On-Line HPLC-UV-mass spectrometry and tandem mass spectrometry for the rapid delineation and characterization of differences in complex mixtures: a case study using toxic oil variants

An integrated differential approach to the characterization of complex mixtures is presented which includes the targeting of liquid chromatography (LC) peaks for identification using characteristic UV adsorption of the LC peak, subsequent molecular weight and formula determination using accurate mass LC mass spectrometry (MS), and structure characterization using accurate mass LC-tandem mass spectrometry. The use of differential UV adsorption aids in narrowing the scope of the study to only specific peaks of interest. Accurate mass measurement of the molecular ion species provides molecular weight information as well as atomic composition information. The tandem MS (MS/MS) spectra provide fragmentation information which allows for structural characterization of each component. Accurate mass assignment of each of the fragment ions in the MS/MS spectrum provides atomic composition for each of the fragment ions and thus further aids in the structural characterization. These experiments are facilitated through the use of on-line LC-MS and LC-MS/MS with in-line UV detection. A synthetic toxic oil (STO) related to Toxic Oil Syndrome is studied with a focus on possible contaminants resulting from the interaction of aniline, used as a denaturant, with the normal components of the oil. A differential analysis between the STO and a control oil is performed. LC peaks were targeted using UV absorbance to indicate the possible presence of the aniline moiety. Further differential analysis was performed through the determination of the MS signals associated with each component separated on the LC. Finally, the MS/MS data was also used to determine if the fragmentation of the targeted components indicated the presence of aniline. The MS/MS and accurate mass data were used to assign the structures for the targeted components.     

Determination of flavonoids and stilbenes in red wine and related biological products by HPLC and HPLC-ESI-MS-MS

To investigate probable health benefits of flavonoids and stilbenes in red wine a new reversed-phase (RP) high-performance liquid-chromatographic (HPLC) method with enhanced separation efficiency and improved selectivity, sensitivity, and speed has been established for determination of the flavonoids quercetin, myricetin and kaempferol and the stilbenes cis- and trans-resveratrol, in a single run . UV-absorbance, fluorescence (FLD), and mass-spectrometric (MS) detection were also evaluated. UV-absorbance detection at 320 nm for stilbenes and 377 nm for flavonoids enables their determination up to the nanogram range with a linearity of R2>0.9999 (linear range 50 ng mL(-1)-50 microg mL(-1)). Calculated values of average recoveries were between 95 and 105% for all analytes. For resveratrol, fluorescence detection was highly selective and twice as sensitive as UV detection, and linearity was satisfactory (R2>0.9996; linear range see UV detection). For the detection of the hydrophilic glycosidic compounds piceid and rutin, which are coeluted with other hydrophilic ingredients, the validated RP HPLC system was coupled to a quadrupole ion-trap mass-spectrometer (MS) via an electrospray interface (ESI) with 25% ammonia solution as sheath liquid. MS detection was, highly linear (R2>0.9878; linear range 50 ng mL(-1)-50 microg mL(-1)) for all investigated analytes and the limits of detection were in the low nanogram range. Compared with UV detection MS detection resulted in a 200% increase in signal intensity for myricetin and 400% increases for quercetin and kaempferol, but equal signal intensity for resveratrol. Calculated values of average recoveries were 102% for myricetin and 79% for piceid. Collision induced dissociation (CID) was also used to obtain characteristic fragmentation fingerprints to facilitate qualitative and quantitative analysis even in complex matrices. Finally, this hyphenated HPLC-ESI-MS method was highly suitable and an essential improvement compared with UV- and fluorescence detection.     

Analytical separation and detection methods for flavonoids

Flavonoids receive considerable attention in the literature, specifically because of their biological and physiological importance. This review focuses on separation and detection methods for flavonoids and their application to plants, food, drinks and biological fluids. The topics that will be discussed are sample treatment, column liquid chromatography (LC), but also methods such as gas chromatography (GC), capillary electrophoresis (CE) and thin-layer chromatography (TLC), various detection methods and structural characterization. Because of the increasing interest in structure elucidation of flavonoids, special attention will be devoted to the use of tandem-mass spectrometric (MS/MS) techniques for the characterization of several important sub-classes, and to the potential of combined diode-array UV (DAD UV), tandem-MS and nuclear magnetic resonance (NMR) detection for unambiguous identification. Emphasis will be on recent developments and trends.     

Determination of protein oxidation by mass spectrometry and method transfer to quality control

Characterization and quantitative analysis of oxidation plays an important role in biopharmaceutical development. This study demonstrates an approach to the assessment of susceptible to oxidation methionine residues in monoclonal antibodies and recombinant proteins. A method for the determination of oxidation levels by peptide mapping with mass spectrometric (MS) detection is described and its advantages compared to the UV detection are presented. Good linearity and reproducibility for determination of oxidation with MS detection are demonstrated (R2 > 0.99; RSDs of 4-9%). Aspects of method transfer to quality control group (QC) are discussed. As well, a quick and easy flow injection/MS method is proposed to substitute for peptide map analysis. Peptide coverage, linearity, reproducibility, robustness, sensitivity and quantitative oxidation results are compared for the flow injection/MS and LC/MS approaches.     

Identification and quantification of flavonoids and chromes in Baeckea frutescens by using HPLC coupled with diode-array detection and quadruple time-of-flight mass spectrometry

This article marks the first report on high-performance liquid chromatography (HPLC) coupled with diode-array detection (DAD) and quadruple time-of-flight mass spectrometry (Q-TOF/MS) for the identification and quantification of main bioactive constituents in Baeckea frutescens. In total, 24 compounds were identified or tentatively characterised based on their retention behaviours, UV profiles and MS fragment information. Furthermore, a validated method with good linearity, sensitivity, precision, stability, repeatability and accuracy was successfully applied for simultaneous determination of five flavonoids and one chromone in different plant parts of B. frutescens collected at different harvest times, and their dynamic contents revealed the appropriate harvest times. The established HPLC-DAD-Q-TOF/MS using multi-bioactive markers was proved to be a validated strategy for the quality evaluation on both raw materials and related products of B. frutescens.     

Simultaneous determination of five coumarins in Angelicae dahuricae Radix by HPLC/UV and LC‐ESI‐MS/MS

Rapid, simple and reliable HPLC/UV and LC‐ESI‐MS/MS methods for the simultaneous determination of five active coumarins of Angelicae dahuricae Radix, byakangelicol (1), oxypeucedanin (2), imperatorin (3), phellopterin (4) and isoimperatorin (5) were developed and validated. The separation condition for HPLC/UV was optimized using a Develosil RPAQUEOUS C₃₀ column using 70% acetonitrile in water as the mobile phase. This HPLC/UV method was successful for providing the baseline separation of the five coumarins with no interfering peaks detected in the 70% ethanol extract of Angelicae dahuricae Radix. The specific determination of the five coumarins was also accomplished by a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source (LC‐ESI‐MS/MS). Multiple reaction monitoring (MRM) in the positive mode was used to enhance the selectivity of detection. The LC‐ESI‐MS/MS methods were successfully applied for the determination of the five major coumarins in Angelicae dahuricae Radix. These HPLC/UV and LC‐ESI‐MS/MS methods were validated in terms of recovery, linearity, accuracy and precision (intra‐ and inter‐day validation). Taken together, the shorter analysis time involved makes these HPLC/UV and LC‐ESI‐MS/MS methods valuable for the commercial quality control of Angelicae dahuricae Radix extracts and its pharmaceutical preparations. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of a series of quinolones in pig plasma using solid-phase extraction and liquid chromatography coupled with mass spectrometric detection Application to pharmacokinetic studies

A simple and sensitive liquid chromatography (LC) method was developed for the simultaneous determination of eight quinolones in pig plasma samples. The following two methods of detection were used: ultraviolet (UV) and mass spectrometry with electrospray ionization (ESI/MS). Sample preparation consisted of solid-phase extraction (SPE) on Strata X cartridges prior to the analysis by LC/UV or LC/ESI/MS. The recovery, linearity, limit of detection (LOD) and limit of quantification (LOQ), precision and accuracy of the method were evaluated using spiked pig plasma samples. The suitability of the method for pharmacokinetic studies was evaluated by determining the concentrations of enrofloxacin (ENR) and ciprofloxacin (CIP) also in pig plasma, after administration of 200mg of enrofloxacin per kilogram of fodder during 5 consecutive days.     

Quantitative determination of acetylcholine in microdialysis samples using liquid chromatography/atmospheric pressure spray ionization mass spectrometry

A fast, simple and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the determination of acetylcholine in rat brain microdialysis samples. The chromatographic separation was achieved in 3 min on a reversed-phase column with isocratic conditions using a mobile phase containing 2% (v/v) of acetonitrile and 0.05% (v/v) of trifluoroacetic acid (TFA). A stable isotope-labeled internal standard was included in the analysis and detection was carried out with a linear ion trap mass spectrometer using selected reaction monitoring (SRM). Analyte ionization was performed with an atmospheric pressure chemical ionization (APCI) source without applying discharge current (atmospheric pressure spray ionization). This special ionization technique offered significant advantages over electrospray ionization for the analysis of acetylcholine with reversed-phase ion-pairing chromatography. The lower limit of quantification was 0.15 nM (1.5 fmol on-column) and linearity was maintained over the range of 0.15-73 nM, providing a concentration range that is significantly wider than that of the existing LC/MS methods. Good accuracy and precision were obtained for concentrations within the standard curve range. The method was validated and has been used extensively for the determination of acetylcholine in rat brain microdialysis samples.     

Determination of cefuroxime lysine in rat brain microdialysates by ultra‐fast liquid chromatography with UV and tandem mass spectrometry: application to an acute toxicokinetic study

Cefuroxime lysine is a new second‐generation cephalosporins, which can penetrate the blood–brain barrier to cure the meningitis. In order to investigate its acute toxicokinetic study after intraperitoneal injection of 675 mg/kg cefuroxime lysine, a sensitive and clean ultra‐fast liquid chromatography–tandem mass spectrometry (UFLC‐MS/MS) method for the determination of cefuroxime lysine in microdialysate samples was developed and validated, which was compared with UFLC‐UV as a reference method. Chromatographic separation was performed on a Shim‐pack XR‐ODS C₁₈ column (75 × 3.0 mm, 2.2 µm), with an isocratic elution of 0.1% formic acid in acetonitrile–0.1% formic acid in water (45:55, v/v) for LC‐MS and acetonitrile–20 mm potassium dihydrogen phosphate (pH 3.0,20:80, v/v) for LC‐UV. The lower limit of detection was 0.01 µg/mL for LC‐MS and 0.1 µg/mL for LC‐UV method, with the same corresponding linearity range of 0.1–50 µg/mL. The intra‐ and inter‐day precisions (relative standard deviation) for both methods were from 1.1 to 8.9%, while the accuracy was all within ±10.9%. The results of both methods were finally compared using paired t‐test; the results indicated that the concentrations measured by the two methods correlated significantly (p < 0.05), which suggested that the two methods based on LC‐MS and LC‐UV were suitable for the acute toxicokinetic study. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of ascorbic acid and its degradation products by high‐performance liquid chromatography‐triple quadrupole mass spectrometry

This study describes application of liquid chromatography coupled with triple quadrupole mass spectrometry (LC‐MS) for evaluation of vitamin C stability, the objective being prediction of the degradation products. Detection was performed with an UV detector (UV‐Vis) in sequence with a triple‐quad mass spectrometer in the multiple reaction mode. The negative ion mode of ESI and MS‐MRM transitions of m/z 175→115 (quantifier) and 175→89 (qualifier) for ascorbic acid was used. All the validation parameters were within the range of acceptance proposed by the Food and Drug Administration. The method was fully validated in terms of linearity, LOD, LOQ, accuracy, and interday precision. Validation experiments revealed good linearity with R² = 0.999 within the established concentration range, and excellent repeatability (9.3%). The LOD of the method was 0.1524 ng/mL whereas the LOQ was 0.4679 ng/mL. LC‐MS methodology proves to be an improved, simple, and fast approach to determining the content of vitamin C and its degradation products with high sensitivity, selectivity, and resolving power within 6 minutes of analysis.     

Development and evaluation of a new method for the determination of the carotenoid content in selected vegetables by HPLC and HPLC-MS-MS

Epidemologic studies have shown inverse correlation between the consumption of carotenoid-rich vegetables and the incidence of cancer. Therefore, analytical techniques for the quantitative determination of carotenoids in complex sample matrices are important. The most used method is reversed-phase (RP)-high-performance liquid chromatography (HPLC). In this study, seventeen mobile-phase systems described in the literature and six RP-HPLC columns with differences in particle size and porosity are evaluated. Derived from these results, a new mobile-phase (acetonitrile, methanol, chloroform, and n-heptane) including solvent modifiers is presented, which allows an improved and more efficient separation of carotenoids. From all columns tested, the best chromatographic parameters are found using a silica C18 column (250 x 2 mm, 5 microm, 100 A). As was found, absorbance detection at 450 nm allows the determination of the carotenoids down to the picogram range with good linearity (R2 > 0.98). For the identification and quantitation of carotenoids in complex sample matrices (containing additionally other ultraviolet-absorbing compounds), the optimized RP chromatographic system is coupled to a mass spectrometer (MS) using an atmospheric pressure ionization interface. The calibration plots show high linearity (R2 > 0.99), and the detection limit is found in the lower nanogram range. Furthermore, collision-induced dissociation in the ion source allows for the identification of carotenoids by their characteristic fragmentation pathways. In this study, a total of nine species of vegetables commonly consumed in Central Europe are analyzed for their contents of carotenoids (namely lutein, zeaxanthin, beta-cryptoxanthin, and beta-carotene) by RP-HPLC and RP-HPLC-MS-MS. It is found that good sources for lutein are spinach, kale, and broccoli, and sources for beta-carotene are broccoli, spinach, kale, carrots, and tomatoes. This new method is an improvement for the identification and quantitation of carotenoids in complex biological tissues.     

Simultaneous determination of nonionic and anionic industrial surfactants by liquid chromatography combined with evaporative light-scattering detection

LC/ELSD was used for the simultaneous and rapid analysis of various anionic and nonionic surfactants. Eight surfactants (APG, LAE7, CDE, NPE7, LAE9-cap, SLS, AOS, AOT) were separated within 30 min in one LC run on C18-bonded silica column with a methanol-water gradient condition, using ELSD detection. The ELSD could be used efficiently for the detection of the surfactants not detectable by UV absorbance. Calibration plots of peak area vs. injected mass were linear in the range of 0.4-140 microg injected per 20 microL, with a log-log slope of 1.4-2.4. Several commercial products were analyzed to demonstrate the practicality of the procedure.     

A comparative study of first-derivative spectrophotometry and column high-performance liquid chromatography applied to the determination of repaglinide in tablets and for dissolution testing

A fast and reliable method for the determination of repaglinide is highly desirable to support formulation screening and quality control. A first-derivative UV spectroscopic method was developed for the determination of repaglinide in tablet dosage form and for dissolution testing. First-derivative UV absorbance was measured at 253 nm. The developed method was validated for linearity, accuracy, precision, limit of detection (LOD), and limit of quantitation (LOQ) in comparison to the U.S. Pharmacopeia (USP) column high-performance liquid chromatographic (HPLC) method. The first-derivative UV spectrophotometric method showed excellent linearity [correlation coefficient (r) = 0.9999] in the concentration range of 1-35 microg/mL and precision (relative standard deviation < 1.5%). The LOD and LOQ were 0.23 and 0.72 microg/mL, respectively, and good recoveries were achieved (98-101.8%). Statistical comparison of results of the first-derivative UV spectrophotometric and the USP HPLC methods using the t-test showed that there was no significant difference between the 2 methods. Additionally, the method was successfully used for the dissolution test of repaglinide and was found to be reliable, simple, fast, and inexpensive.     

Determination of the anthraquinones aloe-emodin and aloin-A by liquid chromatography with mass spectrometric and diode array detection

Methods using liquid chromatography/mass spectrometry (LC/MS) and LC with diode array detection (DAD) in the UV range (LC/UV) were developed for the determination of low levels of the anthraquinones aloe-emodin and aloin-A (barbaloin) in aloe-based products. The methods were used to analyze several commercial products (liquids, semisolids, and solids) for the 2 anthraquinones. The wavelengths used for quantification of aloin-A, aloe-emodin, and emodin (internal standard) by DAD were 357, 257, and 289 nm, respectively. The on-column sensitivities were 0.25 and 0.05 ng by LC/UV and 0.01 and 0.025 ng by LC/MS for aloin-A and aloe-emodin, respectively. The methods are simple and sensitive and provide reproducible results; therefore, they are suitable for the determination of these anthraquinones in various aloe-based products.     

Multiresidue determination of quinolones regulated by the European Union in bovine and porcine plasma. Application of chromatographic and capillary electrophoretic methodologies

This paper presents the multiresidue determination of the series of quinolones regulated by the European Union (marbofloxacin, ciprofloxacin, danofloxacin, enrofloxacin, sarafloxacin, difloxacin, oxolinic acid and flumequine) in bovine and porcine plasma using capillary electrophoresis and liquid chromatography with ultraviolet detection (CE‐UV, LC‐UV), liquid chromatography–mass spectrometry and –tandem mass spectrometry (LC‐MS, LC‐MS/MS) methods. These procedures involve a sample preparation by solid‐phase extraction for clean‐up and preconcentration of the analytes before their injection into the separation system. All methods give satisfactory results in terms of linearity, precision, accuracy and limits of quantification. The suitability of the methods to determine quinolones was evaluated by determining the concentration of enrofloxacin and ciprofloxacin in real samples from pig plasma and cow plasma. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of piperazine in pharmaceutical drug substances using capillary electrophoresis with indirect UV detection

A fast, selective capillary electrophoresis (CE) method was developed for piperazine counter-ion analysis and applied to the analysis of an active pharmaceutical ingredient (API) that exists as a hemipiperazine salt. Due to the poor chromophore, the detection method chosen was indirect UV detection using benzylamine as the UV absorbing probe. Piperazine quantitation was performed using diethylamine as an internal standard and the method was validated for specificity, linearity, precision, and accuracy. The results indicate the method is suitable for piperazine counter-ion analysis in support of salt form characterization.     

Determination of two oxy-pyrimidine metabolites of diazinon in urine by gas chromatography/mass selective detection and liquid chromatography/electrospray ionization/mass spectrometry/mass spectrometry

An analytical method was developed for the determination in urine of 2 metabolites of diazinon: 6-methyl-2-(1-methylethyl)-4(1H)-pyrimidinone (G-27550) and 2-(1-hydroxy-1-methylethyl)-6-methyl-4(1H)-pyrimidinone (GS-31144). Two of the urine sample preparation procedures presented rely on gas chromatography/mass selective detection (GC/MSD) in the selected ion monitoring mode for determination of G-27550. For fast sample preparation and a limit of quantitation (LOQ) of 1.0 ppb, urine samples were purified by using ENV+ solid-phase extraction (SPE) columns. For analyte confirmation at an LOQ of 0.50 ppb, classical liquid/liquid partitioning was used before further purification in a silica SPE column. An SPE sample preparation procedure and liquid chromatography/electrospray ionization/mass spectrometry/mass spectrometry (LC/ESI/MS/MS) were used for both G-27550 and GS-31144. The limit of detection was 0.01 ng for G-27550 with GC/MSD, and 0.016 ng when LC/ESI/MS/MS was used for both G-27550 and GS-31144. The LOQ was 0.50 ppb for G-27550 when GC/MSD and the partitioning/SPE sample preparation procedure were used, and 1.0 ppb for the SPE only sample preparation procedure. The LOQ was 1.0 ppb for both analytes when LC/ESI/MS/MS was used.     

Comparison of LC‐UV and LC–MS methods for simultaneous determination of teriflunomide,dimethyl fumarate and fampridine in human plasma: application to rat pharmacokinetic study

This study describes a comparison between LC‐UV and LC–MS method for the simultaneous analyses of a few disease‐modifying agents of multiple sclerosis. Quantitative determination of fampridine (FAM), teriflunomide (TFM) and dimethyl fumarate (DMF) was performed in human plasma with the recovery values in the range of 85–115%. A reversed‐phase high‐performance liquid chromatography (HPLC) with UV as well as MS detection is used. The method utilizes an XBridge C₁₈ silica column and a gradient elution with mobile phase consisting of ammonium formate and acetonitrile at a flow rate of 0.5 mL min^?1. The method adequately resolves FAM, TFM and DMF within a run time of 15 min. Owing to low molecular weights, the estimation of DMF and FAM is more versatile in UV than MS detection. With LC‐UV, the detection limits of FAM, TFM and DMF were 0.1, 0.05, 0.05 μg and the quantification limit for all the analytes was 1 μg. With LC–MS, the detection and quantification limits for all of the analytes were 1 and 5 ng, respectively. The two techniques were completely validated and shown to be reproducible and sensitive. They were applied to a pharmacokinetic study in rats by a single oral dose. Copyright © 2016 John Wiley & Sons, Ltd.     

Automated interface for hyphenation of planar chromatography with mass spectrometry

A fully automated interface to couple high-performance thin-layer chromatography (HPTLC) with mass spectrometry (MS) is described. This universal hands-free interface connects intact normal-phase plates to any liquid chromatography/mass spectrometry (LC/MS) system without any adjustments or modifications to the mass spectrometer. The interface extracts the complete substance band with its depth profile and thus allows detections in the pg/band range. The high performance of the automated interface was evaluated through caffeine quantification in real samples, viz., energy drinks and pharmaceutical tablets, without internal standard. Following chromatographic separation on silica gel 60 F(254) HPTLC plates, caffeine bands were eluted from the plate by means of the automated interface to the electrospray ionization (ESI) source of a triple-quadrupole mass spectrometer. Since in full scan mode only the protonated molecule [M+H](+) was observed, caffeine quantification was performed using the selected-ion monitoring (SIM) mode at m/z 195. The validation showed highly reliable results for the linear range (R(2) = 0.9973), repeatability (RSD = 5.6%, n = 6) and intermediate precision (RSD = 1.5%, n = 3). Regarding accuracy the results obtained by HPTLC/MS were not statistically different (F-test, t-test) from those obtained by validated HPTLC/UV methods. Hence, this interface proved to be one of the most reliable and universal interfaces for HPTLC/MS.     

A highly automated 96-well solid phase extraction and liquid chromatography/tandem mass spectrometry method for the determination of fentanyl in human plasma

A high-throughput bioanalytical method based on automated sample transfer, automated solid phase extraction, and fast liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis, has been developed for the determination of the analgesic fentanyl in human plasma. Samples were transferred into 96-well plates using an automated sample handling system. Automated solid phase extraction (SPE) was carried out using a 96-channel programmable liquid-handling workstation using a mixed-mode sorbent. The extracted samples were then dried down, reconstituted and injected onto a silica column using an aqueous/organic mobile phase with tandem mass spectrometric detection. The method has been validated over the concentration range 0.05-100 ng/mL fentanyl in human plasma, based on a 0.25-mL sample size. The assay is sensitive, specific and robust. More than 2000 samples have been analyzed using this method. The automation of the sample preparation steps not only increased the analysis throughput, but also facilitated the transfer of the method between different bioanalytical laboratories of the same organization.