Determination of proline in human serum by a robust LC‐MS/MS method: application to identification of human metabolites as candidate biomarkers for esophageal cancer early detection and risk stratification

Altered serum proline levels are related to cancer metabolism. This study developed and validated a LC‐MS/MS method to analyze proline in human serum. Surrogate blank serum, coupled with stable isotope l ‐proline‐¹³C₅,¹⁵ N as internal standard, was used for generating standard curves ranging from 2.5 to 100 μg/mL. Proline was extracted from serum samples using methanol. A Phenomenex Lux 5u Cellulose‐1 column (250 × 4.6 mm) was used for chromatographic separation with 40% methanol in 0.05% formic acid aqueous solution as a mobile phase. Mass detection was performed under positive ionization electrospray. Intra‐ and inter‐day accuracy and precision were <10%. The extraction recovery and matrix factor were 99.17 and 1.47%, respectively. Our study showed that the chiral column had high specificity and selectivity for separating proline from serum components. The assay was successfully applied for the quantification of human serum proline levels from 30 esophageal cancer patients and 30 healthy volunteers. Statistical analyses showed significantly lower levels of serum proline in the patients as compared with the healthy volunteers (p‐value = 0.011). We report here a simple, specific and reproducible LC‐MS/MS method for the quantification of proline in human serum as a potential screening biomarker for esophageal cancer. Copyright © 2014 John Wiley & Sons, Ltd.     

Development and validation of a sensitive LC‐MS/MS method for determination of tacrolimus on dried blood spots

A bioanalytical method for the quantification of tacrolimus (TAC) on dried blood spots (DBS) using liquid chromatography, electrospray ionization coupled with tandem mass spectrometry (LC‐ESI‐MS/MS) was developed and validated. It involves solvent extraction of a punch disk of DBS followed by liquid–liquid extraction. The analyte and the internal standard (IS, ascomycin) were separated on a phenyl column using an isocratic mobile phase elution at a flow rate of 0.3 mL/min. The assay was linear from 1 to 80 ng/mL. The mean recovery of TAC was 76.6%. Intra‐assay, inter‐assay imprecision and biases were all less than 15%. TAC on DBS was stable for at least 10 days at room temperature, and at least 24 h at 50°C. A chromatographic effect of the filter paper (Whatman 903) was not detected. The volume of blood (15–50 μL) and hematocrit of blood (ranging from 23.2 to 48.6%) did not show a significant influence on detection of TAC concentration by DBS‐LC‐MS/MS. Fifty samples from patients were detected by both DBS‐LC‐MS/MS and microparticle enzyme‐linked immunoassay (MEIA). TAC concentrations measured by DBS‐LC‐MS/MS method tended to be lower than those by MEIA. Copyright © 2012 John Wiley & Sons, Ltd.     

General method for extraction of blueberry anthocyanins and identification using high performance liquid chromatography–electrospray ionization-ion trap-time of flight-mass spectrometry

A systematic approach for optimizing the extraction and identification of anthocyanins from blueberries was explored using HPLC-UV and HPLC–ESI-IT-TOF-MS. Sample homogenization effects, extraction solvent selection, type of acid, and amount used in extraction solvent were investigated. A mixture of methanol:water:trifluoroacetic acid (70:30:1, v/v/v) was found to be the best solvent system for blueberry anthocyanin extraction. Differences in total anthocyanin content due to commercial blueberry processing were explored as an application using the optimized extraction technique and HPLC-UV analysis. A methodical system for anthocyanin identification by HPLC–ESI-IT-TOF-MS without the use of standards was also reviewed and applied. Consideration was given to elution order by chromatographic separation with selective detection at 520 nm, high mass accuracy m/z values, tandem MS fragmentation, and previously published literature. Overall, 25 anthocyanins from a wild type highbush blueberry were identified and reported.     

Optimization of chromatography to overcome matrix effect for reliable estimation of four small molecular drugs from biological fluids using LC–MS/MS

The article describes a systematic study to overcome the matrix effect during chromatographic analysis of gemfibrozil, rivastigmine, telmisartan and tacrolimus from biological fluids using LC–ESI–MS/MS. All four methods were thoroughly developed by the appropriate choice of analytical column, elution mode and pH of mobile phase for improved chromatography and overall method performance. Matrix effect was assessed by post-column analyte infusion, slope of calibration line approach and post-extraction spiking. The best chromatographic conditions established were: Acquity BEH C₁₈ (50 × 2.1 mm, 1.7 μm) column with 5.0 mm ammonium acetate, pH 6.0–methanol as the mobile phase under gradient program for gemfibrozil; Luna CN (50 × 2.0 mm, 3 μm) column with a mobile phase consisting of acetonitrile–10 mm ammonium acetate, pH 7.0 (90:10, v/v) for rivastigmine; Inertsustain C₁₈ (100 × 2.0 mm, 5 μm) column using methanol–2.0 mm ammonium formate, pH 5.5 (80: 20, v/v) as the mobile phase for isocratic elution of telmisartan; and Acquity BEH C₁₈ (50 × 2.1 mm, 1.7 μm) with methanol–10 mm ammonium acetate, pH 6.0 (95:5, v/v) as mobile phase for tacrolimus. The methods were thoroughly validated as per European Medicines Agency and US Food and Drug Administration guidance and were successfully applied for pharmacokinetic studies in healthy subjects.     

Development and validation of an LC-MS/MS method for profiling 39 urinary steroids (estrogens,androgens, corticoids,and progestins)

Abnormal production or metabolism of steroid hormones is responsible for the development of endocrine diseases. Thus, accurate quantification of steroid hormones is needed for both research into clinical conditions and diagnostic and monitoring purposes. An improved analytical method for profiling 39 steroids in urine using LC–MS/MS was developed. As a pre-treatment procedure prior to LC–tandem mass spectrometry (LC–MS/MS) analysis, hydrolysis using β-glucuronidase and solid-phase extraction for purifying the samples were performed. Steroids were separated using Waters ACQUITY BEH C₁₈ column (2.1 × 100 mm, 1.7 μm) and a mobile phase consisting of eluent A (0.01% formic acid and 1 mm ammonium formate in water) and eluent B (0.01% formic acid and 1 mm ammonium formate in methanol) with a gradient program at a flow rate of 0.4 mL/min. Under the optimized method, the linearity of calibration curves was higher than 0.992. The limits of detection at signal-to-noise ratio of 3 were 0.03–90 ng/mL. The developed novel LC–MS/MS method can quantitatively profile 39 steroids in a single analytical run. Steroid profiling based on quantitative results could improve the diagnosis and monitoring of hormone-dependent diseases.     

Measurement of fexofenadine concentration in micro‐sample human plasma by a rapid and sensitive LC‐MS/MS employing protein precipitation: application to a clinical pharmacokinetic study

A simple, rapid and sensitive liquid chromatography/positive ion electro‐spray tandem mass spectrometry method (LC‐MS/MS) was developed and validated for the quantification of fexofenadine with 100 μL human plasma employing glipizide as internal standard (IS). Protein precipitation was used in the sample preparation procedure. Chromatographic separation was achieved on a reversed‐phase C₁₈ column (5 μm, 100 × 2.1 mm) with methanol : buffer (containing 10 mmol/L ammonium acetate and 0.1% formic acid; 70 : 30, v/v) as mobile phase. The total chromatographic runtime was approximately 3.0 min with retention time for fexofenadine and IS at approximately 1.9 and 2.1 min, respectively. Detection of fexofenadine and IS was achieved by LC‐MS/MS in positive ion mode using 502.1 → 466.2 and 446.0 → 321.1 transitions, respectively. The method was proved to be accurate and precise at linearity range of 1–600 ng/mL with a correlation coefficient (r) of ≥0.9976. The validated method was applied to a pharmacokinetic study in human volunteers following oral administration of 60 or 120 mg fexofenadine formulations, successfully. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of spinosad at trace levels in bee pollen and beeswax with solid–liquid extraction and LC–ESI‐MS

This paper reports the use of a new LC method with a fused‐core analytical column coupled to ESI‐MS to determine residues of the biopesticide spinosad in bee pollen and beeswax. The method analyzes the active ingredients, spinosyns A and D, with a simple and efficient sample treatment (recovery between 90 and 105%) consisting of a solid–liquid extraction with acetone (bee pollen) or acetonitrile (beeswax). The method was validated in terms of selectivity, LOD, LOQ, linearity, and precision. The LOD and LOQ values ranged between 0.1–0.2 and 0.4–0.7 μg/kg, respectively. Moreover, the precision obtained within the linear concentration range (LOQ 500 μg/kg) was satisfactory (RSD lower than 5%). Finally, the proposed method was applied to analyze bee pollen and beeswax samples collected from apiaries located close to fruit orchards in two Spanish regions.     

Identification and quantification of free radical scavengers in the flower buds of Lonicera species by online HPLC-DPPH assay coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry

Flos Lonicerae, derived from the flower buds of several medicinal Lonicera species, is a commonly used herbal medicine with multiple pharmacological activities, one of the major ones being antioxidant activity. In this study, free radical scavengers in the flower buds of six Lonicera species were screened, identified and quantified by online HPLC-DPPH (1,1-diphenyl-2-picrylhydrazyl) assay coupled with LC quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS). The antioxidants were firstly screened from the complex plant matrix by the online HPLC-DPPH assay. Then the active compounds were identified by LC Q-TOF MS/MS, and the possible fragmentation pathways were proposed. The reactivity of antioxidants available was investigated using an internal standard method by online LC assay. The contents of 12 antioxidants were also determined or estimated by HPLC coupled with diode array detector. The total antioxidant capability determined by the online method was used as the marker to evaluate the quality of Flos Lonicerae. The results were important to clarify the material basis and therapeutic mechanism of Flos Lonicerae.     

Detection of monohydroxylated polycyclic aromatic hydrocarbons in urine and particulate matter using LC separations coupled with integrated SPE and fluorescence detection or coupled with high‐resolution time‐of‐flight mass spectrometry

A high‐performance liquid chromatographic (HPLC) method with integrated solid‐phase extraction for the determination of 1‐hydroxypyrene and 1‐, 2‐, 3‐, 4‐ and 9‐hydroxyphenanthrene in urine was developed and validated. After enzymatic treatment and centrifugation of 500 μL urine, 100 μL of the sample was directly injected into the HPLC system. Integrated solid‐phase extraction was performed on a selective, copper phthalocyanine modified packing material. Subsequent chromatographic separation was achieved on a pentafluorophenyl core–shell column using a methanol gradient. For quantification, time‐programmed fluorescence detection was used. Matrix‐dependent recoveries were between 94.8 and 102.4%, repeatability and reproducibility ranged from 2.2 to 17.9% and detection limits lay between 2.6 and 13.6 ng/L urine. A set of 16 samples from normally exposed adults was analyzed using this HPLC‐fluorescence detection method. Results were comparable with those reported in other studies. The chromatographic separation of the method was transferred to an ultra‐high‐performance liquid chromatography pentafluorophenyl core–shell column and coupled to a high‐resolution time‐of‐flight mass spectrometer (HR‐TOF‐MS). The resulting method was used to demonstrate the applicability of LC‐HR‐TOF‐MS for simultaneous target and suspect screening of monohydroxylated polycyclic aromatic hydrocarbons in extracts of urine and particulate matter.     

Determination of three capsaicinoids in Capsicum annuum by pressurized liquid extraction combined with LC‐MS/MS

A new method based on pressurized liquid extraction followed by LC‐MS/MS analysis has been developed for the identification and quantification of three capsaicinoids (capsaicin, dihydrocapsaicin, and nordihydrocapsaicin) in extracts of Capsicum annuum. For the recovery of three capsaicinoids, the efficiency levels of ultrasonic‐assisted extraction, microwave‐assisted extraction, Soxhlet extraction, and pressurized liquid extraction were compared under different conditions. Pressurized liquid extraction resulted in higher yields. Pressurized liquid extractions were performed using methanol; temperature was set at 100°C and pressure at 1500 psi. LC analysis was performed on a Waters XBridge? C₁₈ column (150 × 2.1 mm, id 3.5 μm) eluted by a mobile phase of 0.1% formic acid and ACN. Data acquisition was carried out in multiple reaction monitoring transitions mode, monitoring two‐reaction monitoring transitions to ensure an accurate identification of target compounds in the samples. The proposed method is rapid, simple, and could be utilized for the routine analysis of three capsaicinoids in C. annuum samples.     

Simultaneous determination of a novel oral Janus kinase inhibitor ASP015K and its sulfated metabolite in rat plasma using LC‐MS/MS

A sensitive and selective liquid chromatography with tandem mass spectrometry (LC‐MS/MS) was developed for determining the concentrations of novel Janus kinase inhibitor ASP015K and its sulfated metabolite M2 in rat plasma. This method involves solid‐phase extraction (SPE) from 25 μL of rat plasma. LC separation was performed on an Inertsil PH‐3 column (100 mm L ×4.6 mm I.D., 5 µm) with a mobile phase consisting of 10 mM ammonium acetate and methanol under linear gradient conditions. Analytes were introduced to the LC‐MS/MS through an electrospray ionization source and detected in positive‐ion mode using selected reaction monitoring. Standard curves were linear from 0.25 to 500 ng/mL (r ≥0.9964). This assay enabled quantification of ASP015K and M2 at a concentration as low as 0.25 ng/mL in rat plasma. Validation data demonstrated that the method is selective, sensitive and accurate. Further, we also successfully applied this method to a preclinical pharmacokinetic study in rats. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of bencycloquidium bromide in dog plasma by liquid chromatography with electrospray ionization tandem mass spectrometry

A rapid, selective and sensitive liquid chromatography/tandem mass spectrometry (LC‐MS/MS) method was developed and validated for determining bencycloquidium bromide (BCQB) in beagle dog plasma. The plasma sample was deproteinized with methanol which contained l‐ethyl‐bencycloquidium bromide as internal standard, and supernantant was assayed by LC‐MS/MS. The chromatographic separation was performed on a Phenomenex C₁₈ column (100 × 2.0 mm, i.d., 3.0 μm) with a gradient programme mobile phase consisting of methanol and ammonium acetate (5 mm) containing 0.15% acetic acid and at a flow rate of 0.3 mL/min. Electrospray ionization in positive ion mode and selective reaction monitoring was used for the quantification of BCQB with a monitored transitions m/z 330.2 → 142.1 for BCQB and m/z 344.2 → 126.2 for IS. Validation results indicated that the lower limit of quantification was 0.05 ng/mL and the assay exhibited a linear range of 0.05–10.0 ng/mL and gave a correlation coefficient of 0.9998. The intra‐ and inter‐run precisions of the assay were 1.7–4.6 and 3.2–15.6%, respectively, and the intra‐ and inter‐day accuracies were ?8.8 to 1.1 and ?5.0 to 4.6%, respectively. The developed method was applied for the pharmacokinetic study of BCQB in beagle dogs following a single intranasal dose. Copyright © 2009 John Wiley & Sons, Ltd.     

A validated LC–MS/MS method for quantitative determination of L-dopa in Fagioli di Sarconi beans (Phaseolus vulgaris L.)

An analytical method based on ultrasound assisted extraction (UAE) and liquid chromatography coupled to electrospray tandem mass spectrometry (LC–ESI/MS/MS) was validated and applied for determining L-dopa in four ecotypes of Fagioli di Sarconi beans (Phaseolus vulgaris L.), marked with the European label PGI (Protected Geographical Indication). The selectivity of the proposed method was ensured by the specific fragmentation of the analyte. Simple isocratic chromatographic conditions and mass spectrometric detection in multiple reaction monitoring (MRM) acquisition mode were used for sensitive quantification. The LC–ESI/MS/MS method was validated within a linear range of 0.001–5.000 μg/mL. Values of 0.4 and 1.1 ng/mL were obtained for the limits of detection and quantification, respectively. The repeatability, inter-day precision, and recovery values ranges were 0.6%–4.5%, 5.4%–9.9%, and 83%–93%, respectively. Fresh and dried beans, as well as pods, cultivated exclusively with organic methods avoiding any synthetic fertilizers and pesticides were analyzed showing an L-dopa content ranging from 0.020 ± 0.005 to 2.34 ± 0.05 μg/g dry weight.     

Analysis of perfluorinated chemicals in sludge: Method development and initial results

A fast, rigorous method was developed to maximize the extraction efficacy for ten perfluorocarboxylic acids and perfluorooctanesulfonate from waste-water-treatment sludge and to quantitate using liquid chromatography, tandem-mass spectrometry (LC/MS/MS). First, organic solvents were tested for extraction efficiency, including acetonitrile (ACN), methanol (MeOH), isopropanol (IPA), tetrahydrofuran (THF), and 50/50 ACN/MeOH (v/v). Among the extractants tested, 50/50 ACN/MeOH yielded the best results for our combined criteria of extraction efficacy and solvent-handling convenience. Second, chemical pretreatment prior to solvent extraction was tested with sodium hydroxide (NaOH), potassium hydroxide (KOH), hydrochloric acid (HCl), and potassium persulfate (K₂S₂O₈). Pretreatment with NaOH and HCl effectively recovered additional PFCs from the sludge, but KOH and K₂S₂O₈ digestion were less effective than no pretreatment. Third, cleanup methods were investigated with solid-phase extraction using HLB (hydrophilic–lipophilic balanced) and WAX (weak-anion exchange) stationary phases, and with ion-pairing. The HLB stationary phase yielded a slight edge over the other two cleanup strategies in terms of recoverable PFCs and chromatographic separation. Finally, the appropriateness of isotopically labeled PFCs for quantitating unlabeled PFCs using isotopic dilution in complex sludge extracts was evaluated by comparison to results obtained with the standard-addition method. A National Institute of Standards and Technology (NIST) domestic sludge (CRM 2781) was analyzed using our finalized method and compared with previously reported results.     

Determination of benzoylurea insecticides in food by pressurized liquid extraction and LC‐MS

A method based on pressurized liquid extraction and LC‐MS/MS has been developed for determining nine benzoylureas (BUs) in fruit, vegetable, cereals, and animal products. Samples (5 g) were homogenized with diatomaceous earth and extracted in a 22 mL cell with 22 mL of ethyl acetate at 80°C and 1500 psi. After solvent concentration and exchange to methanol, BUs were analyzed by LC‐MS/MS using an IT mass analyzer, which achieved several transitions of precursor ions that increase selectivity providing identification. LOQs were between 0.002 and 0.01 mg/kg, which are equal or lower than maximum residue limits established by the Codex Alimentarius. Excellent linearity was achieved over a range of concentrations from 0.01 to 1 mg/kg with correlation coefficients 0.995–0.999 (n=7). Validation of the total method was performed by analyzing in quintuplicate seven different commodities (milk, eggs, meat, rice, lettuce, avocado, and lemon) at three concentration levels (0.01, 0.1, and 1 mg/kg). The recoveries ranged from 58 to 97% and the RSDs from 5 to 19% depending on the compound and the commodity. The combination of pressurized liquid extraction with LC‐MS/MS provides a sensitive and selective method for the determination of BUs in food.     

An LC‐MS/MS method for determination of curculigoside with anti‐osteoporotic activity in rat plasma and application to a pharmacokinetic study

A rapid, simple, selective and sensitive LC‐MS/MS method was developed for the determination of curculigoside in rat plasma. The analytical procedure involves extraction of curculigoside and syringin (internal standard, IS) from rat plasma with a one‐step extraction method by protein precipitation. The chromatographic resolution was performed on an Agilent XDB‐C₁₈ column (4.6 × 50 mm, 5 µm) using an isocratic mobile phase of methanol with 0.1% formic acid and H₂O with 0.1% formic acid (45:55, v/v) at a flow rate of 0.35 mL/min with a total run time of 2.0 min. The assay was achieved under the multiple‐reaction monitoring mode using positive electrospray ionization. Method validation was performed according to US Food and Drug Administration guidelines and the results met the acceptance criteria. The calibration curve was linear over 4.00–4000 ng/mL (R = 0.9984) for curculigoside with a lower limit of quantification of 4.00 ng/mL in rat plasma. The intra‐ and inter‐day precisions and accuracies were 3.5–4.6 and 0.7–9.1%, in rat plasma, respectively. The validated LC‐MS/MS method was successfully applied to a pharmacokinetic study of curculigoside in rats after a single intravenous and oral administration of 3.2 and 32 mg/kg. The absolute bioavailability of curculigoside after oral administration was 1.27%. Copyright © 2013 John Wiley & Sons, Ltd.     

Development of a fast LC‐MS/MS assay for the determination of deferiprone in human plasma and application to pharmacokinetics

A fast and accurate liquid chromatography/tandem mass spectrometric (LC‐MS/MS) assay was first developed and validated for the determination of deferiprone in human plasma. The analytes were extracted with acetonitrile from only 50 μL aliquots of human plasma to achieve the protein precipitation. After extraction, chromatographic separation of analytes in human plasma was performed using a Synergi Fusion‐RP 80A column at 30 °C. The mobile phase consisted of methanol and 0.2% formic acid containing 0.2 mM EDTA (60:40, v/v). The flow rate of the mobile phase was 0.8 mL/min. The total run time for each sample analysis was 4 min. Detection was performed using electrospray ionization in positive ion multiple reaction monitoring mode by monitoring the precursor‐to‐parent ion transitions m/z 140.1 → 53.1 for deferiprone and m/z 143.1 → 98.1 for internal standard. A linear range was established from 0.1 to 20 µg/mL. The limit of detection was determined as 0.05 µg/mL. The validated method was estimated for linearity, recovery, stability, precision and accuracy. Intraday and interday precisions were 4.3–5.5 and 4.6–7.3%, respectively. The recovery of deferiprone was in the range of 80.1–86.8%. The method was successfully applied to a pharmacokinetic study of deferiprone in six thalassemia patients. Copyright © 2012 John Wiley & Sons, Ltd.     

Sensitive, selective and rapid determination of bupropion and its major active metabolite, hydroxybupropion, in human plasma by LC-MS/MS: application to a bioequivalence study in healthy Indian subjects

A sensitive, selective and rapid liquid chromatography tandem mass spectrometry (LC‐MS/MS) method was developed for the simultaneous determination of bupropion (BUP) and its major active metabolite hydroxybupropion (HBUP) in human plasma. Separation of both the analytes and venlafaxine as internal standard (IS) from 50 μL human plasma was carried out by solid‐phase extraction. The chromatographic separation of the analytes was achieved on a Zorbax Eclipse XDB C₁₈ (150 × 4.6 mm, 5 µm) analytical column using isocratic mobile phase consisting of 20 mm ammonium acetate–methanol (10:90, v/v), with a resolution factor of 3.5. The method was validated over a wide dynamic concentration range of 0.1–350 ng/mL for BUP and 0.1–600 ng/mL for HBUP. The matrix effect was assessed by post‐column infusion and the mean process efficiency was 96.08 and 94.40% for BUP and HBUP, respectively. The method was successfully applied to a bioequivalence study of 150 mg BUP (test and reference) extended release tablet formulation in 12 healthy Indian male subjects under fed conditions. Copyright © 2011 John Wiley & Sons, Ltd.     

LC‐MS/MS method for the determination of haemanthamine in rat plasma,bile and urine and its application to a pilot pharmacokinetic study

Evidence gathered in various studies points to the fact that haemanthamine, an isoquinoline alkaloid, has multiple medicinally interesting characteristics, including antitumor, antileukemic, antioxidant, antiviral, anticonvulsant and antimalarial activity. This work presents, for the first time, a universal LC‐MS/MS method for analysis of haemanthamine in plasma, bile and urine which has been verified in a pilot pharmacokinetic experiment on rats. Chromatographic separation was performed on a pentafluorophenyl core–shell column in gradient elution mode with a mobile phase consisting of acetonitrile–methanol–ammonium formate buffer. A sample preparation based on liquid–liquid extraction with methyl tert‐butyl ether was employed with ambelline used as an internal standard. Quantification was performed using LC‐MS‐ESI(+) in Selected Reaction Monitoring mode. The method was validated according to the European Medicines Agency guideline in a concentration range of 0.1–10 μmol/L in plasma, bile and urine. The concentration–time profiles of haemanthamine in plasma, bile and urine after a single i.v. bolus of 10 mg/kg have been described for the first time. The presented study addresses the lack of information on haemanthamine pharmacokinetics and also introduces a new universal method of haemanthamine analysis in complex biological matrices. Copyright © 2015 John Wiley & Sons, Ltd.     

Screening for anthocyanins using high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry with precursor-ion analysis, product-ion analysis, common-neutral-loss analysis, and selected reaction monitoring

A systematic method for anthocyanin identification using tandems mass spectrometry (MS/MS) coupled to high-performance liquid chromatography (HPLC) with photo-diode array detection (PDA) was developed. Scan for the precursor ions of commonly found anthocyanidins (cyanidin, delphinidin, malvidin, pelargonidin, petunidin, and peonidin) using LC/MS/MS on a triple quadrupole instrument allows for the specific determination of each category of anthocyanins. Further characterization of each anthocyanin was performed using MS/MS product-ion analysis, common-neutral-loss analysis, and selected reaction monitoring (SRM). The method was demonstrated for analysis of anthocyanins in black raspberries, red raspberries, highbush blueberries, and grapes (Vitis vinifera). Previous reported anthocyanins in black raspberries and red raspberries are confirmed and characterized. Common-neutral-loss analysis allows for the distinction of anthocyanin glucosides or galactoside and arabinosides in highbush blueberries. Separation and identification of anthocyanin glucosides and galactosides were achieved by LC/MS/MS using SRM. Anthocyanin isomers such as cyanidin sophoroside and 3,5-diglucoside were differentiated by their fragmentation pattern during product-ion analysis. Fifteen anthocyanins (all possible combinations of five anthocyanidins and three sugars) were characterized in highbush blueberries. Pelargonidin 3-glucoside and pelargonidin 3,5-diglucoside were detected and characterized for the first time in grapes. The present approach allows mass spectrometry to be used as a highly selective detector for rapid identification and characterization of anthocyanins and can be used as a sensitive procedure for screening anthocyanins in fruits and vegetables.