Performance of zwitterionic hydrophilic interaction LC for the determination of iodinated X‐ray contrast agents

This study compares the separation performance of a group of iodinated X‐ray contrast media on four different columns. The first three were two stationary phases (SPs) modified with C₁₈ and a polar‐embedded SP (polar amide group bonded to an alkyl chain), all of which worked under RP‐LC mode. The fourth was a zwitterionic sulphoalkylbetaine SP, working under the hydrophilic interaction LC (HILIC) mode. After the optimisation of the different parameters, the zwitterionic column displayed the best separation, which also overcomes the problems encountered when these analytes were separated under RP‐LC. Moreover, when HILIC is coupled to MS/MS, sensitivity is enhanced. However, when sewage samples were analysed by SPE followed by the optimal HILIC–MS/MS, the sensitivity of the method was affected due to the high matrix effect, which had to be solved by dilution of the extract. Finally, the method was preliminarily validated with sewage and the figures of merit were comparable to those of the SPE–RP‐LC–MS/MS.     

A study of ion suppression effects in electrospray ionization from mobile phase additives and solid-phase extracts

Since the wide adoption of liquid chromatography/tandem mass spectrometry (LC/MS/MS), the ion suppression/enhancement phenomenon is the latest barrier to high-throughput analysis. This consequence of a nonoptimized analytical method can lead to adverse effects during quantitation (i.e. poor accuracy and precision). Previous papers have reported that ion suppression is a direct result of endogenous material present in biological samples. However, in the case of a solid-phase liquid chromatography/tandem mass spectrometry (SPE/LC/MS/MS) system, the measured result is the combination of several operating conditions and parameters. Little has been done to effectively monitor and/or choose optimized conditions for the complete sequence of extraction, clean up, separation and analysis. This paper describes a simple setup for quantification of ion suppression/enhancement. Several mobile phase additives, ion-pairing agents and SPE extracts were measured and compared against a standard reference. The results demonstrated that a clean up of plasma extracts based on ion exchange leads to minimal ion suppression/enhancement for the compounds that were investigated.     

Capillary electrochromatography and nano‐liquid chromatography coupled to nano‐electrospray ionization interface for the separation and identification of estrogenic compounds

Nano‐LC and CEC were coupled to MS through a nanospray or a pressurized liquid‐junction interface for the simultaneous separation and determination of 11 estrogenic compounds. Different stationary phases, that is, phenyl, C18, and C18 bidentate silica hydrate, were studied. For both techniques, the phenyl stationary phase was the best option, considering separation efficiency, selectivity, and resolution. Under the optimized conditions, the baseline separation of the target compounds (including estradiol and zearalanol epimers) was achieved in less than 20 min in nano‐LC‐MS and less than 13 min in CEC‐MS. Molecular imprinted polymer SPE was used for extracting the target compounds from mineral water samples with the analysis of nano‐LC‐MS. The whole molecular imprinted polymer SPE nano‐LC‐MS method was validated through a recovery study at two levels of concentration. Sensitivity was improved by on‐column focusing technique obtaining LODs in the range 1.4–55.4 ng/L.     

A new high-performance liquid chromatographic method with evaporative light scattering detector for the analysis of phospholipids. Application to Iberian pig subcutaneous fat

A new method for the analysis of phospholipids by normal-phase HPLC is described using a silica column. Addition of ammonia and triethylamine to a gradient based on chloroform/methanol/water promoted a good and rapid separation of phospholipid classes (20 min run). The use of an evaporative light scattering detector permitted an accurate analysis of a mixture of phospholipids. Calibration curves were linear within different range for each phospholipid class. The LOD and LOQ obtained were below 0.03 and 0.05 mg kg⁻¹ for all cases, respectively. Besides, a new method for the separation of phospholipids from total lipids before HPLC analysis by a solid-phase extraction (SPE) with Si cartridges has been developed. This methodology gave a good recovery ranging from 97 to 117%. The method was validated with a standard mixture of phospholipids. This method has been applied to characterize the phospholipid fraction of subcutaneous fat from Iberian pig. Cardiolipin, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylcholine, and sphingomyelin have been described for first time in these samples. The fatty acid composition of the different phospholipid classes and their HPLC electrospray ionization mass spectrometry have been used for characterizing the molecular species present in each one.     

Determination of phospholipids in dairy products by SPE/HPLC/ELSD

The aim of this work was to evaluate the performance of different methods for both milk lipid extraction and phospholipids separation. As far as the lipid extraction procedure is concerned, the Folch method showed a higher phospholipid recovery with respect to the Rose-Gottlieb method. Different SPE cartridges and solvent phases were tested to carry out the separation of phospholipids from fat. The yield of extraction was evaluated by isolating phospholipids from both milk fat and synthetic fat; Standard Addition Method was applied as well. The isolation of the phospholipids by SPE silica column and subsequent analysis by HPLC/ELSD was shown to be an accurate and reproducible analytical method for the determination of phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylcholine and sphingomyelin in milk fat extracted by Folch method.     

A novel on-line solid-phase extraction approach integrated with a monolithic column and tandem mass spectrometry for direct plasma analysis of multiple drugs and metabolites

An on-line solid-phase extraction liquid chromatography/tandem mass spectrometry (SPE LC/MS/MS) assay using a newly developed SPE column and a monolithic column was developed and validated for direct analysis of plasma samples containing multiple analytes. This assay was developed in an effort to increase bioanalysis throughput and reduce the complexity of on-line SPE LC/MS/MS systems. A simple column-switching configuration that requires only one six-port valve and one HPLC pumping system was employed for on-line plasma sample preparation and subsequent gradient chromatographic separation. The resulting analytical method couples the desired sensitivity with ease of use. The method was found to perform satisfactorily for direct plasma analysis with respect to assay linearity, specificity, sensitivity, precision, accuracy, carryover, and short-term stability of an eight-analyte mixture in plasma. A gradient LC condition was applied to separate the eight analytes that cannot be distinctly differentiated by MS/MS. With a run time for every injection of 2.8 min, a minimum of 300 direct plasma injections were made on one on-line SPE column without noticeable changes in system performance. Due to the ruggedness and simplicity of this system, generic methods can be easily developed and applied to analyze a wide variety of compounds in a high-throughput manner without laborious off-line sample preparation.     

Determination of penicillins in milk using LC‐UV,LC‐MS and LC‐MS/MS

The aim of this work is to establish a method for the simultaneous determination of eight penicillins in milk samples by LC‐UV, LC‐MS and LC‐MS/MS. The procedure involves a step for clean‐up and to preconcentrate the analytes by SPE and a subsequent chromatographic analysis. LC‐UV, LC‐MS and LC‐MS/MS have been used for the simultaneous quantification of penicillins in milk. The proposed methods have been validated according to the EU guideline and present LOQ below the maximum limits of residues (MRLs) established by the European Union for penicillins in milk. The developed methods were applied to different milk samples obtained from cows medicated with penicillins.     

Determination of total oncopterin,neopterin and biopterin in human urine by high performance liquid chromatography with solid phase extraction

A new, sensitive method for the determination of oncopterin, biopterin, and neopterin in human urine has been developed using SPE with 6,7‐dimethylpterin as internal standard and gradient HPLC with fluorescence detection. SPE was tested for the pre‐treatment of urine samples on different types of sorbents (strong ion exchange resins, polar adsorbents, and reversed‐phase sorbents). RP‐SPE with subsequent evaporation of eluate has been found to be the most appropriate. The extraction efficiency exceeded 95% for all selected pterins. The extracted pterins were subsequently analyzed on a Purospher RP‐18 RP column. The LOD of oncopterin was 1.43 nmol/L of urine. The intra‐day and inter‐day imprecision at a physiological oncopterin concentration never exceeded 10%. The potential of this method was tested using urine samples of healthy volunteers and cancer patients without methotrexate therapy.     

A simplified protein precipitation/mixed-mode cation-exchange solid-phase extraction, followed by high-speed liquid chromatography/mass spectrometry, for the determination of a basic drug in human plasma

A simplified protein precipitation/mixed-mode cation-exchange solid-phase extraction (PPT/SPE) procedure has been investigated. A mixture of acetonitrile and methanol along with formic acid was used to precipitate plasma proteins prior to selectively extracting the basic drug. After vortexing and centrifugation, the supernatants were directly loaded onto an unconditioned Oasis MCX microElution 96-well extraction plate, where the protonated drug was retained on the negatively charged sorbent while interfering neutral lipids, steroids or other endogenous materials were washed away. Normal wash steps were deemed unnecessary and not used before sample elution. The sample extracts were analyzed under both conventional and high-speed liquid chromatography/tandem mass spectrometry (LC/MS/MS) conditions to examine the feasibility of the PPT/SPE procedure for human plasma sample clean-up. For the conventional LC/MS/MS method, chromatographic separation was achieved on a C18, 2.1 x 50 mm column with gradient elution (k' = 5.5). The mobile phase contained 0.1% formic acid in water and 0.1% formic acid in acetonitrile. For the high-speed LC/MS/MS method, chromatographic separation was achieved on a C18, 2.1 x 10 mm guard column with gradient elution (k' = 2.2, Rt = 0.26 min). The mobile phase contained 0.1% formic acid in water and 0.001% trifluoroacetic acid in acetonitrile. Detection for both conventional and high-speed LC/MS/MS methods was by positive ion electrospray tandem mass spectrometry on a ThermoElectron Finnigan TSQ Quantum Ultra, where enhanced resolution (RP 2000; 0.2 amu) was used for high-speed LC/MS/MS. The standard curve, ranging from 0.5 to 100 ng/mL, was fitted to a 1/x weighted quadratic regression model.This combined PPT/SPE procedure effectively eliminated time-consuming sorbent conditioning and wash steps, which are essential for a conventional mixed-mode SPE procedure, but retained the advantages of both PPT (removal of plasma proteins) and mixed-mode SPE (analyte selectivity). The validation results demonstrated that this PPT/SPE procedure was well suited for both conventional and high-speed LC/MS/MS analyses. In comparison with a conventional mixed-mode SPE procedure, the simplified PPT/SPE process provided comparable sample extract purity. This simple sample clean-up procedure can be applied to other basic compounds with minor modifications of PPT solvents.     

Fractionation and proteomic analysis of the Walterinnesia aegyptia snake venom using OFFGEL and MALDI‐TOF‐MS techniques

Animal venoms are complex mixtures of more than 100 different compounds, including peptides, proteins, and nonprotein compounds such as lipids, carbohydrates, and metal ions. In addition, the existing compounds show a wide range of molecular weights and concentrations within these venoms, making separation and purification procedures quite tedious. Here, we analyzed for the first time by MS the advantages of using the OFFGEL technique in the separation of the venom components of the Egyptian Elapidae Walterinnesia aegyptia snake compared to two classical methods of separation, SEC and RP‐HPLC. We demonstrate that OFFGEL separates venom components over a larger scale of fractions, preserve respectable resolution with regard to the presence of a given compound in adjacent fractions and allows the identification of a greater number of ions by MS (102 over 134 total ions). We also conclude that applying several separating techniques (SEC and RP‐HPLC in addition to OFFGEL) provides complementary results in terms of ion detection (21 more for SEC and 22 more with RP‐HPLC). As a result, we provide a complete list of 134 ions present in the venom of W. aegyptia by using all these techniques combined.     

Efficiency of a miniaturized silica monolithic cartridge in reducing matrix ions as demonstrated in the simultaneous extraction of morphine and codeine from urine samples for quantification with liquid chromatography-tandem mass spectrometry (LC-MS/MS)

Presence of matrix ions could negatively affect the sensitivity and selectivity of liquid chromatography‐tandem mass spectrometer (LC‐MS/MS). In this study, the efficiency of a miniaturized silica monolithic cartridge in reducing matrix ions was demonstrated in the simultaneous extraction of morphine and codeine from urine samples for quantification with LC‐MS. The miniaturized silica monolith with hydroxyl groups present on the largely exposed surface area function as a weak cation exchanger for solid phase extraction (SPE). The miniaturized silica cartridge in 1 cm diameter and 0.5 cm length was housed in a 2‐ml syringe fixed over a SPE vacuum manifold for extraction. The cleaning effectiveness of the cartridge was confirmed by osmometer, atomic absorption spectrometer, LC‐MS and GC‐TOFMS. The drugs were efficiently extracted from urine samples with recoveries ranging from 86% to 114%. The extracted analytes, after concentration and reconstitution, were quantified using LC‐MS/MS. The limits of detection for morphine and codeine were 2 ng/ml and 1 ng/mL, respectively. The relative standard deviations of measurements ranged from 3% to 12%. The monolithic sorbent offered good linearity with correlation coefficients > 0.99, over a concentration range of 50–500 ng/ml. The silica monolithic cartridge was found to be more robust than the particle‐based packed sorbent and also the commercial cartridge with regards to its recyclability and repeated usage with minimal loss in efficiency. Our study demonstrated the efficiency of the miniaturized silica monolith for removal of matrix ions and extraction of drugs of abuse in urinary screening. Copyright © 2011 John Wiley & Sons, Ltd.     

Identification and quantitation of pyrethroid pesticide residues in vegetables by solid-phase extraction and liquid chromatography/electrospray ionization ion trap mass spectrometry

A quantitative method consisting of solid-phase extraction (SPE) followed by liquid chromatography/electrospray ionization ion trap mass spectrometry (LC/ESI-ITMS) analysis was developed for the identification and quantitation of ten pyrethroid pesticides commonly used in vegetables. The best HPLC separation was achieved using a gradient program of methanol/water mixture. For the vegetable samples, an SPE procedure to clean up the matrices was carried out prior to LC/MS analysis. Under the optimum conditions, the limits of quantification of the pyrethroid pesticides (tetramethrin, allethrin, fenpropathrin, lambda-cyhalothrin, cypermethrin, deltamethrin, fenvalerate, bioresmethrin, permethrin and bifenthrin) ranged from 0.03 to 0.1 mg kg-1 with relative standard deviations<20%, and the mean recoveries ranged from 69.5 to 102.5%. The proposed method has been successfully applied to the determination of pyrethroids in six vegetables with satisfactory results.     

Better understanding of carbon nanoparticles via high‐performance liquid chromatography‐fluorescence detection and mass spectrometry

RP‐HPLC coupled with fluorescence detection for separation of carbon nanoparticles (CNP) synthesized with microwave‐assisted pyrolysis of citric acid and 1,2‐ethylenediamine is presented. The influence of methanol content and pH of mobile phase on the separation of CNP has been investigated. Under optimal mobile phase and elution gradient conditions, the effect of mole ratio of amine to carboxylic groups (NH₂/COOH) in the initial reagents on CNP product is studied. At NH₂/COOH = 0.67, the strongest fluorescence CNP sample is obtained. The separated CNP fractions are collected and further characterized by UV‐visible absorption and photoluminescence (PL) spectroscopy, CE, transmission electron microscopy (TEM), and MALDI‐TOF MS. The absorption and PL emission bands of the fractions are bathochromatically shifted with the elution order of CNP on RP‐HPLC. The TEM images prove that CNP are eluted from the smallest to the largest. The MS data show that CNP undergo fragmentations, closely relating to their surface‐attached carboxylic acid and amide/amine moieties. This work highlights the merit of RP‐HPLC coupled with fluorescence detection, TEM, and MS for isolation and characterization of individual CNP species present in a CNP sample.     

SPE/RIA vs LC/MS for measurement of low levels of budesonide in plasma

A radioimmunoassay is described that measures budesonide in plasma after solid-phase extraction (SPE/RIA) of the analyte. The performance of the assay was compared with that of a selective LC/MS method. The limit of quantitation of budesonide determined for the LC/MS and SPE/RIA assay was 50 pg/mL and 120 pg/mL, respectively. Based on quality control samples, a higher variability was observed for the SPE/RIA (CV between 4.5 and 23.0%) than for the LC/MS method (CV between 7.5 and 12.5%). Plasma samples obtained from healthy volunteers after administration of budesonide rectal foam were assayed by both methods. In a subset of samples, these results were compared with those measured by direct RIA to evaluate the selectivity of two assays. About two times higher budesonide levels were measured with the direct RIA (lacking the extraction step), presumably because of cross-reactivity with budesonide metabolites, indicating that the extraction step in SPE/RIA is necessary for selectivity. Both SPE/RIA and LC/MS methods were found to be selective, sensitive and suitable for pharmacokinetic studies. Results obtained from the two methods were compared with a number of statistical methods. Ratios of results obtained for the clinical samples were close to 1 (ratio LC-MS/ SPE/RIA = 0.98 +/- 0.27). Linear regression indicated a slope of 1.17 +/- 0.0378. The concordance correlation (r = 0.91) indicated that the agreement between both methods was fair while the Bland-Altman plot indicated that the agreement was less pronounced at higher concentrations (1-3 ng/mL). In summary, the results confirm that the SPE/RIA is an alternative to HPLC/MS and that among the statistical methods tested the concordance correlation analysis was judged to be the most informative test to assess the comparability of two methods.     

Highly sensitive and accurate profiling of carotenoids by supercritical fluid chromatography coupled with mass spectrometry

We attempted to establish a high‐speed and high‐resolution profiling method for a carotenoid mixture as a highly selective and highly sensitive detection method; the analysis was carried out by supercritical fluid chromatography (SFC) coupled with mass spectrometry (MS). When an octadecyl‐bonded silica (ODS) particle‐packed column was used for separation, seven carotenoids including structural isomers were successfully separated within 15 min. This result indicated not only improved separation but also improved throughput compared to the separation and throughput in RP‐HPLC. The use of a monolithic ODS column resulted in additional improvement in both the resolution and the throughput; the analysis time was reduced to 4 min by increasing the flow rate. Furthermore, carotenoids in biological samples containing the complex matrices were separated effectively by using several monolithic columns whose back pressure was very low. The mass spectrometer allowed us to perform a more sensitive analysis than UV detection; the detection limit of each carotenoid was 50 pg or below. This is the first report of carotenoid analysis carried out by SFC‐MS. The profiling method developed in this study will be a powerful tool for carrying out accurate profiling of biological samples.     

Detection of 20‐hydroxyecdysone in calf urine by comparative liquid chromatography/high‐resolution mass spectrometry and liquid chromatography/tandem mass spectrometry measurements: application to the control of the potential misuse of ecdysteroids in cattle

Ecdysteroids, which are steroid hormones in invertebrates, but which are also present in plants, could potentially be used as anabolic agents in food‐producing animals. The control of ecdysteroid misuse in cattle relies on the development of an efficient method for their detection in biological matrices at trace levels (µg L⁻¹). In order to propose an analytical procedure dedicated to the identification of excreted 20‐hydroxyecdysone (20E) in urine and faecal samples of breeding animals, a comparative study of the spectrometric behaviour of these compounds was carried out both by LC/(ESI‐)/HRMSⁿ (hybrid linear ion trap – orbital trap) and by LC/(ESI+)/MS/MS (triple quadrupole). This study revealed the formation of a large number of product ions both in positive and negative ion mode, corresponding to losses of water molecules and specific cleavages on the side chain. The sample preparation consisted of sequential purification on two solid‐phase extraction cartridges (SPE octadecylsilyl and SPE silica). The detection limits were around 0.5 µg L⁻¹ in the selected reaction monitoring (SRM) mode and recoveries above 60% were obtained. The method was successfully applied to the analysis of real samples collected from calves treated with 60 mg 20E over 4 days. Analysis of the samples allowed the investigation of the kinetics of elimination of 20E in calf urine and determination of the time‐frame for the control of potential abuse. Copyright © 2008 John Wiley & Sons, Ltd.     

Characterization and quantification of triacylglycerols in peanut oil by off‐line comprehensive two‐dimensional liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometry

The complexity of natural triacylglycerols (TAGs) in various edible oils is prodigious due to the hundreds of set is of TAG compositions, which makes the identification of TAGs quite difficult. In this investigation, the off‐line 2D system coupling of nonaqueous RP and silver‐ion HPLC with atmospheric pressure chemical ionization MS detection has been applied to the identification and quantification of TAGs in peanut oil. The method was successful in the separation of a high number of TAG solutes, and the TAG structures were evaluated by analyzing their atmospheric pressure chemical ionization mass spectra information. HPLC and MS conditions have been optimized and the fragmentation mechanisms of isomers have been validated. In addition, an internal standard approach has been developed for TAG quantification. Then this system was applied in peanut oil samples and there was a total of 48 TAGs including regioisomers that have been determined and quantified.     

Two‐dimensional LC‐MS/MS to enhance ceramide and phosphatidylcholine species profiling in mouse liver

A two‐dimensional (2D) hydrophilic interaction liquid chromatography (HILIC) and reverse‐phase (RP) liquid chromatography (LC) system coupled with triple‐quadrupole mass spectrometry (MS) was developed to comprehensively profile ceramides and phosphatidylcholine in extracted biological samples. Briefly, the 2D HILIC‐RPLC system used a silica HILIC column operated in the first dimension to distinguish the lipid classes and a BEH C₁₈ column operated in the second dimension to separate the lipid species of the same class. The regression linearity of each lipid was satisfactory in both systems; however, the absolute matrix effect factor was reduced in 2D LC‐MS/MS system. Limits of detection of 2D LC‐MS/MS system were 2‐ to 3‐fold lower compared with one‐dimensional RPLC‐MS/MS. The recovery from the sample ranged from 84.5 to 110%. To summarize, the developed method was proven to be accurate and producible, as relative standard deviations remained <20% at three spiked levels. The efficiency of this newly developed system was applied to measure changes of lipids in the liver of mice after naphthalene treatment. Orthogonal projection to latent structures‐discriminant analysis discriminated the lipids from control and the treatment group. We concluded that 2D LC‐MS/MS is a promising method to assist lipidomic studies of complex biological samples. Copyright © 2014 John Wiley & Sons, Ltd.     

Heart‐cut nano‐LC–CZE–MS for the characterization of proteins on the intact level

Multidimensional separation techniques play an increasingly important role in separation science, especially for the analysis of complex samples such as proteins. The combination of reversed‐phase liquid chromatography in the nanoscale and CZE is especially beneficial due to their nearly orthogonal separation mechanism and well‐suited geometries/dimensions. Here, a heart‐cut nano‐LC–CZE–MS setup was developed utilizing for the first time a mechanical 4‐port valve as LC–CE interface. A model protein mixture containing four different protein species was first separated by nano LC followed by a heart‐cut transfer of individual LC peaks and subsequent CZE–MS analysis. In the CZE dimension, various glycoforms of one protein species were separated. Improved separation capabilities were achieved compared to the 1D methods, which was exemplarily shown for ribonuclease B and its different glycosylated forms. LODs in the lower μg/mL range were determined, which are considerably lower compared to traditional CZE–MS. In addition, this study represents the first application of an LC–CE–MS system for intact protein analysis. The nano‐LC–CZE–MS system is expected to be applicable to various other analytical challenges.     

Derivative spectrum chromatographic method for the determination of trimethoprim in honey samples using an on-line solid-phase extraction technique

A simple, selective and rapid analytical method for determination of trimethoprim (TMP) in honey samples was developed and validated. This method is based on a SPE technique followed by HPLC with photodiode array detection. After dilution and filtration, aliquots of 500 μL honey samples were directly injected to an on-line SPE HPLC system. TMP was extracted on an RP SPE column, and separated on a hydrophilic interaction chromatography column during HPLC analysis. At the first detection step, the noise level of the photodiode array data was reduced with two-dimensional equalizer filtering, and then the smoothed data were subjected to derivative spectrum chromatography. On the second-derivative chromatogram at 254 nm, the limit of detection and the limit of quantification of TMP in a honey sample were 5 and 10 ng/g, respectively. The proposed method showed high accuracy (60-103%) with adequate sensitivity for TMP monitoring in honey samples.