout samples (Salmo trutta) stored in ice for 15 days. The results showed that on day 15 quite large amounts of putrescine (76.530 mg/kg), cadaverine (85.530 mg/kg), tryptamine (25.210 mg/kg), and histamine (15.975mg/kg) were detected, while the other BAs remained low (ß-phenylethylamine: 3.230 mg/kg, tyramine: 0.165mg/kg). Furthermore, microbiological data (Total Vial Count- TVC, Pseudomonas spp, and Shewanella putrefaciens) showed that trout samples became organoleptically unacceptable on day 12, while volatile compound analysis showed a significant increase in total amounts of alcohols, aldehydes, and ketones on days 12 and 15.

UHPLC-MS/MS method for simultaneously detecting 16 tryptamines and their metabolites in human hair and applications to real forensics cases.

Tryptamines are hallucinogenic substances many of which have appeared recently as novel psychoactive substances (NPS). Herein, we describe the establishment of a rapid UHPLC-MS/MS quantitative method for the targeted screening of 16 tryptamines of abuse in hair. Twenty milligram pieces of hair were pulverized below 4 °C in 0.5 mL of deionized water containing 0.1% formic acid and an internal standard (2 ng/mL psilocin-d10 and psilocybin-d4). After subsequent centrifugation, 5 µL of the supernatant was injected into a LC-MS/MS system fitted with a Waters Acquity UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 µm). The column was gradient eluted at 0.3 mL/min with mobile phases composed of 20 mmol/L ammonium acetate, 5% acetonitrile, and 0.1% formic acid in water (solvent A) and acetonitrile (solvent B). Limits of detection ranged between 0.1 and 20 pg/mg, with limits of quantitation ranging from 3 to 50 pg/mg. The calibration curves for all analytes were linear (r > 0.992). Accuracies varied between 91% and 114%, with intraday precision RSDs < 14% and interday precision RSDs of between 1.3% and 14%. The recoveries of all tryptamines were in the 85-115% range, with the matrix effect ranging from 95% to 112%. The validated method was successfully used to analyse 191 hair samples from suspected tryptamine users, 77 of which were 5-MeO-DiPT-positive, while the 16 tryptamines and their metabolites were not detected in the remaining 114 hair samples. 5-MeO-DiPT and its 5-MeO-NiPT, 5-OH-DiPT, and 4-OH-DiPT metabolites were concurrently detected in 34 hair samples. 5-MeO-DiPT, as the parent drug, was the parent substance found in the hair samples.

Investigation of Ayahuasca ß-Carboline Alkaloids and Tryptamine in Sweat Samples from Religious Community Participants by GC-MS.

Ayahuasca, a hallucinogenic beverage used in religious rituals in South America, has become a global phenomenon. Its main active components are the ß-carbolines alkaloids, harmine (HRM) and harmaline (HRL), as well as the potent hallucinogen N,N-dimethyltryptamine (DMT). Despite its rising consumption, information regarding possible clinical applications and toxicological effects of ayahuasca is still limited. This study presents the first investigation of the use of sweat for the determination of DMT, HRM and HRL in ayahuasca users during a religious ritual. Sweat is an alternative matrix with advantages over many conventional biological samples, mainly because the collection procedure is non-invasive, easy and simple and samples can be collected without disturbing the religious ritual. In the study, solid-phase extraction was performed under basic conditions. Linearity was observed ranging from 20 to 1500 ng/patch with coefficients of determination (R2) higher than 0.99 for all analytes. The results indicated high selectivity for all investigated analytes, with extraction efficiency exceeding 70%, accuracy ranging from 87.5 to 102.4%, intra-assay precision of 1.85-9.44% and inter-assay precision between 3.34 and 9.85%. The limits of detection were 15 ng/patch for HRM and HRL and 10 ng/patch for DMT. The sweat proved to be a viable option to monitor ayahuasca use.

Highly sensitive carbon-based nanohybrid sensor platform for determination of 5-hydroxytryptamine receptor agonist (Eletriptan).

Highly sensitive nanosensors such as graphene oxide/ platinum-iridium nanohybrid, carboxylic acid functionalized multiwalled carbon nanotubes (GO/Pt-Ir/MWCNT-COOH) and amine functionalized multiwalled carbon nanotubes (GO/Pt-Ir/MWCNT-NH2) modified glassy carbon electrode were developed for the determination of 5-hydroxytryptamine receptor agonist, Eletriptan. Graphene oxide/platinum-iridium nanohybrid was synthesized using sonication method and then characterized by spectroscopic and microscopic methods such as Raman, TEM, HRTEM, XPS, and XRD. The prepared nanohybrids modified on glassy carbon electrodes were well characterized and applied for electrochemical determination of Eletriptan. The significant enhancement of the oxidation peak current of Eletriptan was observed in GO/Pt-Ir/MWCNT-COOH as a best nanosensor in all prepared ones. The pH, scan rate and the amount of GO/Pt-Ir/MWCNT-COOH were also optimized for Eletriptan analysis. After obtaining of the optimum conditions, the identification of Eletriptan was performed between the linear range of 1 × 10-7 M and 4 × 10-6 M with a detection limit of 6.1 × 10-9 M. The developed method was successfully applied for the determination of the drug in tablets with acceptable recoveries. Moreover, it can be elicited that, in electrochemical studies, electroactive interferences from the tablet excipients did not interfere with the results.

Microbial diversity and chemical analysis of Shuidouchi, traditional Chinese fermented soybean.

Shuidouchi is a traditional Chinese fermented soybean product and its quality is largely affected by the microbes involved in the fermentation. In this study, eleven Shuidouchi samples were collected from southwest China and the microbial diversity and its correlations with chemical characteristics were investigated. Bacterial community was detected using 16S rRNA sequencing, along with bacterial and fungal viable plate counts. Biogenic amines and other chemical characteristics were determined by HPLC and corresponding chemical reaction methods. Among eleven Shuidouchi samples, 21 phyla and 356 genera were identified. Firmicutes, Bacteroidetes and Proteobacteria were the predominant phyla while Bacillus, Bacteroides and Lactobacillus were the main genera. The average cell number of bacteria, lactic acid bacteria and fungi were 1.6 × 106, 5.9 × 104 and 7.6 × 103 CFU/g, respectively. HPLC results showed that the mean concentration of tryptamine, ß-phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine and spermine were 23.11, 3.66, 12.21, 7.12, 8.13, 22.98, 24.72, and 39.00 mg/kg, respectively. The average content of other characteristics including amino acid nitrogen, titratable acidity, and reducing sugar were 2.08, 3.44, and 25.78 g/kg, respectively. Shuidouchi samples were slightly acidic or neutral. Fibrinolytic enzyme activity was detected only in one sample. Among top 52 identified genera, 9 genera showed positive correlations with the chemical characteristics of Shuidouchi while 15 genera were negatively associated. Our results indicated that Shuidouchi contained rich microbial resources and were edible safety based on the tested indexes. The associations identified between microbes and chemical characteristics could be further utilized in the food fermentation industry.

Investigating the ability of the microbial model Cunninghamella elegans for the metabolism of synthetic tryptamines.

Tryptamines can occur naturally in plants, mushrooms, microbes, and amphibians. Synthetic tryptamines are sold as new psychoactive substances (NPS) because of their hallucinogenic effects. When it comes to NPS, metabolism studies are of crucial importance, due to the lack of pharmacological and toxicological data. Different approaches can be taken to study in vitro and in vivo metabolism of xenobiotica. The zygomycete fungus Cunninghamella elegans (C. elegans) can be used as a microbial model for the study of drug metabolism. The current study investigated the biotransformation of four naturally occurring and synthetic tryptamines [N,N-Dimethyltryptamine (DMT), 4-hydroxy-N-methyl-N-ethyltryptamine (4-HO-MET), N,N-di allyl-5-methoxy tryptamine (5-MeO-DALT) and 5-methoxy-N-methyl-N-isoporpoyltryptamine (5-MeO-MiPT)] in C. elegans after incubation for 72 hours. Metabolites were identified using liquid chromatography-high resolution-tandem mass spectrometry (LC-HR-MS/MS) with a quadrupole time-of-flight (QqTOF) instrument. Results were compared to already published data on these substances. C. elegans was capable of producing all major biotransformation steps: hydroxylation, N-oxide formation, carboxylation, deamination, and demethylation. On average 63% of phase I metabolites found in the literature could also be detected in C. elegans. Additionally, metabolites specific for C. elegans were identified. Therefore, C. elegans is a suitable complementary model to other in vitro or in vivo methods to study the metabolism of naturally occurring or synthetic tryptamines.

Analysis of bacterial diversity and biogenic amines content during the fermentation processing of stinky tofu.

Fermented foods may confer several benefits to human health and play an important role in a healthy and balanced diet. Stinky tofu is an important traditional fermented soy product in Asia. In this study, a comprehensive analysis of bacterial diversity in terms of rarefaction abundance, rank abundance, alpha diversity, and principal coordinates analysis was conducted using next-generation sequencing. The results obtained were further verified by culture-dependent methods. In addition, eight biogenic amines were also quantified to determine their levels in stinky tofu during conventional production. Our study revealed that lactobacillus was the major lactic acid bacteria throughout the fermentation process. Further, some potentially harmful bacterial belonging to the genera Halomonas and Solobacterium, were also found existing in the stinky tofu. In case of biogenic amines, the concentration of putrescine, cadaverine and histamine finally increased to high levels while spermine disappeared after the fermentation was completed. After the soaking step, putrescine, tryptamine and ß-phenethylamine increased sharply. This work provided an improved understanding of microbes and biogenic amines content associated with the traditionally consumed fermented food. However, there is a need to understand microbial interactions related to biogenic amines during the fermentation, and the origin and survival strategies of two important pathogens detected in our study.

Competitive fluorescent pseudo-immunoassay exploiting molecularly imprinted polymers for the detection of biogenic amines in fish matrix.

We developed a competitive fluorescent molecularly imprinted polymer (MIP) assay to detect biogenic amines in fish samples. MIPs synthesized by precipitation polymerization using histamine as template were used in a batch binding assay analogous to competitive fluoroimmunoassays. Introducing a complex sample matrix, such as fish extract, into the assay changes the environment and the binding conditions, therefore the importance of the sample preparation is extensively discussed. Several extraction and purification methods for fish were comprehensively studied, and an optimal clean-up procedure for fish samples using liquid-liquid extraction was developed. The feasibility of the competitive MIP assay was shown in the purified fish extract over a broad histamine range (1 - 430µM). The MIP had the highest affinity towards histamine, but recognized also the structurally similar biogenic amines tyramine and tryptamine, as well as spermine and spermidine, providing simultaneous analysis and assessment of the total amount of biogenic amines.

Stability-Indicating Reversed-Phase UHPLC Method Development and Characterization of Degradation Products of Almotriptan Maleate by LC-QTOF-MS/MS.

Almotriptan maleate (ALMT), a highly selective 5-hydroxy tryptamine 1B/1D (5-HT1B/1D) receptor agonist used in the treatment of migraine headache was subjected to various ICH (Q1A (R2)) specified guidelines. The drug underwent significant degradation under hydrolytic (acid, base and neutral), oxidative and photolytic stress conditions, while it was stable under thermal stress condition. A total of seven significant degradation products (DPs) were obtained. A simple, selective and reliable UPLC method has been developed for the separation of ALMT and its DPs using Acquity UPLC HSS Cyano (100 × 2.1 mm, 1.8 µm) column with mobile phase consisting of ammonium acetate (10 mM, pH 4.4) buffer and acetonitrile in gradient elution mode. Chromatographic analysis was performed at a flow rate of 0.3 mL/min using a PDA detector at a wavelength of 230 nm. All the DPs (DP-1 to DP-7) were characterized using UHPLC-ESI-QTOF based on mass fragmentation pattern and accurate m/z values. The developed UPLC method was validated in terms of specificity, linearity, precision and accuracy. The developed stability-indicating method helps in quantification of drug in the presence of DPs.

Evaluation of electrochemical, UV/VIS and Raman spectroelectrochemical detection of Naratriptan with screen-printed electrodes.

Naratriptan, active pharmaceutical ingredient with antimigraine activity was electrochemically detected in untreated screen-printed carbon electrodes (SPCEs). Cyclic voltammetry and differential pulse voltammetry were used to carry out quantitative analysis of this molecule (in a Britton-Robinson buffer solution at pH 3.0) through its irreversible oxidation (diffusion controlled) at a potential of +0.75V (vs. Ag pseudoreference electrode). Naratriptan oxidation product is an indole based dimer with a yellowish colour (maximum absorption at 320nm) so UV-VIS spectroelectrochemistry technique was used for the very first time as an in situ characterization and quantification technique for this molecule. A reflection configuration approach allowed its measurement over the untreated carbon based electrode. Finally, time resolved Raman Spectroelectrochemistry is used as a powerful technique to carry out qualitative and quantitative analysis of Naratriptan. Electrochemically treated silver screen-printed electrodes are shown as easy to use and cost-effective SERS substrates for the analysis of Naratriptan.

The impacts of temperature, alcoholic degree and amino acids content on biogenic amines and their precursor amino acids content in red wine.

The aim was to study how factors such as temperature, alcoholic degree, and amino acids supplementation are able to influence the content of tyramine, histamine, 2-phenylethylamine, tryptamine and their precursor amino acids in winemaking process. Biogenic amines and amino acids were quantified at the beginning, middle and end of alcoholic fermentation, and at the end of malolactic fermentation. In general, samples produced with amino acid supplementation did not show the highest concentrations of biogenic amines, except for histamine, which content increased with the addition of the four amino acids. The synthesis of tyramine was mainly affected by the temperature and alcoholic degree, the formation of phenylethylamine was largely influenced by alcoholic degree, and tryptamine synthesis principally depended on temperature. Interestingly, there was interaction between these three factors for the biogenic amines studied. In conclusion, winemaking conditions should be established depending on the biogenic amine which synthesis is required to be controlled.

Optimal Hotspots of Dynamic Surfaced-Enhanced Raman Spectroscopy for Drugs Quantitative Detection.

Surface-enhanced Raman spectroscopy (SERS) as a powerful qualitative analysis method has been widely applied in many fields. However, SERS for quantitative analysis still suffers from several challenges partially because of the absence of stable and credible analytical strategy. Here, we demonstrate that the optimal hotspots created from dynamic surfaced-enhanced Raman spectroscopy (D-SERS) can be used for quantitative SERS measurements. In situ small-angle X-ray scattering was carried out to in situ real-time monitor the formation of the optimal hotspots, where the optimal hotspots with the most efficient hotspots were generated during the monodisperse Au-sol evaporating process. Importantly, the natural evaporation of Au-sol avoids the nanoparticles instability of salt-induced, and formation of ordered three-dimensional hotspots allows SERS detection with excellent reproducibility. Considering SERS signal variability in the D-SERS process, 4-mercaptopyridine (4-mpy) acted as internal standard to validly correct and improve stability as well as reduce fluctuation of signals. The strongest SERS spectra at the optimal hotspots of D-SERS have been extracted to statistics analysis. By using the SERS signal of 4-mpy as a stable internal calibration standard, the relative SERS intensity of target molecules demonstrated a linear response versus the negative logarithm of concentrations at the point of strongest SERS signals, which illustrates the great potential for quantitative analysis. The public drugs 3,4-methylenedioxymethamphetamine and α-methyltryptamine hydrochloride obtained precise analysis with internal standard D-SERS strategy. As a consequence, one has reason to believe our approach is promising to challenge quantitative problems in conventional SERS analysis.

Determination of Tryptamines and ß-Carbolines in Ayahuasca Beverage Consumed During Brazilian Religious Ceremonies.

Ayahuasca is a potent hallucinogenic beverage prepared from Banisteriopsis caapi in combination with other psychoactive plants. N,N-dimethyltryptamine, tryptamine, harmine, harmaline, harmalol, and tetrahydroharmine were quantified in ayahuasca samples using a simple and low-cost method based on SPE and LC with UV diode-array detection. The experimental variables that affect the SPE method, such as type of solid phase and nature of solvent, were optimized. The method showed good linearity (r > 0.9902) and repeatability (RSD < 0.8%) for alkaloid compounds, with an LOD of 0.12 mg/L. The proposed method was used to analyze 20 samples from an ayahuasca cooking process from a religious group located in the municipality of Fortaleza, state of Ceará, Brazil. The results showed that concentrations of the target compounds ranged from 0.3 to 36.7 g/L for these samples.