LC–MS/MS coupled with immunoaffinity extraction for determination of estrone, 17β-estradiol and estrone 3-sulfate in human plasma

Determination of estrogens in plasma is important in evaluation of effects of some anticancer drugs, such as aromatase inhibitors. However, as reported previously, high performance liquid chromatography–radio immunoassay (HPLC–RIA) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) with chemical derivatization require complicated sample preparation. In this study, a highly sensitive and simple method for determination of estrone (E1), 17β-estradiol (E2) and estrone 3-sulfate (E1S) in human plasma has been developed. Following diethylether extraction from plasma, analytes were purified by immunosorbents and then determined by LC–MS/MS using electrospray ionization (ESI). Immunosorbents were prepared by immobilization of specific antibodies raised against each analyte onto solid support. Use of selective immunosorbents in sample preparation removed interference in plasma samples that would cause ionization suppression, and markedly improved the sensitivity of LC–MS/MS for these analytes, without derivatization. Calibration curves of each analyte showed good linearity and reproducibility over the range of 0.05–50 pg/injection for E1, 0.2–50 pg/injection for E2 and 0.05–300 pg/injection for E1S, respectively. The mean values of lower limits of quantification (LLOQ) in human plasma corrected by recovery of deuterated estrogens (internal standard, I.S.) were 0.1892 pg/mL for E1, 0.7064 pg/mL for E2 and 0.3333 pg/mL for E1S, respectively. These LLOQ values were comparable to those previous reported using HPLC–RIA and LC–MS/MS. Using this method, the normal levels of three estrogens in healthy female plasma (n = 5) were determined. The mean values of E1, E2 and E1S were 38.0 pg/mL (range 24.8–53.0), 34.3 pg/mL (22.6–46.6) and 786 pg/mL (163–2080), respectively. The immunoaffinity LC–MS/MS described here allows sensitive and accurate quantification of E1, E2 and E1S without laborious sample preparation.     

LC–ESI-Q-TOF-MS for faster and accurate determination of microcystins and nodularins in serum

Microcystins (MC) and nodularins (Nod) are cyclic peptide hepatotoxins and tumour promoters produced by cyanobacteria. This study deals with liquid chromatography–mass spectrometry (LC–MS) analyses of 9 major cyanobacterial peptide toxins, starting with a comparison of six small particle size reversed-phase HPLC columns, from which one, Phenomenex Synergi Hydro-RP, was chosen for further chromatography with accurate mass MS studies in a complex biological fluid, serum. The instrumentation used for the serum sample analysis included a Bruker micrO-TOF-Q-MS coupled to an Agilent 1200RR LC system. Total analysis run time per sample was 8.5 min. The Q-TOF-MS instrument was operated on auto MS–MS mode to obtain fragment ions (such as the characteristic fragment m/z 135 from Adda amino acid residue) for toxin identification purposes. Detected mass errors in serum samples were in the range of from 0.3 mDa to 9.1 mDa. The narrow mass window (±20 mDa) for mass chromatograms used in quantitation gave benefits by background noise reduction. We conclude that a LC–ESI-Q-TOF-MS instrumentation is a powerful tool for identification and quantitation of cyanobacterial peptide toxins in a biological matrix.     

Development of a coupled-column liquid chromatographic–tandem mass spectrometric method for the direct determination of betamethasone in urine

Different hyphenated liquid chromatographic (LC) and mass spectrometric (MS) techniques were investigated in order to set-up a method for the fast, direct analysis of betamethasone in hydrolysed and non-hydrolysed urine using large-volume sample injection. After the optimisation of the LC parameters using a traditional UV detector and of the thermospray and mass spectrometric parameters by flow injection, urine samples (0.5 ml) were submitted to analysis by either LC combined with tandem mass spectrometry (MS–MS), coupled-column LC (LC–LC) combined with single quadrupole MS, and LC–LC–MS–MS. Both the three-step configurations (LC–MS–MS and LC–LC–MS) did not provide satisfactory results: loss of sensitivity was noted in the case of LC–MS–MS (likely due to reduced efficiency in the ionisation of betamethasone in the thermospray owing to the presence of large amounts of matrix interference), while in the case of LC–LC–MS a high chemical noise resulting in insufficient selectivity of detection was observed. On the contrary, LC–LC–MS–MS analysis proved to meet the demand of high speed of analysis (sample throughput, 4.5 h⁻¹), selectivity, and sensitivity (LOQ, 1 ng/ml; LOD, 0.2 ng/ml). Notwithstanding the complex analytical system adopted, the developed procedure was manageable and very robust, provided that at the beginning of each analytical session the performance of the system was controlled by checking the retention time of the analytes on the first analytical column with UV detection and by optimising vaporiser temperature of the thermospray by flow injection.     

Quantitative analysis of 3α,6α,24-trihydroxy-24,24-di(trifluoromethyl)-5β-cholane,a potent synthetic steroidal liver X receptor agonist in plasma using liquid chromatography–tandem mass spectrometry

The steroidal liver X receptor agonist, 3α,6α,24-trihydroxy-24,24-di(trifluoromethyl)-5β-cholane (ATI-829) is a potential therapeutic agent for the treatment of atherosclerosis. A sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS–MS) method for the quantification of ATI-829 in mouse plasma was developed and validated. Proteins in a 25 μL aliquot of mouse plasma were precipitated, and ATI-829 was extracted from the precipitate by the addition of 125 μL methanol. The overall extraction efficiency was greater than 99%. LC–MS–MS with negative ion electrospray and selected reaction monitoring was used for the quantitative analysis of ATI-829. The lower limit of quantitation of ATI-829 corresponded to 5.0 ng/mL (9.7 nM) plasma. Interference from matrix was negligible. The calibration curve was linear over the range 5–2000 ng/mL. The intra-day precision and inter-day precision of the analyses were <4.5% and <6%, respectively, and the accuracy ranged from 92% to 103%. ATI-829 in plasma was stable for at least 6 h at room temperature, 1 week at 4 °C, and 3 weeks at −20 °C. The validated method was then utilized for pharmacokinetic studies of ATI-829 administered to mice.     

Highly sensitive method for the determination of tamsulosin hydrochloride in human plasma dialysate, plasma and urine by high-performance liquid chromatography–electrospray tandem mass spectrometry

A highly sensitive method for the determination of tamsulosin hydrochloride, a structurally new type of sulphamoile derivative, in human plasma dialysate, plasma and urine has been developed by using liquid chromatography–electrospray tandem mass spectrometry (LC–MS–MS). Plasma dialysate, plasma and urine samples were extracted by brief liquid-phase extraction and analyzed using an HPLC system coupled to a mass spectrometer via an electrospray ionization interface. Selected reaction monitoring was used for the detection of tamsulosin and its internal standard. This method was validated in the concentration range 10–1000 pg/ml in plasma dialysate, 0.5–50 ng/ml in plasma, and 1–100 ng/ml in urine with sufficient specificity, accuracy and precision. The in vivo protein binding study demonstrated that the unbound tamsulosin in human plasma obtained by the equilibrium dialysis after 0.4-mg oral dosing was measurable. In addition, the percentage of unbound tamsulosin in an in vitro study (0.71–0.91%) obtained by using spiked ¹⁴C-labelled tamsulosin was slightly larger than that of the in vivo study (0.68–0.86%), indicating that the unbound concentration calculated by the product of the plasma concentration and the in vitro unbound fraction (fu) was unfavorably overestimated. These results suggest that the combination of LC–MS–MS and equilibrium dialysis method has enough sensitivity to determine the unbound concentration in clinical use and gives the concentration more exactly than the in vitro fu.     

Improved liquid chromatography–tandem mass spectrometry method for the analysis of eptifibatide in human plasma

A rapid, selective and highly sensitive high performance liquid chromatography–tandem mass spectrometry method (LC–MS/MS) was developed and validated for the determination and pharmacokinetic investigation of eptifibatide in human plasma. Eptifibatide and the internal standard (IS), EPM-05, were extracted from plasma samples using solid phase extraction. Chromatographic separation was performed on a C₁₈ column at a flow rate of 0.5 mL/min. Detection of eptifibatide and the IS was achieved by tandem mass spectrometry with an electrospray ionization (ESI) interface in positive ion mode. Traditional multiple reaction monitoring (MRM) using the transition of m/z 832.6 → m/z 646.4 and m/z 931.6 → m/z 159.4 was performed to quantify eptifibatide and the IS, respectively. The calibration curves were linear over the range of 1–1000 ng/mL with the lower limit of quantitation validated at 1 ng/mL. The intra- and inter-day precisions were within 13.3%, while the accuracy was within ±7.6% of nominal values. The validated LC–MS/MS method was successfully applied for the evaluation of pharmacokinetic parameters of eptifibatide after intravenous (i.v.) administration of a 45 μg/kg bolus of eptifibatide to 8 healthy volunteers.     

An automated screening method for drugs and toxic compounds in human serum and urine using liquid chromatography–tandem mass spectrometry

A fully automated screening using liquid chromatography–mass spectrometric method applying data-dependent acquisition was developed to identify toxicologically relevant substances in serum and urine. A library including more than 405 spectra of about 365 compounds (main drugs and important metabolites) was established. An easy to use program was created to automate and accelerate library search. Drugs were identified based on their relative retention times, molecular ions and fragment ions. Limits of detection were tested with 100 of the 365 compounds the majority of these were lower than 100 μg/l (67%). The developed LC–MS–MS system seems to be a valuable alternative to other general unknown screening methods allowing fast and specific identification of drugs in serum and urine samples.     

Quantitation of fatty acid ethyl esters in human meconium by an improved liquid chromatography/tandem mass spectrometry

This paper reports the development and validation of an improved assay for quantitation of fatty acid ethyl esters (FAEEs) in human meconium using liquid chromatography/tandem mass spectrometry (LC–MS/MS). FAAEs (ethyl laurate, ethyl myristate, ethyl palmitate, ethyl palmitoleate, ethyl stearate, ethyl oleate, ethyl linoleate, ethyl linolenate, and ethyl arachidonate) and the internal standard (I.S.), ethyl heptadecanoate, were separated by reverse phase HPLC and quantified by MS/MS using electrospray ionization (ESI) and multiple reaction monitoring (MRM) in the positive ionization mode. The absolute recovery of FAEEs varied from 55 ± 10% for 0.33 nmol/g (100 ng/g) of ethyl linoleate up to 86 ± 8% for 1.55 nmol/g (500 ng/g) of ethyl miristate. The LODs and LOQs varied from 0.01 to 0.08 nmol/g and from 0.02 to 0.27 nmol/g, respectively. The assay has been successfully applied to examine the FAEE levels in 81 meconium samples from babies born to mothers reporting alcohol consumption, to varying degrees, during pregnancy.     

Liquid chromatography–tandem mass spectrometry quantification of levosulpiride in human plasma and its application to bioequivalence study

An improved method for determining levels of levosulpiride in human plasma using ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) was developed and validated. The protein precipitation method was used for plasma sample preparation. Levosulpiride and an internal standard (IS) were isocratically separated on a UPLC BEH C₁₈ column with a mobile phase of ammonium formate buffer (1 mM, adjusted to pH 3 with formic acid) and acetonitrile (60:40, v/v). MS/MS detection was performed by monitoring the parent → daughter pair of levosulpiride and the IS at m/z 342 → 112 and 329 → 256, respectively. The method was linear from 2.5 to 200 ng/mL and exhibited acceptable precision and percent recovery. The method was successfully demonstrated in pharmacokinetic and bioequivalence studies of two levosulpiride oral formulations administered to healthy volunteers. When compared to the previous LC–MS methods, the proposed method is faster, well-validated, and uses lesser plasma volume and a similar sensitivity. The use of UPLC allowed rapid and sensitive quantification of levosulpiride, making this method suitable for high-throughput clinical applications.     

Methyl vinyl ketone—Identification and quantification of adducts to N-terminal valine in human hemoglobin

Adducts to N-terminal valines in Hb have been shown useful as biomarkers of exposure to electrophilic compounds. Adducts from many compounds have earlier been measured with a modified Edman degradation method using a GC–MS/MS method. A recently developed method, the adduct FIRE procedure™, adopted for analysis by LC–MS/MS, has been applied in this study. With this method a fluorescein isothiocyanate (FITC) reagent is used to measure adducts (R) from electrophiles with a modified Edman procedure. By using LC–MS/MS in product ion scan mode, a new peak was identified and the obtained MS data indicated that this adduct could originate from methyl vinyl ketone (MVK). Incubation of human-, sheep- and bovine blood with MVK increased the signal of the identified peak. By comparing the LC–MS/MS data from the unknown background peak with data obtained from synthesized fluorescein thiohydantoin (FTH) standards of the MVK adduct to valine and d₈-valine, the identity of this adduct was confirmed. The MVK adduct was shown present in human blood (∼35 pmol/g globin, n = 3) and only just above LOD in bovine blood, n = 1 (LOD = 2 pmol/g globin). MVK reacts, in similarity with acrylamide, via Michael addition. MVK is known to occur in the environment and has earlier been observed in biological samples, which means that there are possible natural and anthropogenic exposure sources. Analysis of an Hb adduct from MVK in humans has to our knowledge not been described before.     

Determination of esmolol and metabolite enantiomers within human plasma using chiral column chromatography

A high performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed for the simultaneous determination of each of esmolol's enantiomers at the 25–1000 ng/ml concentrations observed in human plasma upon intravenous administration of this rapidly metabolized beta-adrenergic receptor blocking agent. Alternatively, a high performance liquid chromatography (HPLC) UV detection method has been developed for the simultaneous determination of each of the enantiomers for esmolol's metabolite which, in turn, achieve 2.5–50 μg/ml concentrations in human plasma. Utilizing chiral columns, these methods do not require a precolumn asymmetric derivatization step. Linearity in all cases was >0.99. Precision and accuracy at all but the lowest concentrations were within ±6% for the esmolol enantiomers and within ±2.5% for the esmolol metabolite enantiomers. These values should be suitable for performing thorough pharmacokinetic studies for all of the stereoisomers of this prototypical soft drug and its corresponding metabolite.     

Development and validation of sensitive and selective LC–MS/MS methods for the determination of BMS-708163, a γ-secretase inhibitor,in plasma and cerebrospinal fluid using deprotonated or formate adduct ions as precursor ions

BMS-708163 is a γ-secretase inhibitor that is being developed for the treatment of Alzheimer's disease. Several LC–MS/MS methods have been developed for the determination of BMS-708163 in both plasma and cerebrospinal fluid in support of dog, rat, mouse and human studies. To support non-clinical studies, an LC–MS/MS method with a lower limit of quantitation (LLOQ) of 5 ng/mL, was developed and validated in dog, rat, and mouse plasma by using the deprotonated ion as the precursor ion. To support clinical studies, an LC–MS/MS method with LLOQ of 0.1 ng/mL, was developed and validated in human plasma by using the formate adduct as the precursor ion. Formic acid (0.01%) in water and acetonitrile was found to be the most favorable mobile phases for both deprotonated and formate adduct ions in negative electrospray ionization mode. A combination of a 3M Empore™ C18 plate for SPE and a Waters Atlantis dC18 analytical column for separation was used to achieve a highly selective solid phase extraction and chromatographic procedure from plasma without dry down and reconstitution steps. In the development of an assay for BMS-708163 in cerebral spinal fluid (CSF), significant non-specific binding of BMS-708163 was observed and resolved with pre- or post-spike of 0.2% Tween 20 into CSF samples. A dilute-and-shoot LC–MS/MS method with LLOQ of 0.1 ng/mL was developed and validated to assess BMS-708163 exposure in human CSF.     

Quantification of corticosteroids in bovine urine using selective solid phase extraction and reversed-phase liquid chromatography/tandem mass spectrometry

This paper presents the development of a simple liquid chromatography–tandem mass spectrometry (LC–MS/MS) method to determine corticosteroids in bovine urine sample matrices. This method uses a single phase extraction (SPE) for cleaning of the sample with an Oasis MAX cartridge at pH 9.0–9.5 and elution by a neutral organic solvent (acetonitrile/dichloromethane), followed by separation on a GEMINI C18 column in the gradient mode with acetate buffer (pH 4.1)/methanol. A triple quadrupole mass spectrometer equipped with a multimode ion source, set to negative atmospheric pressure chemical ionization (APCI) in the multiple reaction monitoring mode was used for detection. The main advantage of this method over other commonly used methods includes the use of SPE with a low volume cartridge for sample preparation and no ion suppression effects from matrix components of the urine samples in the LC–MS/MS analysis. This allowed a reduction the quantification limits (decision limits, CCα) for the first time to 0.1 μg/L (1 and 0.2 μg/L for triamcinolone and flumethasone, respectively). The developed method was validated in accordance with the European Union Commission Decision 2002/657 EC. The recoveries and within-laboratory reproducibility varied from 77% to 115% and 87% to 107.5%, respectively, at 2, 3, and 4 μg/L levels of corticosteroids. The relative standard deviation (RSD) of the measurements was lower than 30%. The decision limit was calculated by multiplying the signal-to-noise ratio by 3 and the obtained values were in the range of 0.1–1.0 μg/L, confirmed by the analysis of twenty blank samples, which were spiked at the desired concentrations. The detection capability was calculated by the addition of the decision limit and the standard deviation followed by multiplication by 1.64 of the within-laboratory reproducibility at 2 μg/L of corticosteroids. The method was applied to four urine samples, giving concentrations of prednisolone (PRED) residues in the range from 0.3 to 0.9 μg/L.     

A new modified Edman procedure for analysis of N-terminal valine adducts in hemoglobin by LC–MS/MS

A rapid and sensitive method using liquid chromatography–tandem mass spectrometry (LC–MS/MS) for simultaneous determination of adducts from acrylamide, glycidamide and ethylene oxide to N-terminal valines in hemoglobin (Hb) was developed. This new procedure is based on the same principles as the N-alkyl Edman procedure for analysis of adducts from electrophilic agents to N-terminal valines in Hb. The N-substituted valines can be detached, enriched and measured selectively as thiohydantoins by the use of an Edman reagent, in this case fluorescein isothiocyanate (FITC). This procedure is denoted as the “adduct FIRE procedure” as the FITC reagent is used for measurement of adducts (R) formed from electrophilic compounds with a modified Edman procedure. In this study, fluorescein thiohydantoin (FTH) analytes of N-substituted valines from acrylamide, glycidamide and ethylene oxide, as well as their corresponding hepta- and tri-deuterium-substituted analogues, were synthesized. These analytes (n = 8) were then characterized by LC–MS/MS (ESI, positive ion mode) and obtained product ions were interpreted. A considerable work with optimization of the FIRE procedure™, resulted in a procedure in which low background levels of the studied adducts could be measured from 250 μL lyzed whole blood samples (human non-smokers). The analytes were enriched and purified with solid phase extraction columns and analyzed by LC–MS/MS with LOQ down to 1 pmol adduct/g Hb. Compared to other procedures for determination of N-terminal Hb adducts, the introduction of FITC has led to a simplified procedure, where whole blood also can be used, giving new opportunities and reduced hand on time with increased sample throughput.     

Simultaneous quantification of intracellular and secreted active and inactive glucagon-like peptide-1 from cultured cells

Glucagon-like peptide-1 (GLP-1) is an incretin peptide that regulates islet hormone secretion. During recent years, incretin-based therapies have been widely used for patients with type 2 diabetes. GLP-1 peptides undergo N- and C-terminal processing for gain or loss of functions. We developed a method to quantify picomolar quantities of intact GLP-1 peptides using liquid chromatography–tandem mass spectrometry (LC–MS/MS). By employing this label-free selected reaction monitoring (SRM) method, we were able to analyze secreted GLP-1_1–37, GLP-1_7–37, and GLP-1_{7–36 amide} from human enteroendocrine NCI-H716 cells after stimulation with nateglinide, glucose, and sucralose. The absolute total concentrations of secreted GLP-1 peptides at baseline and after stimulation with nateglinide, glucose, and sucralose were 167.3, 498.9, 238.3, and 143.1 pM, respectively. Meanwhile, the ratios of GLP-1_1–37, GLP-1_7–37, and GLP-1_{7–36 amide} to total GLP-1 peptides were similar (6 ± 3, 26 ± 3, and 78 ± 5%, respectively). The SRM assay can analyze the concentrations of individual GLP-1 peptides and, therefore, is a tool to investigate the physiological roles of GLP-1 peptides. Furthermore, the molecular species secreted from NCI-H716 cells were unknown. Therefore, we performed a secretopeptidome analysis of supernatants collected from cultured NCI-H716 cells. Together with GLP-1 peptides, we detected neuroendocrine convertase 1, which regulates peptide hormones released from intestinal endocrine L-cells.     

Development,validation and comparison of LC–MS/MS and RIA methods for quantification of vertebrates-like sex-steroids in prosobranch molluscs

The role of vertebrate-like sex-steroids (testosterone, T, progesterone, P, and 17β-estradiol, E2) in molluscs is still debated, but they could represent potential biomarkers of endocrine disruption. A radioimmunoassay (RIA) and a liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) methods have been developed and compared to measure their levels in a gastropod snail Potamopyrgus antipodarum. Both methods showed a good reproducibility despite the complex matrix and the very low levels of vertebrate-like sex-steroids. Only T and P were detected using the LC–MS/MS method, while the RIA method reached lower detection limits and enabled the detection of all three steroids. Results indicated that T and P were mainly present as unconjugated forms. Both methods were compared in the analysis of snails exposed to waste water treatment plant effluents and led to the same conclusions concerning the modulation of steroids levels. Moreover, they both were in agreement concerning T measurements. On the other hand, LC–MS/MS appeared to be more suitable when analyzing P levels due to a low sensitivity of the RIA method. As E2 was not measured using the LC–MS/MS method because of a higher detection limit compared to the other steroids, the results obtained with the RIA method should be interpreted with caution. LC–MS/MS remains the gold standard for sex-steroid determinations, however a relevant and alternative method based on RIA was developed, requiring fewer organisms. RIA seems a promising method as a screening tool for experimental use, allowing comparison of sex-steroid levels in the mudsnail both in laboratory and in field experiments.     

Modified method for determination of sulfur metabolites in plant tissues by stable isotope dilution-based liquid chromatography–electrospray ionization–tandem mass spectrometry

A wide variety of sulfur metabolites play important roles in plant functions. We have developed a precise and sensitive method for the simultaneous measurement of several sulfur metabolites based on liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) and ³⁴S metabolic labeling of sulfur-containing metabolites in Arabidopsis thaliana seedlings. However, some sulfur metabolites were unstable during the extraction procedure. Our proposed method does not allow for the detection of the important sulfur metabolite homocysteine because of its instability during sample extraction. Stable isotope-labeled sulfur metabolites of A. thaliana shoot were extracted and utilized as internal standards for quantification of sulfur metabolites with LC–MS/MS using S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), methionine (Met), glutathione (GSH), and glutathione disulfide (GSSG) as example metabolites. These metabolites were detected using electrospray ionization in positive mode. Standard curves were linear (r² > 0.99) over a range of concentrations (SAM 0.01–2.0 μM, SAH 0.002–0.10 μM, Met 0.05–4.0 μM, GSH 0.17–20.0 μM, GSSG 0.07–20.0 μM), with limits of detection for SAM, SAH, Met, GSH, and GSSG of 0.83, 0.67, 10, 0.56, and 1.1 nM, respectively; and the within-run and between-run coefficients of variation based on quality control samples were less than 8%.     

Sensitive and rapid quantification of the cannabinoid receptor agonist naphthalen-1-yl-(1-pentylindol-3-yl)methanone (JWH-018) in human serum by liquid chromatography–tandem mass spectrometry

The current paper describes a validated method for the detection and quantification of naphthalen-1-yl-(1-pentylindol-3-yl)methanone (JWH-018), an ingredient of a herbal mixture called “Spice”, by means of HPLC–ESI–MS–MS in serum. Lower limit of detection and lower limit of quantification were 0.07 and 0.21 ng/ml, respectively. In 2 subjects who consumed ca. 50 μg/kg of JWH-018 by smoking, the active ingredient was detected by means of the described method. Thereby, the serum concentrations reached values of approx. 10 ng/ml and dropped within 3 h very fast (<10% of the measured maximum concentrations).     

Regioselective solvent-phase deuteration of polyphenolic compounds informs their identification by mass spectrometry

Liquid chromatography–mass spectrometry (LC–MS) is a highly sensitive tool for the analysis of polyphenolic compounds in complex food and beverage matrices. However, the high degree of isomerism among polyphenols in general often complicates this approach, especially for identification of novel compounds. Here, we explore the utility of mild acid-catalyzed deuterium (MACD) labeling via electrophilic aromatic substitution as a complementary method for informing polyphenolic compound structure elucidation. To prevent hydrolysis of acid-labile glycosidic linkages, optimal reaction conditions that maximize regioselective hydrogen/deuterium (H/D) exchange of aromatic protons while preserving compound integrity were characterized (60 °C, pH 3.0, 72 h). Under these conditions, standard compounds varying in the number and position of hydroxyl, glycosyl, and methyl groups about their aromatic core structure produced distinguishable H/D exchange patterns. The applicability of this method for the analysis of complex mixtures was demonstrated in red wine where the extent of deuterium exchange, together with accurate mass information, led to the putative identification of an unknown compound. The identification was further supported by tandem MS (MS/MS) data, which matched conclusively to the same compound in the Metlin LC–MS/MS library. With the capacity to discriminate between select isomeric forms, MACD labeling provides structural information that complements accurate mass and tandem mass spectral measurements for informing the identification of polyphenolics by MS.