Phosphate-functionalized magnetic microspheres for immobilization of Zr ions for selective enrichment of the phosphopeptides

In this work, we developed phosphate functionalized magnetic Fe₃O₄@C microspheres to immobilize Zr⁴⁺ ions for selective extraction and concentration of phosphopeptides for mass spectrometry analysis. Firstly, we synthesized Fe₃O₄@C magnetic microspheres as our previous work reported. Then, the microspheres were functionalized with phosphate groups through a simple hydrolysis reaction using 3-(trihydroxysilyl)propyl methylphosphate. And the Zr⁴⁺ ions were immobilized on phosphate-functionalized magnetic microspheres by using phosphate chelator. Finally, we successfully employed Zr⁴⁺-phosphate functionalized magnetic microspheres to selectively isolate the phosphopeptides from tryptic digests of standard protein and real samples including rat brain. All the experimental results demonstrate the enrichment efficiency and selectivity of the method we reported here.     

Enrichment and analysis of phosphopeptides under different experimental conditions using titanium dioxide affinity chromatography and mass spectrometry

Titanium dioxide metal oxide affinity chromatography (TiO₂‐MOAC) is widely regarded as being more selective than immobilized metal‐ion affinity chromatography (IMAC) for phosphopeptide enrichment. However, the widespread application of TiO₂‐MOAC to biological samples is hampered by conflicting reports as to which experimental conditions are optimal. We have evaluated the performance of TiO₂‐MOAC under a wide range of loading and elution conditions. Loading and stringent washing of peptides with strongly acidic solutions ensured highly selective enrichment for phosphopeptides, with minimal carryover of non‐phosphorylated peptides. Contrary to previous reports, the addition of glycolic acid to the loading solution was found to reduce specificity towards phosphopeptides. Base elution in ammonium hydroxide or ammonium phosphate provided optimal specificity and recovery of phosphorylated peptides. In contrast, elution with phosphoric acid gave incomplete recovery of phosphopeptides, whereas inclusion of 2,5‐dihydroxybenzoic acid in the eluant introduced a bias against the recovery of multiply phosphorylated peptides. TiO₂‐MOAC was also found to be intolerant of many reagents commonly used as phosphatase inhibitors during protein purification. However, TiO₂‐MOAC showed higher specificity than immobilized gallium (Ga³⁺), immobilized iron (Fe³⁺), or zirconium dioxide (ZrO₂) affinity chromatography for phosphopeptide enrichment. Matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) was more effective in detecting larger, multiply phosphorylated peptides than liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS), which was more efficient for smaller, singly phosphorylated peptides. Copyright © 2009 Crown in the right of Canada. Published by John Wiley & Sons, Ltd.     

In situ derivatization–liquid liquid extraction as a sample preparation strategy for the determination of urinary biomarker prolyl‐4‐hydroxyproline by liquid chromatography–tandem mass spectrometry

Polar analytes that possess protic functional groups have often been treated with alkyl chloroformates to decrease their polarity and increase their volatility prior to gas chromatography–mass spectrometry analysis. This derivatization reaction has two distinct advantages. It proceeds smoothly in aqueous media, and the desired reaction products are efficiently separated from interfering ionic components by their extraction into a water‐immiscible organic phase. In the present work, the derivatization–liquid liquid sample preparation was examined in detail for analysis of a potential urinary dipeptide biomarker l ‐prolyl‐4‐l ‐hydroxyproline (PHP) by downstream liquid chromatography coupled to electrospray mass spectrometry. PHP was treated with a series of alkyl and fluoroalkyl chloroformates in aqueous media, and the detected reaction products were investigated. Smooth conversion of PHP into the N‐isobutyloxycarbonyl isobutyl ester was accomplished by the coupled action of isobutanol, isobutyl chloroformate and the pyridine catalyst. This derivative afforded a highest detector response from all the derivatized forms examined, including the nonderivatized PHP. A simple isocratic elution on a common RP‐C18 HPLC column coupled with tandem mass spectrometry, and use of the synthesized heptadeuterated analog (D7‐PHP) as an internal standard, enabled validation of the method and determination of PHP in human urine in less than 5 min. The in situ derivatization–liquid liquid extraction has thus been demonstrated to be a useful sample preparation strategy for the analysis of polar metabolites by liquid chromatography–tandem mass spectrometry in the complex urine matrix. Copyright © 2012 John Wiley & Sons, Ltd.     

Base‐deactivated and alkaline‐resistant chromatographic stationary phase based on functionalized polymethylsilsesquioxane microspheres

Vinyl, chloropropyl, and mercaptopropyl functionalized particles were prepared by a two‐step acidic/alkaline catalyzed co‐hydrolysis/condensation of methyltrimethoxysilane with a different silane precursor that carries chemically reactive functional group including vinyl, chloropropyl, and mercaptopropyl, respectively. The morphology, pore structure, and functional groups of the synthesized packings were studied by SEM, nitrogen adsorption‐desorption measurements, and solid‐state ¹³C ²⁹Si NMR spectroscopy, respectively. The particles show ordered sphere, narrow particle size distribution, and mesoporous structure. The carbon contents of the microspheres are in the range of 17–19%, comparable to those of octadecyl‐bonded silica packings. The three‐kind of microspheres were directly used as packing materials for high‐performance liquid chromatography without size classification. The chromatographic performance of the columns was evaluated and compared with a commercially available C₁₈ phase. The results revealed that these columns possess typical reversed‐phase chromatographic properties with increased hydrophobicity than polymethylsilsesquioxane and symmetric peaks for basic compounds. They were applied to the simultaneous separation of combination bendazol hydrochlorothiazide capsules containing polar and basic drugs with peaks identified by tandem with mass spectrometry. In general, a novel method is provided for the synthesis of different methyltrimethoxysilane‐derived microspheres for high‐performance liquid chromatography, which are advantageous for separating basic compounds.     

Rapid identification of efavirenz metabolites in rats and humans by ultra high performance liquid chromatography combined with quadrupole time‐of‐flight tandem mass spectrometry

An in vivo study of efavirenz metabolites in rats and human patients with ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry combined with MetabolitePilot^MT software is reported for the first time. Considering the polarity differences between the metabolites, solid‐phase extraction and protein precipitation were both applied as a part of the sample preparation method. The structures of the metabolites and their fragment ions were identified or tentatively characterized based on the accurate mass and MS² data. As a result, a total of 15 metabolites, including 11 from rat samples and 13 from human samples, were identified or tentatively characterized. Two metabolites and several new metabolism pathways are reported for the first time. This study provides a practical approach for identifying complicated metabolites through the rapid and reliable ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry technique, which could be widely used for the investigation of drug metabolites.     

Two‐dimensional solid‐phase extraction strategy for the selective enrichment of aminoglycosides in milk

An orthogonal two‐dimensional solid‐phase extraction strategy was established for the selective enrichment of three aminoglycosides including spectinomycin, streptomycin, and dihydrostreptomycin in milk. A reversed‐phase liquid chromatography material (C₁₈) and a weak cation‐exchange material (TGA) were integrated in a single solid‐phase extraction cartridge. The feasibility of two‐dimensional clean‐up procedure that experienced two‐step adsorption, two‐step rinsing, and two‐step elution was systematically investigated. Based on the orthogonality of reversed‐phase and weak cation‐exchange procedures, the two‐dimensional solid‐phase extraction strategy could minimize the interference from the hydrophobic matrix existing in traditional reversed‐phase solid‐phase extraction. In addition, high ionic strength in the extracts could be effectively removed before the second dimension of weak cation‐exchange solid‐phase extraction. Combined with liquid chromatography and tandem mass spectrometry, the optimized procedure was validated according to the European Union Commission directive 2002/657/EC. A good performance was achieved in terms of linearity, recovery, precision, decision limit, and detection capability in milk. Finally, the optimized two‐dimensional clean‐up procedure incorporated with liquid chromatography and tandem mass spectrometry was successfully applied to the rapid monitoring of aminoglycoside residues in milk.     

Analysis of bioactive components and multi‐component pharmacokinetics of saponins from the leaves of Panax notoginseng in rat plasma after oral administration by LC–MS/MS

In this study, simple ultra‐high performance liquid chromatography coupled with quadrupole time‐of‐flight mass tandem mass spectrometry is used to characterize the absorbed components in rat plasma after the oral administration of saponins from the leaves of Panax notoginseng. Seventeen prototype compounds are structurally characterized. Furthermore, a simple and sensitive liquid chromatography with tandem mass spectrometry method is also used for the simultaneous determination of notoginsenoside Fc, ginsenoside Rb1, ginsenoside Rc, ginsenoside Rb3, ginsenoside Rd, and notoginsenoside Fe in rat plasma within 5 min. After n‐butanol mediated liquid–liquid extraction, all analytes were separated on a C₁₈ column and monitored in negative ion mode. Linearity, sensitivity, intra‐ and inter‐assay precision, accuracy, recovery, matrix effect, and stability were all within acceptable ranges. The validated liquid chromatography with tandem mass spectrometry method is successfully applied to the pharmacokinetic study of saponins from the leaves of Panax notoginseng in rats after oral administration. The results suggest that notoginsenoside Fc and ginsenoside Rb3 showed relatively higher exposure compared with other saponins. All saponins showed a long duration in plasma with a t_1/2 longer than 15 h, except notoginsenoside Fe (t_1/2 = 2.78 h). This study provides important information about the metabolism of saponins from the leaves of Panax notoginseng, which is useful for completely understanding its mechanism of action.     

Qualitative screening of absorbed indoloquinazoline alkaloids and their metabolites in rat plasma after the oral administration of Wu‐Zhu‐Yu decoction by high‐resolution mass spectrometry with multiple data mining algorithms

A rapid and sensitive liquid chromatography with high‐resolution mass spectrometry method with multiple data processing algorithms was developed and applied for the metabolite profiling of evodiamine and its analogous alkaloids in rat plasma after the administration of Wu‐Zhu‐Yu decoction. All samples were purified using hydrophilic‐lipophilic balanced solid‐phase extraction cartridges and analyzed by a Sciex TripleTOF 5600⁺ mass spectrometer with a 35 min liquid chromatography gradient elution. High‐resolution full‐scan mass spectrometry and information‐dependent acquisition tandem mass spectrometry data were analyzed using multiple data processing approaches. The results indicated that the detected eight prototype alkaloids could be metabolized to 58 metabolites through both phase I and phase II reactions. Oxidation was demonstrated to be the principle metabolic pathway of the parent compounds. The study contributes to the understanding of the absorption and metabolism of the alkaloids in Wu‐Zhu‐Yu decoction and provides a detailed analysis of scientific data.     

One‐step synthesized magnetic MIL‐101(Cr) for effective extraction of triazine herbicides from rice prior to determination by liquid chromatography‐tandem mass spectrometry

The magnetic metal‐organic framework MIL‐101(Cr) material‐based solid‐phase extraction method coupled with high‐performance liquid chromatography and tandem mass spectrometry was applied to extract seven triazine herbicides in rices. Fe₃O₄/MIL‐101(Cr) was synthesized using reduction‐precipitation method, in which steps including pre‐synthesis and modification of Fe₃O₄ nanoparticles were by‐passed. Various parameters including extraction solvent type and volume, ultrasonic extraction time, amount of Fe₃O₄/MIL‐101(Cr) microspheres, adsorption time, desorption volume and time were investigated. Under optimal conditions, the proposed method had the limit of detection (S/N = 3) and the limit of quantification (S/N = 10) of 1.08–18.10 and 3.60–60.20 pg/g, respectively. Relative standard deviations calculated for all herbicides with concentrations of 2 and 20 ng/g were in the range of 0.5 to 13% (n = 3). In addition, at the two above‐mentioned concentrations, the method achieved relative recoveries percentages of 79.3 to 116.7% when applied to determine the triazine herbicides in real samples spiked. This rapid, green, non‐polluting, pre‐concentrated extraction method was successfully developed and applied to analyze herbicides in rice samples.     

Analysis by liquid chromatography‐tandem mass spectrometry of biophenolic compounds in virgin olive oil,Part II

In this study the qualitative and quantitative content of the biophenolics in virgin olive oils is evaluated by liquid chromatography‐tandem mass spectrometry. The extraction and purification method for these compounds from virgin oils was optimised. After liquid‐liquid and solid‐phase extraction the separation of 35 of these compounds was achieved on reversed phase in gradient mode. The detection was preliminarily by UV and fluorescence, but then the final choice was ion‐spray tandem mass spectrometry in multiple reaction monitoring mode in negative ionization, acquiring two diagnostic product ions from the chosen precursor [M—H]^–. Using this last approach we obtained the best sensitivity, selectivity, and specificity. The recovery of the method ranged from 70–90% and detection limits were less than 1 ng for all the analyzed compounds.     

Simultaneous determination of pharmaceuticals and some of their metabolites in wastewaters by high performance liquid chromatography with tandem mass spectrometry

The present work describes the development of a sensitive and reliable analytical method based on solid‐phase extraction followed by analysis using liquid chromatography with tandem mass spectrometry for the simultaneous determination of pharmaceuticals from antibiotics (fluoroquinolones, sulfonamides, and their N⁴‐acetyl metabolites, and trimethoprim as sulfonamides synergist) and anthelmintics groups. SPE was optimized using different cartridges (Strata‐X, Oasis HLB, Strata C₁₈‐E, Isolute C₁₈, SampliQ C₈/Si‐SCX). The highest recovery was achieved using Strata X cartridge (>80%) with good reproducibility (RSDs < 5%) despite various physicochemical properties of the compounds. Investigated analytes were identified and quantitatively determined by liquid chromatography with tandem mass spectrometry using multiple reaction monitoring. The method was shown to be linear over the concentration range of 0.05–30 μg/L for febantel and albendazole, and 0.10–60 μg/L for all other pharmaceuticals. Correlation coefficients were >0.99 for all compounds except for sulfamethazine (0.98). In order to demonstrate the applicability of the developed method, wastewater from the veterinary industry was analyzed. Results evidenced the presence of febantel, praziquantel, albendazole, enrofloxacin, sulfamethazine, and sulfadiazine.     

Simultaneous analysis of ten phytohormones in Sargassum horneri by high‐performance liquid chromatography with electrospray ionization tandem mass spectrometry

Phytohormones have attracted wide attention due to their important biological functions. However, their detection is still a challenge because of their complex composition, low abundance and diverse sources. In this study, a novel method of high‐performance liquid chromatography with electrospray ionization tandem mass spectrometry was developed and validated for the simultaneous determination of ten phytohormones including indole‐3‐acetic acid, isopentenyladenine, isopentenyl adenosine, trans‐zeatin riboside, zeatin, strigolactones, abscisic acid, salicylic acid, gibberellin A3, and jasmonic acid in Sargassum horneri (S. horneri). The phytohormones were extracted from freeze‐dried S. horneri with methanol/water/methanoic acid (15:4:1, v/v/v) analyzed on a Hypersil Gold C₁₈ column and detected by electrospray ionization tandem triple quadrupole mass spectrometry in the multiple reaction monitoring mode. The experimental conditions for the extraction and analysis of phytohormones were optimized and validated in terms of reproducibility, linearity, sensitivity, recovery, accuracy, and stability. Distributions of the phytohormones in the stems, blades, and gas bladder of the S. horneri in drift, fixed, and semi‐fixed growing states were investigated for the first time. The observed contents of the phytohormones in S. horneri range from not detected to 5066.67 ng/g (fresh weight). Most phytohormones are distributed mainly in the stems of S. horneri in drift and semi‐fixed states.     

A validated LC‐MS/MS method for the determination of canagliflozin,a sodium–glucose co‐transporter 2 (SGLT‐2) inhibitor,in a lower volume of rat plasma: application to pharmacokinetic studies in rats

Canagliflozin is a novel, orally selective inhibitor of sodium‐dependent glucose co‐transporter‐2 (SGLT2) for the treatment of patients with type 2 diabetes mellitus. In this study, a validated liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method for the quantitative analysis of canagliflozin in a lower volume of rat plasma (0.1 mL) was established and applied to a pharmacokinetic study in rats. Following liquid–liquid extraction by tert‐butyl methyl ether, chromatographic separation of canagliflozin was performed on a Quicksorb ODS (2.1 mm i.d. × 150 mm, 5 µm size) using acetonitrile–0.1% formic acid (90:10, v/v) as the mobile phase at a flow rate of 0.2 mL/min. The detection was carried out using an API 3200 triple‐quadrupole mass spectrometer operating in the positive electrospray ionization mode. Selected ion monitoring transitions of m/z = 462.0 [M + NH₄]⁺ → 191.0 for canagliflozin and m/z = 451.2 [M + H]⁺ → 71.0 for empagliflozin (internal standard) were obtained. The validation of the method was investigated, and it was found to be of sufficient specificity, accuracy and precision. Canagliflozin in rat plasma was stable under the analytical conditions used. This validated method was successfully applied to assess the pharmacokinetics of canagliflozin in rats using 0.1 mL rat plasma. Copyright © 2016 John Wiley & Sons, Ltd.     

l‐Cysteine‐modified magnetic microspheres for extraction and quantification of saxitoxin in rat plasma with liquid chromatography and tandem mass spectrometry

Saxitoxin, which is one of the most typical paralytic shellfish poisoning toxins, ranks the highest intoxication rate of marine biological poisoning cases globally. Efficient clean‐up and extraction of saxitoxin from complex biological matrices are imperative for the analysis and concentration monitoring of the toxin when correlative poisoning cases happen. Herein, l ‐cysteine‐modified magnetic microspheres based on metal‐organic coordination were synthesized by a facile approach and applied for magnetic solid‐phase extraction of saxitoxin from rat plasma samples before liquid chromatography–tandem mass spectrometry detection. Parameters, including adsorbent amount, extraction time, desorption solution, and desorption time that could affect the extraction efficiency, were respectively investigated. The developed method demonstrated good linearity in the range of 5–300 ng/mL (R² = 0.9985) with a limit of quantification of 5 ng/mL and a limit of detection of 0.5 ng/mL, acceptable accuracy. and precision of within‐run and between‐run.     

Study of the oxidation dynamics of ethyl cysteinate dimer in solution by ultra‐performance liquid chromatography with tandem mass spectrometry

Diaminodithiol (N₂S₂)‐type compounds readily oxidize to produce disulfides. We found that some ligands failed to produce a prospective protonated molecular ion peak but gave a peak of [M–2+H]⁺, whereas others produced both [M+H]⁺ and [M–2+H]⁺ peaks in electrospray ionization mass spectra. In this study, an important N₂S₂ ligand, the ethyl cysteinate dimer (ECD), was investigated with high‐resolution accurate mass measurements and tandem mass spectrometric analysis. The elemental compositions of ECD and its oxidized product were analyzed. The oxidation of ECD was confirmed. An ultra‐performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method in multiple reaction monitoring mode was developed, and ECD and its oxidized product were quantitated in solution. The dynamic oxidation process of ECD in solution was studied in detail. The full time course of the decrease in ECD and the increase in its oxide was observed; the oxidation procedure followed first‐order kinetics, and the half‐life time of ECD was 51 min. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of Microcystins in Cyanobacteria by Supercritical Fluid Extraction and Liquid Chromatography‐Tandem Mass Spectrometry

Abstract

A method for the detection of microcystins (microcystin LR, RR, and YR) in cyanobacteria by supercritical fluid extraction (SFE) and liquid chromatography‐mass spectrometry (LC/MS) has been developed. Supercritical fluids for the analytical extraction of nonvolatile, higher molecular weight compound, and microcystins from cyanobacteria were investigated. The microcystins included in this study are sparsely soluble in neat supercritical fluid CO₂. However, the microcystins was successfully extracted with a ternary mixture (90% CO₂, 9.5% methanol, 0.5% water) at 40°C and 250 atm. The polar carbon dioxide‐aqueous methanol fluid system gave high extraction efficiency for the extraction of the polar microcystins from cyanobacteria. The microcystins were determined by liquid chromatography‐tandem mass spectrometry (LC/MS/MS).     

Simultaneous determination of phenolic compounds in Equisetum palustre L. by ultra high performance liquid chromatography with tandem mass spectrometry combined with matrix solid‐phase dispersion extraction

A method based on matrix solid‐phase dispersion extraction followed by ultra high performance liquid chromatography with tandem mass spectrometry is presented for the extraction and determination of phenolic compounds in Equisetum palustre. This method combines the high efficiency of matrix solid‐phase dispersion extraction and the rapidity, sensitivity, and accuracy of ultra high performance liquid chromatography with tandem mass spectrometry. The influential parameters of the matrix solid‐phase dispersion extraction were investigated and optimized. The optimized conditions were as follows: silica gel was selected as dispersing sorbent, the ratio of silica gel to sample was selected to be 2:1 (400/200 mg), and 8 mL of 80% methanol was used as elution solvent. Furthermore, a fast and sensitive ultra high performance liquid chromatography with tandem mass spectrometry method was developed for the determination of nine phenolic compounds in E. palustre. This method was carried out within <6 min, and exhibited satisfactory linearity, precision, and recovery. Compared with ultrasound‐assisted extraction, the proposed matrix solid‐phase dispersion procedure possessed higher extraction efficiency, and was more convenient and time saving with reduced requirements on sample and solvent amounts. All these results suggest that the developed method represents an excellent alternative for the extraction and determination of active components in plant matrices.     

两类三组份反应产物：1,3-恶嗪及嘧啶-2-酮类化合物的质谱学行为研究

电喷雾质谱被应用于分辨2-氨基-1,3-恶嗪及六氢化-4-苯基-吡喃[2,3-d]嘧啶-2-酮的杂环结构。两类化合物均为三组份反应的产物,且其杂环的结构很难用NMR判断。实验首次系统研究了两类化合物的质谱学行为（包括氘代实验和高分辨质谱研究）,发现前者在CID实验中丢失CH2N2和HCNO,而后者为直接丢失尿素。这些特征丢失为该类衍生物的结构判断,尤其是高通量的合成产物分析提供了重要的依据。     

High‐efficiency sample preparation approach to determine acrylamide levels in high‐fat foods

An improved sample preparation method was developed to enhance acrylamide recovery in high‐fat foods. Prior to concentration, distilled deionized water was added to protect acrylamide from degradation, resulting in a higher acrylamide recovery rate from fried potato chips. A Chrome‐Matrix C₁₈ column (2.6 μm, 2.1 × 100 mm) was used for the first time to analyze acrylamide levels using ultra high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry, displaying good separation of acrylamide from interference. A solid‐phase extraction procedure was avoided, and an average recovery of >89.00% was achieved from different food matrices for three different acrylamide spiking levels. Good reproducibility was observed, with an intraday relative standard deviation of 0.04–2.38%, and an interday relative standard deviation of 2.34–3.26%. Thus, combining the improved sample preparation method for acrylamide analysis with the separation on a Chrome‐Matrix C₁₈ column (2.6 μm, 2.1 × 100 mm) using ultra high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry is highly useful for analyzing acrylamide levels in complex food matrices.     

A supported liquid extraction LC–MS/MS method for determination of concentrations of GDC‐0425, a small molecule Checkpoint kinase 1 inhibitor,in human plasma

A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the determination of GDC‐0425 concentrations in human plasma has been developed and validated. Supported liquid extraction was used to extract plasma samples (50 μL) and the resulting samples were analyzed using reverse‐phase chromatography and mass spectrometry coupled with a turbo‐ionspray interface. The mass analysis of GDC‐0425 was performed using multiple reaction monitoring transitions in positive ionization mode. The method was validated over the calibration curve range of 1.00–1000 ng/mL using linear regression and 1/x² weighting. Within‐run relative standard deviation ranged from 0.8 to 5.1%, while between‐run RSD varied from 1.9 to 4.7% for QCs. The accuracy ranged from 90.0 to 101.0% of nominal for within‐run and from 94.0 to 100.0% of nominal for between‐run. Overall extraction recovery was 87.4% for GDC‐0425 and 87.9% for GDC‐0425‐d₉. Stability of GDC‐0425 was established in human plasma for 374 days at ?20 and ?70 °C and established in reconstituted sample extracts for 88 h when stored at 2–8 °C. Stable‐labeled internal standard was used to minimize matrix effects. This assay was used to characterize the pharmacokinetics of GDC‐0425 in cancer patients.