Direct coupling of sample preparation with mass spectrometry (MS) can speed up analysis, enabling faster decision-making. In such combinations, where the analysis time is mainly defined by the extraction procedure, magnetic dispersive solid-phase extraction emerges as a relevant technique because of its rapid workflow. The dispersion and retrieval of the magnetic sorbent are typically uncoupled stages, thus reducing the potential simplicity. Stir bar sorptive dispersive microextraction (SBSDME) is a novel technique that integrates both stages into a single device. Its miniaturization (mSBSDME) makes it more portable and compatible with low-availability samples. This article reports the direct combination of mSBSDME and MS using a needle-based electrospray ionization (NESI) emitter as the interface. This combination is applied to determine tetrahydrocannabinol in saliva samples, a relevant societal problem if the global consumption rates of cannabis are considered. The coupling requires only the transference of the magnet (containing the sorbent and the isolated analyte) from the mSBSDME to the hub of a hypodermic needle, where the online elution occurs. The application of 5 kV on the needle forms an electrospray on its tip, transferring the ionized analyte to the MS inlet. The excellent performance of mSBSDME-NESI-MS/MS relies on the sensitivity (limits of detection as low as 2.25 ng mL-1), the precision (relative standard deviation lower than 15%), and the accuracy (relative recoveries ranged from 87 to 127%) obtained. According to the results, the mSBSDME-NESI-MS/MS technique promises faster and more efficient chemical analysis in MS-based applications.
This article describes the synthesis of sorptive phases for bioanalysis based on the modification of cellulose paper with natural beeswax as sorbent, resulting in a substrate completely renewable and sustainable. The preparation of the sorptive phases consisted of the dissolution of beeswax in hexane, followed by its drop-casting on cellulose paper and subsequent evaporation of the solvent. The beeswax modification of paper renders it hydrophobic, enabling the extraction of the target analytes, i.e., imipramine, desipramine, amitriptyline and trimipramine, via hydrophobic interactions. The main variables affecting the extraction performance were investigated (e.g., pH, ionic strength, extraction time, eluent composition, agitation speed). The analytical workflow combines a straightforward sampling, simultaneous extraction of 30 samples in 1 h, and the rapid (<2 min) determination of the analytes via direct infusion mass spectrometry. The method provided limits of detection in the range 2.0 and 3.2 µg L-1, and the precision, expressed as relative standard deviation, was better than 5.4 % and 8.5 % for intra and inter-day analyses, respectively. The accuracy, in terms of relative recovery, ranged from 90 % to 121 % using saliva as model biofluid.
Antidepressive Agents, Tricyclic , Cellulose , Cellulose/chemistry , Amitriptyline , Waxes
BACKGROUND: Opioids are effective painkillers used for medical purposes. Their prolonged ingestion can provoke some side effects (including overdose or constipation) that are minimized by using opioid antagonists (e.g., naloxone). The rapid determination of opioids and their antagonists in biosamples is essential for an effective medical treatment. The direct combination of sample preparation and mass spectrometry (MS) fits well in this scenario. It can speed up the analysis achieving a good selectivity, which relies on the sample preparation and MS, and sensitivity levels. RESULTS: This article presents a novel substrate-spray mass spectrometry interface based on a polydopamine-cotton (PDA-Cel) composite hosted inside the inner diameter of a 14-gauge blunt needle to determine oxycodone and naloxone in saliva samples. The needle is used as a microextraction device and a substrate for mass spectrometric analysis. The lack of sharpness of the 14-gauge (14G) blunt needles challenges the formation of the electrospray (ESI), and a commercial 10 µL pipette tip is proposed as a simple solution to this shortcoming. Under the optimum parameters, the proposed method was validated, obtaining limits of detection lower than 0.6 µg L-1, linear range up to 200 µg L-1, and linearity better than 0.9915. Relative standard deviation (RSD) and relative recoveries (RR) were studied at three different concentration levels (2, 40, and 200 µg L-1). RSD values were better than 20.7 %, and RR ranged from 90 to 114 %. Finally, a positive sample from a patient under medical treatment was analyzed. SIGNIFICANCE AND NOVELTY: 14G blunt needles have been demonstrated as effective extraction devices due to their low price (<0.15 per extraction unit), their better safety (avoiding finger pricking), and their higher hosting capacity (up to 8 mg of sorbent). The conductivity of stainless steel permits their use as electrospray emitters, making their direct combination to MS easier. The large variety of fibrous sorbents makes this approach versatile enough to be adapted to other analytical problems.
Naloxone , Oxycodone , Humans , Saliva , Analgesics, Opioid , Mass Spectrometry
Direct mass spectrometry (MS) is an exciting strategy in bioanalysis, enabling rapid decision-making in different scenarios. Its application is usually hindered by matrix effects and the typically low concentration of the target compounds in the biofluids. In this sense, combining a previous sample preparation step minimizes or removes these shortcomings. This article describes sorptive tape-spray tandem mass spectrometry (STS-MS/MS) based on mixed-mode particles as a strategy to combine sample preparation and MS analysis in a single device. The technique uses a sorptive tape (ST) consisting of mixed-mode polymeric microparticles (combining ionic exchange and hydrophobic interactions) coated over aluminum foil in a spatial controlled way. The tapes act as the sorptive phases to isolate the analytes from the sample matrix and substrates for STS-MS/MS. The performance of the technique has been evaluated by developing a method to determine codeine in saliva as proof of concept. The affordability of the STs elements allows the preparation of many individual phases at low cost so that several samples can be extracted simultaneously, thus increasing the sample throughput. The extraction variables were optimized following a multivariate approach. Working under the optimum conditions, the limit of detection was 0.3 µg L-1, while the intraday precision, calculated as relative standard deviation (RSD) at three concentration levels, was better than 9.4 %. The accuracy, expressed as relative recovery, was in the range of 78-98 %. The method was also applied to the analysis of real samples. Despite being a powerful strategy, the direct combination of microextraction to MS is not always affordable in all laboratories. For this reason, the STs were also combined with commercial liquid chromatography-MS working under the direct infusion mode to demonstrate the usefulness of the ST in classical extraction workflows.
Aluminum , Tandem Mass Spectrometry , Tandem Mass Spectrometry/methods , Chromatography, High Pressure Liquid/methods
Sample preparation is a constantly evolving step in the measurement process with a positive effect on its performance. Its evolution has been marked by an underlying environmental commitment, with simplification, miniaturization, and automation being three of its driving forces. This trends article deepens how the sample preparation can go sustainable through the efficient design of new sorptive materials, either liquid or solid. This objective can be achieved by using natural and/or biodegradable materials as precursors of the functional sorptive phases and by designing materials that simplify the procedures (thus reducing the energy or resources required). Although environmental performance is a crucial aspect of a new material, its applicability is what really defines its incorporation into the sample preparation toolbox. For this reason, their characteristics and more relevant applications will be briefly presented to conclude with the tendency of their use in the very near future.
Psychiatric disorders are usually treated with antipsychotic agents belonging to different pharmacological and chemical classes, the most recent ones collectively known as "third-generation antipsychotics", such as cariprazine, approved in 2015 for the treatment of patients affected by schizophrenia. For these patients, a frequent therapeutic drug monitoring (TDM) becomes essential to assess compliance and to optimise and personalise their therapy, also due to cariprazine interindividual variability and narrow therapeutic range. In this study, a bioanalytical method featuring miniaturised sampling and pretreatment was developed, based on volumetric absorptive microsampling (VAMS) for TDM of psychiatric patients under cariprazine treatment and compared to a reference method based on fluid plasma analysis. Minimally invasive whole blood VAMS was coupled to an original instrumental method based on ultra-high performance liquid chromatography hyphenated to mass spectrometry (UHPLC-MS). A feasible and streamlined, yet reliable VAMS pretreatment protocol was carefully optimised and the VAMS-UHPLC-MS methodology was validated with satisfactory results in terms of linearity (r2 > 0.9970 in the 1.5-100 ng/mL range), precision (%RSD < 11.7), extraction yield (> 90.0 %) and matrix effect (8.2 ≤ %RE ≤ 10.9). Finally, the microsampling approach coupled to UHPLC-MS was successfully applied to the TDM of psychiatric patients treated with cariprazine and compared with standard fluid plasma analysis, providing reliable quali-quantitative results, and proving to be readily applicable to the clinical practice in TDM programs as a useful alternative to cariprazine plasma analysis. This is the first report of a successful microsampling application, and in particular the first report of VAMS application, for the TDM of cariprazine.
This article describes the synthesis of mixed-mode cationic exchange (MCX) tapes as sorptive phases in bioanalysis, and it faces the determination of methadone and tramadol in saliva as the model analytical problem. The tapes are synthesized using aluminum foil as substrate, which is subsequently covered with double-sided adhesive tape where the MCX particles (ca. 1.4 ± 0.2 mg) finally adhere. MCX particles allow the extraction of the analytes at the physiological pH, where both drugs are positively charged, minimizing the potential co-extraction of endogenous matrix compounds. The extraction conditions were studied considering the main variables (e.g. ionic strength, extraction time, sample dilution). Under the optimum conditions and using direct infusion mass spectrometry as the instrumental technique, detection limits as low as 3.3 µg·L-1 were obtained. The precision calculated at three different levels, and expressed as relative standard deviation, was better than 3.8%. The accuracy, expressed as relative recoveries, ranged from 83 to 113%. The method was finally applied to determine tramadol in saliva samples from patients under medical treatment. This approach opens the door to easily preparing sorptive tapes based on commercial (or ad-hoc synthesized) sorbent particles.
Analgesics, Opioid , Tramadol , Humans , Saliva , Mass Spectrometry , Solid Phase Extraction/methods
Paper-based sorptive phases have gained attention recently due to the low-cost and sustainable character of the cellulosic substrate. However, the sustainability of the resulting phase can be limited by type of coating used for analytes isolation. In this article, this limitation is overcome by using deep eutectic solvents (DES) as coating. To this aim, a Thymol-Vanillin DES is synthesized and deposited on pre-cut cellulose paper strips. The paper-supported DES is employed as sorptive phase for the isolation of selected triazine herbicides for environmental waters analysis. The isolated analytes are finally determined by gas chromatography-mass spectrometry using selected ion monitoring. The method is optimized according to the critical variables that potentially affect its analytical performance such as sample volume, extractant amount, extraction time and sample ionic strength. The method was characterized in terms of sensitivity, accuracy and precision and its applicability was evaluated for the analysis of real environmental water samples. Good linearity values (R2>0.995) were obtained for all the analytes. Limits of detection (LODs) ranged from 0.4 to 0.6 µg L-1 and the precision, expressed as relative standard deviation (RSD) was better than 14.7%. The relative recoveries, calculated in spiked well and river samples, were in the range 90-106%.
Deep Eutectic Solvents , Liquid Phase Microextraction , Solvents/chemistry , Liquid Phase Microextraction/methods , Gas Chromatography-Mass Spectrometry , Mass Spectrometry , Limit of Detection
The development of synthetic particles that emulate real viruses in size, shape, and chemical composition is vital to the development of imprinted polymer-based sorbent materials (molecularly imprinted polymers, MIPs). In this study, we address surrogates for adenovirus type 5 (Adv 5) via the synthesis and subsequent modification of icosahedral gold nanoparticles (iAuNPs) decorated with the most abundant protein of the Adv 5 (i.e., hexon protein) at the surface. CTAB-capped iAuNPs with dimensions in the range of 40-90 nm were synthesized, and then CTAB was replaced by a variety of polyethylene glycols (PEGs) in order to introduce suitable functionalities serving as anchoring points for the attachment of the hexon protein. The latter was achieved by non-covalent linking of the protein to the iAuNP surface using a PEG without reactive termination (i.e., methoxy PEG thiol, mPEG-SH, Mn=800). Alternatively, covalent anchoring points were generated by modifying the iAuNPs with a bifunctional PEG (i.e., thiol PEG amine, SH-PEG-NH2) followed by the addition of glutaraldehyde. X-ray photoelectron spectroscopy (XPS) confirmed the formation of the anchoring points at the iAuNP surface. Next, the amino groups present in the amino acids of the hexon protein interacted with the glutaraldehyde. iAuNPs before and after PEGylation were characterized using dynamic light scattering (DLS), XPS, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and UV-Vis spectroscopy, confirming the CTAB-PEG exchange. Finally, the distinct red shift obtained in the UV-Vis spectra of the pegylated iAuNPs in the presence of the hexon protein, the increase in the hydrodynamic diameter, the change in the zeta potential, and the selective binding of the hexon-modified iAuNPs towards a hexon-imprinted polymer (HIP) confirmed success in both the covalent and non-covalent attachment at the iAuNP surface.
Gold , Metal Nanoparticles , Adenoviridae , Cetrimonium , Glutaral , Gold/chemistry , Metal Nanoparticles/chemistry , Polyethylene Glycols/chemistry , Polymers/chemistry , Serogroup , Sulfhydryl Compounds/chemistry
This study proposes a hypodermic needle (HN) as a sorbent holder and an electrospray (ESI) emitter, thus combining extraction and analysis in a single device. A novel nylon 6-cellulose (N6-Cel) composite sorbent is proposed to extract methadone from oral fluid samples. The cellulosic substrate provides the composite with high porosity, permitting the flow-through of the sample, while the polyamide contributes to the extraction of the analyte. The low price of the devices (considering the holder and the sorbent) contributes to the affordability of the method, and their small size allows easy transportation, opening the door to on-site extractions. Under the optimum conditions, the analyte can be determined by high-resolution ambient ionization mass spectrometry at a limit of detection (LOD) as low as 0.3 µg/L and precision (expressed as relative standard deviation, RSD) better than 9.3%. The trueness, expressed as relative recovery (RR), ranged from 90% to 109%. As high-resolution mass spectrometers are not available in many laboratories, the method was also adapted to low-resolution spectrometers. In this sense, the direct infusion of the eluates in a triple quadrupole-mass spectrometry provided an LOD of 2.2 µg/L. The RSD was better than 5.3%, and the RR ranged from 96% to 121%.
In this article, polydopamine inner wall-coated hypodermic needles (PDA-HNs) are evaluated as both microextraction devices and electrospray ionization (ESI) emitters for determining selected illicit drugs (methamphetamine, cocaine, and methadone) in oral fluid samples. The PDA film, located in the inner wall of the needle, allows the extraction of the analytes at alkaline pH, where their hydrophobic character is promoted. The extracted analytes are finally eluted in a methanol/formic acid mixture that also acts as ESI solution. For this purpose, a dedicated interface based on the connection of a PEEK tube with the needle hub is proposed. This assembly allows delivering the ESI solution by the infusion syringe pump of the mass spectrometer, providing an efficient ESI on the tip of the needle. The double use of the PDA-HNs as microextraction devices and ESI emitters permits the determination of the target analytes with limits of detection and precision (expressed as relative standard deviation) values better than 2.4 µg/L and 17.6%, respectively. The accuracy was evaluated by analyzing independent spiked oral fluid samples, obtaining good results.
Illicit Drugs , Illicit Drugs/analysis , Indoles , Needles , Polymers , Spectrometry, Mass, Electrospray Ionization/methods
This review critically summarizes the latest contributions in the preparation of advanced nano/microparticle-contained polymers from emulsions. The nano- or micro-particles can be dispersed in the phase where the polymerization reaction takes place and, consequently, once the solid is formed, the particles are embedded in the final polymeric structure. This results in the formation of hybrid materials, which combine the unique properties of nano/micro-particles with the inherent properties of the polymers (which depend mainly on the selected monomers). In addition to this, some nano- and micro-particles can be used as solid stabilizers in emulsions. This kind of emulsions, called 'Pickering emulsions', presents some additional advantages over those prepared with conventional surfactants (e.g., non-ionic polymers) such as higher emulsion stability. In this way, the nano/micro-particles fulfill a double mission. On the one hand, they are responsible for forming and stabilizing the emulsion. On the other, they are part of the final solid, obtaining polymeric materials with new functionalities. In this context, this review aims to describe the most innovative strategies for the incorporation of nano- and micro-particles in polymers through the direct addition of them to the emulsion in which the polymerization is carried out. Also, the effect of the addition of these nano/micro-particles in the emulsions (e.g., size of droplets, type of emulsion and stability), the type of solids obtained (e.g., monolithic polymers or individual particles), morphology (e.g., open- or closed-cell polymers) and functionality of the final solid will be also commented on.
Polymers , Surface-Active Agents , Emulsions/chemistry , Polymerization , Polymers/chemistry , Surface-Active Agents/chemistry
Paper-based sorptive phases (PSPs) are functional planar materials with a demonstrated potential in analytical sample preparation. This article describes the synthesis of a polyimide coated paper by an in-situ imidization at a high temperature. Polyimides (PI) are synthesized in two subsequent steps where a hydrophilic polymer, in this case, poly(amic acid) (PAA), is formed as an intermediate product. PAA is finally transformed into hydrophobic PI by thermal curing at 180 °C. The synthesis of PI-paper takes advantage of this two-step procedure. In the first stage, a segment of filter paper is immersed into an aqueous PAA solution. After the solvent evaporation, the paper is heated at 180 °C for 1â¯h inducing the formation of the hydrophobic PI over the cellulose fibers. Infrared spectroscopy has been used to characterize the synthesized materials by defining a coverage factor F. The hydrophobicity of the materials has been studied using an aqueous methylene blue solution as a marker. To fully demonstrate the usefulness of the material in the sample preparation field, the extraction of methadone from oral fluid (OF) samples has been considered as a model analytical problem. The main variables affecting the synthesis (PAA concentration on the precursor solution and number of dips) and the extraction (elution and extraction times) have been fully evaluated. Working under the optimum conditions, a limit of quantification of 9⯵g/L, intraday and interday precision better than 14.6%, and accuracy in the range of 87-108% were obtained.
Methadone , Polymers , Hydrophobic and Hydrophilic Interactions , Polymers/chemistry , Solvents , Water
This article deepens the potential of pre-cleaned bare wooden toothpicks (pb-WTs) for extracting drugs (antidepressants and acetaminophen) from oral fluid samples. The leaching of the intrinsic compounds from the wood matrix is identified as the main challenge for the final determination of the targets, even when a very selective instrumental technique, such as mass spectrometry, is employed. The pre-cleaning of the WTs is proposed for improving the analytical performance. The number of cleaning cycles depends on the injection mode (direct infusion or chromatography) into the mass spectrometer. The different variables affecting the extraction of selected antidepressant drugs were studied in detail, and the optimum procedure was validated using the two mentioned injection modes. The limits of detection were in the ranges 0.1-0.5 ng/mL and 0.1-0.3 ng/mL for direct infusion and liquid chromatography, respectively. The intra-day precision (expressed as relative standard deviation) was better than 12.1% and 8.6%, for direct infusion and liquid chromatography, respectively. Single-blind samples were used to study the applicability of the method. Finally, as a proof-of-concept, the potential of pb-WTs for in vivo sampling was outlined.
Specimen Handling , Chromatography, High Pressure Liquid/methods , Chromatography, Liquid/methods , Mass Spectrometry , Single-Blind Method
Lignocellulosic materials have arisen as a sustainable alternative in microextraction techniques during the last 10 years. As they are natural materials, their use fits into some of the principles of Green Analytical Chemistry. Their inherent porosity, narrow shape, and rigidity permit their use in ambient ionization mass spectrometry techniques. In particular, the combination of wooden-based materials and direct analysis gives birth to the so-called wooden-tip electrospray ionization mass spectrometry technique. This approach has been used for the direct analysis of complex samples, and as a streamlined tool for fingerprint quality analysis. Also, wooden-based materials can be superficially modified to boost the interaction with target compounds, allowing their isolation from complex samples. This review describes the potential and applicability of direct analysis using lignocellulosic materials, as well as other alternatives related to their use in microextraction.
Lignin/chemistry , Solid Phase Microextraction/instrumentation , Spectrometry, Mass, Electrospray Ionization/instrumentation , Wood/chemistry , Green Chemistry Technology , Porosity , Solid Phase Microextraction/methods , Spectrometry, Mass, Electrospray Ionization/methods
The spatial heterogeneity of environmental systems makes sampling at multiple locations crucial to provide complete and representative information. The on-site application of an extraction technique simplifies the sampling logistics, increasing sample integrity during transportation and storage. This article presents a portable, simple, and low-cost device capable of performing the simultaneous on-site extraction of several environmental water samples. The device consists of a small electric motor integrated into the plastic cap of a conventional glass bottle and operated with a portable battery. The electric motor provides stirring to a novel magnetic sorptive phase based on the deposition of hydrophilic-lipophilic balance (HLB) particles over a magnetic tape. The use of open technology makes the device globally affordable. In this first approach, the isolation and preconcentration of atrazine and simazine have been selected as proof of concept. Using an internal standard made unnecessary the adjustment of the ionic strength before the extraction, thus simplifying the analytical procedure. Under the optimum conditions and using direct infusion mass spectrometry as the instrumental technique, detection limits as low as 15 ng/L were obtained. The precision calculated at three different levels was better than 8.3%. The accuracy, calculated with spiked samples, indicates the applicability of the approach for environmental water analysis.
Atrazine , Water Pollutants, Chemical , Hydrophobic and Hydrophilic Interactions , Magnetic Phenomena , Mass Spectrometry , Water Pollutants, Chemical/analysis
A new mix-mode cellulose-based sorptive phase is described that combines two different polymeric domains (i.e., nylon and polystyrene), thus providing simultaneous hydrophilic and hydrophobic features as a result. By analogy with Janus materials, the new paper-based sorptive phase has been named paper-based polystyrene/nylon Janus-platform (P-Ps/Ny-JP). The main advantages of the proposed P-Ps/Ny-JP are the sustainability, simplicity in synthesis, and low cost of this extraction device. The main parameters affecting the synthesis (i.e., coating procedure and polymers proportion) have been studied, and the resulting material has been characterized via scanning electron microscopy and infrared spectroscopy. As a proof-of-concept, the simultaneous extraction of fourteen UV filters of a wide range of polarity, with log P values ranging from - 0.234 to 16.129, from water samples and their determination by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been performed. The proposed methodology enables the determination of these chemicals with limits of detection from 12 to 71 ng L-1, and the precision, expressed as a relative standard deviation, was below 15%. The extraction device was applied to the analysis of real water samples likely to contain UV filters (i.e., two private swimming pool water and one seawater) and the recovery values were in the range 73-121%.
Isoflavones are a group of phytoestrogens of important environmental concern due to their endocrine disrupting effects. This article presents a rapid, green, and sustainable method for determining four isoflavones (daidzein, genistein, formononetin, and biochanin A) in environmental waters complying with current trends in Analytical Chemistry. The method consists of in-syringe dispersive solid-phase extraction (DSPE) as the extraction approach, using carbon fibers as extraction material. The synthesis of carbon fibers is simple and sustainable, since it only requires a natural product such as raw cotton as precursor, which is thermally treated (600 °C for 30 min) in an inert (Ar) atmosphere to convert it into carbon fibers. After extraction, the final eluate is analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The proposed methodology allows the determination of the four isoflavones in water samples at the ng L-1 range, with limits of detection in the range from 17 to 25 ng L-1, relative standard deviations (RSD) from 2.8 to 10.1%, and good batch-to-batch repeatability (RSD < 13%). The method was finally applied to six environmental water samples from different sources and two swimming pool waters, and concentrations of all analytes up to 490 ng L-1 were found. The highest concentrations were found in those samples close to crop fields. Relative recovery values (80-121%) showed that the aqueous matrices considered in this work did not significantly affect the extraction process. This method overcomes the drawbacks of the previous works with the same purpose, such as consuming large volumes of organic solvents or prolonged extraction times. Moreover, this procedure would allow the extraction stage to be carried out in situ, since only the sorbent material (previously synthesized in the laboratory) and disposable syringes are required.
Isoflavones , Water Pollutants, Chemical , Carbon Fiber , Chromatography, Liquid , Isoflavones/analysis , Solid Phase Extraction , Tandem Mass Spectrometry , Water Pollutants, Chemical/analysis
Dry-cured Iberian ham is officially classified into different commercial categories according to the pig's breed and feeding regime. These reach very different prices, thus promoting labelling fraud and causing great damage to the food sector. In this work, a method based on Raman spectroscopy was explored as a rapid in situ screening tool for Iberian ham samples. A total of 110 samples were analyzed to assess the potential of this technique to differentiate purebred, crossbred, acorn-fed and feed-fed dry-cured Iberian ham. A continuous signal probably due to sample fluorescence was obtained, which hid the Raman scattering signal. Therefore, chemometric treatment was applied in order to extract non-apparent information. High validated classification rates were obtained for feeding regime (83.3%) and breed (86.7%). In addition, an interlaboratory study was carried out to confirm the applicability of the method with 52 samples, obtaining a validated rate above 80%.
