Enhanced flow injection analysis for measurements of S-nitrosothiols species in biological samples using highly selective amperometric nitric oxide sensor

A highly selective nitric oxide(NO) sensor is fabricated and applied to devise an enhanced flow injection analysis(FIA) system for S-nitrosothiols(RSNOs) measurement in biological samples.The NO sensor is prepared using a polytetrafluoroethylene(PTFE) gas-permeable membrane loaded with Teflon AF? solution,a copolymer of tetrafluoroethylene and 2,2-bis(trifluoroethylene)-4,5-difluoro -l,3-dioxole,to improve selectivity.This method is much simpler and possesses good performance over a wide range of RSNOs concentrations.Standard deviation for three parallel measurements of blood plasma is 4.0%.The use of the gas sensing configuration as the detector enhances selectivity of the FIA measurement vs.using less selective electrochemical detectors that do not use PTFE/Teflon type outer membranes.     

Glutathione Peroxidase‐Based Amperometric Biosensor for the Detection of S‐Nitrosothiols

A new biosensor is described for the detection of S‐nitrosothiols (RSNOs) based on their decomposition by immobilized glutathione peroxidase (GPx), an enzyme containing selenocysteine residue that catalytically produces nitric oxide (NO) from RSNOs. The enzyme is entrapped at the distal tip of a planar amperometric NO sensor. The new biosensor shows good sensitivity, linearity, reversibility, and response times towards various RSNO species in PBS buffer, pH 7.4 . In most cases, the response time is less than 5 min, and the response is linear up to 6 μM of the tested RSNO species. The lowest detection limit is obtained for S‐nitrosocysteine (CysNO), at approx. 0.2 μM. The biosensor's sensitivity is not affected by the addition of EDTA as a chelating agent; an advantage over other potential catalytic enzymes that contain copper ion centers, such as CuZn‐superoxide dismutase and xanthine oxidase. However, lifetime of the new sensor is limited, with sensitivity decrease of 50% after two days of use. Nonetheless, the new amperometric GPx based RSNO sensor could prove useful for detecting relative RSNO levels in biological samples, including whole blood.     

Quantitation of Cu+‐catalyzed Decomposition of S‐Nitrosoglutathione Using Saville and Electrochemical Detection: a Pronounced Effect of Glutathione and Copper Concentrations

S‐nitrosothiols (RSNOs) are composed of nitric oxide (NO) bound to the sulfhydryl group of amino acids of peptides or proteins. There is a great interest for their quantitation in biological fluids as they have a crucial impact on physiological and pathophysiological events. Most analytical methodologies for quantitation of RSNOs are based on their decomposition followed by the detection of the released NO. In order to obtain the optimal sensitivity for each detection method, the total decomposition of RSNOs is highly desired. The decomposition of RSNOs can be obtained by using catalytically active metal ions, such as Cu⁺, obtained from CuSO₄ in presence of a reducing agent such as glutathione (GSH) that is naturally present in biological environment. In this work, we have re‐investigated the decomposition of S‐nitrosoglutathione (GSNO) which is the most abundant in vivo low molecular weight RSNO, with a special emphasis on the effect of CuSO₄, GSH, and GSNO concentrations and of their ratio. To this aim, GSNO decomposition optimization was performed by both indirect (Griess assay) and direct (real time electrochemical detection of NO at NO‐microsensor) quantitation methods. Our results show that the ratio between CuSO₄, GSH and GSNO should be adjusted to tune the highest decomposition rate of GSNO and the most efficient electrochemical detection of released NO; also it shows the deleterious effect of very high GSH concentration on the detection of GSNO.     

Flow-injection analysis based on a membrane separation module and a bulk acoustic wave impedance sensor – determination of the volatile acidity of fermentation products

A novel flow-injection analysis (FIA) system has been developed for the rapid determination of the volatile acidity of some fermentation products like vinegars and juices. The proposed method is based on the diffusion of volatile acids, mainly acetic acid, across a PTFE gas-permeable membrane from an acid stream into an alkaline stream, and the acids trapped in the acceptor solution are determined online by a bulk acoustic wave impedance sensor based on changes in the conductivity of the solution. It exhibited a linear frequency response up to 10 mmol · L^–1 acetic acid with a detection limit of 50 μmol · L^–1, and the precision was better than 1% (RSD) at a through-put of 72 h^–1. The effects of operating voltage for the detector, cell constant of the electrode, composition of acceptor stream, flow rates and temperature on the FIA performance were also investigated. Received: 2 June 1997 / Revised: 7 July 1997 / Accepted: 12 July 1997     

Determination of iodide and bromide by flow methods with amperometric detection

Determination of bromide and iodide in real samples (water, pharmaceutical preparations, and biological material) was performed using modified flow injection analyses (FIA) with amperometric detection on platinum electrode. As an additional confirmation of FIA experiments, cyclic voltammetry was employed. Iodide was determined by the kinetic method, its limit of detection was 1.0 nM, and the linearity was 0.1–100 μM. The limit of detection for bromide determination was 50.0 nM and the calibration was linear for 2.5–100 μM and 0.1–10 mM. The relative standard deviation for 1 μM of iodide was 3.03% and, for 5 μM of bromide, it was 1.23% (n = 6). Both methods enable 60 analyses per hour to be performed. The text was submitted by the authors in English.     

Comparison of analytical methods for quality control of pharmaceutical formulations containing glutathione

Summary The aim of this investigation was the study and development of analytical procedures suitable for the assay of glutathione (GSH) in pharmaceutical formulations. Two are based on isocratic HPLC with a 250 mm×4.6 mm i.d., 5 μm C₁₈ column and UV detection. In the first procedure sample solutions were injected without pretreatment whereas in the second the samples were injected after derivatization with Ellman’s reagent which forms an easily detectable adduct with GSH. Good linearity was obtained over the range 0.12–6.00×10⁻⁴M for the direct procedure and 0.25–3.00×10⁻⁴M for the derivatization procedure. The precision and rapidity of analysis were also good for both methods. The third method is based on capillary zone electrophoresis (CZE) in a 27 cm×75 μm i.d untreated fused silica capillary containing pH 7 phosphate buffer. All results are in good agreement with a spectrophotometric procedure used as reference method.     

GC–MS analysis of low-molecular-weight dicarboxylic acids in atmospheric aerosol: comparison between silylation and esterification derivatization procedures

This paper describes methods for the determination of low-molecular-weight (LMW) dicarboxylic acids in atmospheric aerosols as important chemical tracers for source apportionment of aerosol organics and for studying atmospheric processes leading to secondary organic aerosol formation. The two derivatization procedures most widely used in GC analysis of dicarboxylic acids were compared: esterification using BF₃/alcohol reagent and silylation using N,O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA). The advantages and drawbacks of the two methods are investigated and compared in terms of (1) precision and accuracy of the results and (2) sensitivity and detection limit of the procedure. The comparative investigation was performed on standard solutions containing target C₃–C₉ dicarboxylic acids and on experimental particulate matter (PM) samples. Attention was focused on low-volume sampling devices that collect small amounts of sample for organic speciation. The results show that, overall, both the techniques appear suitable for the analysis of LMW dicarboxylic acids in atmospheric aerosols since they provide low detection limits (≤4 ng m⁻³) and satisfactory reproducibility (RSD% ≤ 15%). Between them, BSTFA should be the reagent of choice under the most limiting conditions of PM filters collected by low-volume air samplers: It provides determination of all the target C₃–C₉ dicarboxylic acids with lower detection limits (≤2 ng m⁻³) and higher reproducibility (RSD% ≤ 10%)      

LC–MS–MS Assay for Simultaneous Quantification of Fexofenadine and Pseudoephedrine in Human Plasma

A rapid, sensitive, and simple HPLC–MS–MS method, with electro-spray ionization and cetirizine as internal standard (IS), has been developed and validated for simultaneous quantification of fexofenadine and pseudoephedrine in human plasma. The analytes were isolated from plasma by solid-phase extraction (SPE) on Oasis HLB cartridges. The compounds were chromatographed on an RP 18 column with a mixture of ammonium acetate (10 mm, pH 6.4) and methanol as mobile phase. Quantification of the analytes was based on multiple reaction monitoring (MRM) of precursor-to-product ion pairs m/z 502 → 466 for fexofenadine, m/z 166 → 148 for pseudoephedrine, and m/z 389 → 201 for cetirizine. The linear calibration range for both analytes was 2–1,700 ng mL⁻¹ (r = 0.995), based on analysis of 0.1 mL plasma. Extraction recovery was 91.5 and 80.88% for fexofenadine and pseudoephedrine, respectively. The method was suitable for analysis of human plasma samples obtained 72 h after administration of a drug containing both fexofenadine and pseudoephedrine.     

LC–ESI–MS for Rapid and Sensitive Determination of Aripiprazole in Human Plasma

A rapid, sensitive, and accurate high-performance liquid-chromatographic–mass spectrometric (HPLC–MS) method, with estazolam as internal standard, has been developed and validated for determination of aripiprazole in human plasma. After liquid–liquid extraction the compound was analyzed by HPLC on a C₁₈ column, with acetonitrile—30 mm ammonium acetate containing 0.1% formic acid, 58:42 (v/v), as mobile phase, coupled with electrospray ionization mass spectrometry (ESI-MS). The protonated analyte was quantified by selected-ion recording (SIR) with a quadrupole mass spectrometer in positive-ion mode. Calibration plots were linear over the concentration range 19.9–1119.6 ng mL⁻¹. Intra-day and inter-day precision (CV%) and accuracy (RE%) for quality-control samples (37.3, 124.4, and 622.0 ng mL⁻¹) ranged between 2.5 and 9.0% and between 1.3 and 3.5%, respectively. Extraction recovery of aripiprazole from plasma was in the range 75.8–84.1%. The method enables rapid, sensitive, precise, and accurate measurement of the concentration of aripiprazole in human plasma.     

A versatile electrophoric derivatisation reagent for negative ion chemical ionisation mass spectrometry: <Emphasis Type="Italic">o</Emphasis>-(pentafluorobenzyloxycarbonyl)-2,3,4,5-tetrafluorobenzoyl chloride

The synthesis of a novel electrophoric derivatisation reagent, o-(pentafluorobenzyloxycarbonyl)-2,3,4,5-tetrafluorobenzoyl chloride, is described. The reagent was tested against selected primary and secondary amino compounds, as well as phenolic and aliphatic hydroxyl compounds as analytical targets. The derivatives exhibit excellent mass spectral properties under negative ion chemical ionisation, i.e. reduced fragmentation and thus high ion current for the targeted m/z during analysis. Since the reagent bears a pentafluorobenzyl ester group, resulting negative ion chemical ionisation mass spectra were expectedly dominated by dissociative resonance electron capture typically observed with these compounds, additionally showing neutral loss of carbon dioxide and ammonia (in the case of primary amines). The reagent is suitable for detecting the target compounds with high sensitivity, as exemplified for the analysis of amphetamine and methylphenidate from human plasma where chromatographic background is drastically reduced by a shift in detected m/z and retention time and lower limits of quantification at 7.8 pg/mL (amphetamine) and 4.5 pg/mL (methylphenidate) can be obtained. The choice of two or three target quantification masses allows selective detection and adjustment of lowest background interference. No carryover effect was observed for the derivatives of amphetamine and methylphenidate.     

Cobalt oxide nanostructure-modified glassy carbon electrode as a highly sensitive flow injection amperometric sensor for the picomolar detection of insulin

Glassy carbon electrode modified with electrodeposited cobalt oxide nanostructure shows an excellent electrocatalytic activity toward insulin oxidation at a wide pH range. Cyclic voltammetry, hydrodynamic amperometry, and flow injection analysis (FIA) were used for insulin determination at a picomolar and higher-concentration range. Amperometric determination of insulin at this modified electrode yielded a calibration curve with the following characteristics; linear range, 100 pM–15 nM; sensitivity of 83.9 nA nM⁻¹ and detection limit 10 pM. FIA yielded the calibration curve with sensitivity and detection limit of 2.0 nA nM⁻¹ and 25 pM, respectively. Furthermore, the RSD of repetitive FIA for 200 pM insulin (n = 13) is 2%. In addition, the interference effect of electroactive existing species (lactic acid, cholesterol, ascorbic acid, uric acid, and glucose) was eliminated by covering the surface of the modified electrode with nafion film. Fast response time, signal stability, high sensitivity, low cost, and ease of preparation are the advantages of the proposed insulin sensor.     

Direct Mass Spectrometric Study of the Ionic Species Content of a C<Subscript>2</Subscript>HCl<Subscript>3</Subscript>/He/O<Subscript>2</Subscript> Microwave Discharge Plasma

Direct on-line studies of a C₂HCl₃/He/O₂ microwave discharge plasma made possible the evolution and detection of many unfamiliar ionic species. Numerous ionic chlorocarbons, chlorohydrocarbons, oxygenated chlorohydrocarbons, oxygenated hydrocarbon radicals, and simple hydrocarbon species were identified mass spectrometrically as by-products: C _m Cl _n (m = 1–4, 6, 8; n = 1–8), C _m H _n Cl _x (m = 1–4, 6, 7, 10; n, x = 1–6), C _m H _n Cl _x O _y (m = 1–5, 12; n = 1–7; x = 1, 2, 4, 6; y = 1–3), C _n H_2n−1O (n = 2, 3), C _m H _n (m = 2, 4, 6, 8; n = 2, 4), and so on. The studies clearly showed the presence of various unfamiliar positive ionic O-containing species such as C₂ClO₂, CCl₃CO, C₂H₂Cl₄O₂, and C₄H₂Cl₆O₃. It is apparent that positive-ion reactions play a significant role in producing many ionic species in the chemistry of C₂HCl₃ plasmas.     

Quantitative determination of isoniazid in biological samples by cation-selective exhaustive injection-sweeping-micellar electrokinetic chromatography

Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis, infects approximately one third of the current world population. Isoniazid is one of the most frequently used first-line anti-TB drugs. In this study, we developed a sensitive cation-selective exhaustive injection–sweeping–micellar electrokinetic chromatography method (CSEI-Sweep-MEKC) for analyzing isoniazid in human plasma. Parameters including acetonitrile (ACN) percentage in the separation buffer; the injection time, and concentration of the high-conductivity buffer; sodium dodecyl sulfate (SDS) concentration; phosphate concentration in the sample matrix; and the sample injection time were all optimized to obtain the best analytical performance. The optimal background electrolyte comprised 50 mM phosphate buffer, 100 mM SDS, and 15% ACN. Non-micelle background electrolyte, containing 75 mM phosphate buffer and 15% ACN, was first injected into the capillary, followed by a short plug of 200 mM phosphate (high-conductivity buffer). Run-to-run repeatability (n = 3) and intermediate precision (n = 3) of peak area ratios were found to be lower than 8.7% and 11.4% RSD, respectively. The accuracy of the method was within 98.1–106.9%. The limit of detection of isoniazod in human plasma was 9 ng mL⁻¹. Compared with conventional MEKC, the enhancement factor of the CSEI-Sweep-MEKC method was 85 in plasma samples. The developed method was successfully used to determine isoniazid concentration in patient plasma. The results demonstrated that CSEI-Sweep-MEKC has the potential to analyze isoniazid in human plasma for therapeutic drug monitoring and clinical research.     

GC–ICP–MS determination of dimethylselenide in human breath after ingestion of <Superscript>77</Superscript>Se-enriched selenite: monitoring of in-vivo methylation of selenium

The amount of volatile dimethylselenide (DMSe) in breath has been monitored after ingestion of sub-toxic amounts of selenium (300 μg ⁷⁷Se, as selenite) by a healthy male volunteer. The breath samples were collected in Tedlar bags every hour in the first 12 h and then at longer intervals for the next 10 days. The samples were subjected to speciation analysis for volatile selenium compounds by use of cryotrapping–cryofocussing–GC–ICP–MS. Simultaneously, all urine was collected and subjected to total selenium determination by use of ICP–MS. By monitoring m/z 82 and 77, background or dietary selenium and selenium from the administered selenite were simultaneously determined in the urine and in the breath—dietary selenium only was measured by monitoring m/z 82 whereas the amount of spiked ⁷⁷Se (99.1% [enriched spike]) and naturally occurring selenium (7.6% [natural abundance]) were measured by monitoring m/z 77. Quantification of DMSe was performed by using DMSe gas samples prepared in Tedlar bags (linear range 10–300 pg, R ²=0.996, detection limit of Se as DMSe was 10 pg Se, or 0.02 ng L⁻¹, when 0.5 L gas was collected). Dimethylselenide was the only selenium species detected in breath samples before and after the ingestion of ⁷⁷Se-enriched selenite. Additional DM⁷⁷Se was identified as early as 15 min after ingestion of the isotopically-labelled selenite. Although the maximum concentration of ⁷⁷Se in DMSe was recorded 90 min after ingestion, the natural isotope ratio for selenium in DMSe (77/82) was not reached after 20 days. The concentration of DMSe correlated with the total Se concentration in the urine during the experiment (R ²=0.80). Furthermore, the sub-toxic dose of 300 μg selenium led to a significant increase of DMSe and renal excretion of background selenium, confirming that selenium ingested as selenite is homeostatically controlled by excretion. The maximum concentration of DMSe resulting from the spiked selenite was 1.4 ng Se L⁻¹ whereas the dietary background level was less than 0.4 ng Se L⁻¹. Overall excretion as DMSe was calculated to be 11.2% from the ingested selenite within the first 10 days whereas urinary excretion accounts for nearly 18.5%.     

Development and validation of a selective HPLC-ESI-MS/MS method for the quantification of glyoxal and methylglyoxal in atmospheric aerosols (PM<Subscript>2.5</Subscript>)

This study concerns the development and validation of a complete method for the analysis of two highly reactive α-dicarbonyl compounds, glyoxal (Gly) and methylglyoxal (Mgly), in atmospheric fine particulate matter (PM_2.5). Method development included optimization of sample preparation procedures, e.g., filter extraction, concentration of extracts, derivatization and solid-phase extraction (SPE) of derivatives, as well as reversed-phase liquid chromatography coupled to electrospray ion-trap mass spectrometry (HPLC-ESI-IT/MS/MS) measurement parameters. Selectivity of detection was enhanced using tandem mass spectrometric analysis in ESI positive ion mode via two multiple reaction monitoring channels, m/z 433 → m/z 250 and m/z 419 → m/z 236 for Mgly and Gly. Retention times were 9.5 and 12.5 min for Gly- and Mgly-bis-hydrazone derivatives. Calibration ranged from 0.5 to 50 ng/mL. Inter-batch precision, expressed as relative standard deviation, was <15%. The method was shown to be unaffected by the sample matrix and to have recoveries of 100% and 60% for Gly and Mgly, respectively. Improved instrumental detection limits of 0.51 and 0.62 ng/mL for Gly and Mgly were achieved using a SPE method for the purification of 2,4-dinitrophenylhydrazine derivatization reagent solutions. This permitted the method to be used for analysis of filter samples obtained during a field study at the Taunus Observatory (mount Kleiner Feldberg, Germany). PM_2.5 concentrations ranged from 0.81 to 1.18 ng/m³ for Gly and from 0.83 to 1.92 ng/m³ for Mgly. PM concentrations correlated to the concentration of NO with coefficients (R ²) of 0.67 (Gly) and 0.78 (Mgly).     

An integrated reaction-retention and spectrophotometric detection flow system for the determination of nickel

An integrated flow-through photometric sensor for the determination of nickel in real samples of various origins has been developed. The sensor is based on the reaction of Ni(II) with 1-(2-pyridylazo)-2-naphthol (PAN) immobilized on a cationic resin which was placed in a flow-cell using a spectrophotometer tuned at 566 nm as detector. The Ni(II) ion from the sample injected into the carrrier stream (pH = 5.0) of a monochannel continuous flow system reacts with the immobilized chromogenic reagent to form a red chelate which remains on the active solid support and generates the analytical signal. When this reached its maximum value the Ni(II)-PAN chelate was destroyed using 1 M H₂SO₄ as eluents, leaving the sorbed PAN untouched. The response of the sensor was linear in the three concentration ranges assayed: 0.3–4.0, 0.1–1.6 and 0.05–0.8 μg mL^–1 for sample volumes of 100, 400 and 800 μL, respectively, and the R.S.D.(%) (n = 10) were 1.80(100 μL), 3.04(400 μL) and 2.29(800 μL). The sensor showed an excellent selectivity which could also be increased with a simple on-line modification to avoid interference from copper. It was applied to a variety of real samples with very good results in all cases. Received: 15 April 1998 / Revised: 29 June 1998 / Accepted: 3 July 1998     

Nitric oxide measurement in biological and pharmaceutical samples by an electrochemical sensor

A nitric oxide (NO) electrochemical sensor was developed via one-step construction of gold nanoparticles (GNPs)–chitosan (CS) nanocomposite sensing film on a glassy carbon electrode (GCE) surface. This method is very simple and convenient. The GNPs–CS film which is controllable and stable exhibits catalytic activity to NO oxidation. The anodic peak potential significantly shifted negatively compared with that at bare GCE. The high sensitivity and good stability of developed method have been coupled to a wide linear range from 3.60 × 10⁻⁸ to 4.32 × 10⁻⁵ M for the quantitative analysis of NO. The detection limit of 7.20 nM is much lower than the vast majority of reported methods. This NO sensor has been successfully applied to NO measurement in biological and pharmaceutical samples. Real-time amperometric data show that the addition of L-arginine (L-Arg) can cause a slow release of NO from a whole rat kidney with a maximum concentration of ca. 150 nM. The concentration of NO monitoring from the drug sample was calculated to be ca. 1.60 μM.     

Enantioselective analysis of (<Emphasis Type="Italic">R</Emphasis>)- and (<Emphasis Type="Italic">S</Emphasis>)-carvedilol in human plasma by high-performance liquid chromatography

Summary An HPLC column-switching method has been developed and validated for the enantioselective determination of (R)- and (S)-carvedilol in human plasma. Sample preparation was performed either off-line, by extraction with trichloromethane and back-extraction into 0.01m aqueous citric acid which was injected on to a LiChrosorb RP 8 column, or on-line, by injecting diluted (0.1m formic acid) plasma on to a LiChrosorb ADS column. In both instances separation was performed by gradient elution and on-line transfer of the fraction containing, the carvedilol on to an enantioselective Teicoplanin column. The enantiomers of carvedilol were separated isocratically by use of methanol-acetonitrile-triethylammonium acetate, 70:30:0.05 (v/v/w), as mobile phase. With fluorescence detection the limits of quantitation were 0.30 ng mL⁻¹ for (R)-carvedilol and 0.26 ng mL⁻¹ for (S)-carvedilol; these were sufficient to enable investigation of the effect of exercise on plasma concentrations of (R)- and (S)-carvedilol after oral administration of either the racemate or the pure enantiomers. Although the operating conditions were optimized for sample preparation by on-line deproteination on a LiChrospher RP 18 ADS column, the complete method was insufficiently rugged for analysis of large numbers of plasma samples—the enantioselectivity of the Teicoplanin column deteriorated too rapidly because of the transfer of enantioselectivity-poisoning interferences which could not be suppressed sufficiently. In contrast the liquid-liquid sample-extraction procedure combined with column switching resulted in a analytical method with long-term stability. Presented at Balaton Symposium '01 on High-Performance Separation Methods, Siófok, Hungary, September 2–4, 2001     

Development of automated amperometric detection of antibodies against <Emphasis Type="Italic">Bacillus anthracis</Emphasis> protective antigen

Picogram levels of antibodies against the protective antigen (PA) of Bacillus anthracis were detected in an automated electrochemical sandwich-type enzyme-linked immunosorbant assay. The antibodies were captured and detected using an 8 × 3 array of 50-μm-diameter cavities. The reagent and sample volumes were as low as 200 nL in a less than 25-min assay from capture to signal generation. The electrochemical detection of the antibodies was demonstrated at 0.05–10 μg/mL containing only 10–5,000 pg antibodies. The limit of detection is 10 fg for a 200-nL sample. Detection of anti-PA immunoglobulin G performed in spiked normal human serum and fresh whole human blood did not show a significant difference from detection in a buffer. The initial automation of the assay involved the use of a digital syringe pump for the delivery of reagents to the capture surface.     

Determination of (E)-3,5,4′-Trimethoxystilbene in Rat Plasma by LC with ESI-MS

(E)-3,5,4′-trimethoxystilbene (BTM-0512) is a resveratrol analog with a variety of pharmacological action, including anti-cancer properties, anti-allergic activity, estrogenic activity, antiangiogenic activity, and vascular-targeting activity against microtubule-destabilization. There is, however, no validated analytical method for quantification of (E)-3,5,4′-trimethoxystilbene in biological matrices, so pharmacokinetic data and suitable methods for determination of the compound in plasma are currently lacking. A rapid and sensitive liquid chromatographic–mass spectrometric method for determination of (E)-3,5,4′-trimethoxystilbene in rat plasma, using carbamazepine as internal standard, has been developed and validated. Plasma samples were treated with acetonitrile to precipitate proteins. Samples were then analyzed by HPLC on a 250mm × 4.6 mm i.d., 5-μm particle, C₁₈ column with methanol–water, 80:20 (v/v), containing 10 mm ammonium acetate and 0.2% formic acid (pH 3.0), as mobile phase, delivered at 0.85 mL min⁻¹. A single-quadrupole mass spectrometer with an electrospray interface operated in selected-ion monitoring mode was used to detect [M + H]⁺ ions at m/z 271.3 for (E)-3,5,4′-trimethoxystilbene and m/z 237.5 for the internal standard. (E)-3,5,4′-trimethoxystilbene and the internal standard eluted as sharp, symmetrical peaks with retention times of 8.9 and 4 min, respectively. Calibration plots for (E)-3,5,4′-trimethoxystilbene in rat plasma at concentrations ranging from 0.01 to 5.0 μg mL⁻¹ were highly linear. Intra-day and inter-day precision, as RSD, was <12.9%, and accuracy was in the range 94.8–104.7%. The limit of detection in plasma was 0.005 μg mL⁻¹. The method was successfully used to determine the concentration of (E)-3,5,4′-trimethoxystilbene after oral administration of 86 mg kg⁻¹ of the drug to Sprague–Dawley rats and can be used to investigate the pharmacokinetics of the compound.