Two dimensional assisted liquid chromatography – a chemometric approach to improve accuracy and precision of quantitation in liquid chromatography using 2D separation,dual detectors,and multivariate curve resolution

Comprehensive two-dimensional liquid chromatography (LC × LC) is rapidly evolving as the preferred method for the analysis of complex biological samples owing to its much greater resolving power compared to conventional one-dimensional (1D-LC). While its enhanced resolving power makes this method appealing, it has been shown that the precision of quantitation in LC × LC is generally not as good as that obtained with 1D-LC. The poorer quantitative performance of LC × LC is due to several factors including but not limited to the undersampling of the first dimension and the dilution of analytes during transit from the first dimension (¹D) column to the second dimension (²D) column, and the larger relative background signals. A new strategy, 2D assisted liquid chromatography (2DALC), is presented here. 2DALC makes use of a diode array detector placed at the end of each column, producing both multivariate ¹D and two-dimensional (2D) chromatograms. The increased resolution of the analytes provided by the addition of a second dimension of separation enables the determination of analyte absorbance spectra from the ²D detector signal that are relatively pure and can be used to initiate the treatment of data from the first dimension detector using multivariate curve resolution–alternating least squares (MCR–ALS). In this way, the approach leverages the strengths of both separation methods in a single analysis: the ²D detector data is used to provide relatively pure analyte spectra to the MCR–ALS algorithm, and the final quantitative results are obtained from the resolved ¹D chromatograms, which has a much higher sampling rate and lower background signal than obtained in conventional single detector LC × LC, to obtain accurate and precise quantitative results. It is shown that 2DALC is superior to both single detector selective or comprehensive LC × LC and 1D-LC for quantitation of compounds that appear as severely overlapped peaks in the ¹D chromatogram – this is especially true in the case of untargeted analyses. We also anticipate that 2DALC will provide superior quantitation in targeted analyses in which unknown interfering compounds overlap with the targeted compound(s). When peaks are significantly overlapped in the first dimension, 2DALC can decrease the error of quantitation (i.e., improve the accuracy by up to 14-fold compared to 1D-LC and up to 3.8-fold compared to LC × LC with a single multivariate detector). The degree of improvement in performance varies depending upon the degree of peak overlap in each dimension and the selectivities of the spectra with respect to one another and the background, as well as the extent of analyte dilution prior to the ²D column.     

Optimization of antibiotic analysis in water by solid-phase extraction and high performance liquid chromatography–mass spectrometry/mass spectrometry

This paper describes the development of an optimized method based on solid-phase extraction (SPE) followed by liquid chromatography–electrospray ionization tandem mass spectrometry (LC–MS/MS) for the simultaneous analysis of ten antibiotic compounds including tetracyclines, sulfonamides, macrolides and quinolones. LC–MS/MS sensitivity has been optimized by alterations to both LC and MS operations. Of the two high resolution columns tested, Waters Symmetry C₁₈ endcapped and Agilent Zorbax Bonus-RP, the latter was found to show better performance in producing sharp peaks and clear separation for most of the target compounds. Optimization of the MS fragmentation collision and cone energy enhanced the peak areas of the target analytes. The recovery of the target compounds from water samples was most efficient on Waters Oasis HLB SPE cartridge, while methanol was shown to be the most suitable solvent for desorbing the compounds from SPE. In addition, acidification of samples prior to SPE was shown to enhance the recovery of the compounds. To ensure a satisfactory recovery, the flow rate through SPE should be maintained at ≤10 mL min⁻¹. The method was successfully applied to the analysis of antibiotics from environmental water samples, with concentrations being <LOD in tap water, between <LOD to 28 ng L⁻¹ in river water and between <LOD to 230 ng L⁻¹ in sewage effluent.     

A critical look at the definition of multidimensional separations

Multidimensional (MD) separations, especially comprehensive two-dimensional (2D) separations such as comprehensive 2D LC (LC × LC), and comprehensive 2D GC (GC × GC), are potentially powerful separation techniques. It is important to have a clear definition of MD techniques to better understand the scope and boundaries of the subject. Widely accepted definitions of MD Separations have their roots in the definition proposed by Giddings. Giddings also added several comments that clarified the scope of his definition. However, some researchers extend Giddings’ definitions beyond their intended scope. Doing so disqualifies such comprehensive 2D techniques as LC × LC, GC × GC and 2D TLC from being considered as 2D techniques. In other instances, extended treatment of Giddings’ definition is used as a basis to justify design-parameters of comprehensive 2D separations despite the fact that these parameters lead to sub-optimal implementations. We believe that the shortcomings in the definition and its popular interpretations are serious enough to warrant attention, especially by those interested in designing optimal instrumentation for MD separations like comprehensive 2D GC. After discussion of the weaknesses in the currently used definitions, we propose to define n-dimensional analysis as one that generates n-dimensional displacement information. We believe that this definition captures the spirit of Giddings’ definition while avoiding the problems associated with its popular interpretations.     

Development of a rapid and sensitive method for determination of cysteine/cystine ratio in chemically defined media

Two rapid, sensitive and quantitative methods for the determination of the cysteine and cystine ratio in complex defined media feedstock using monolithic reversed-phase liquid chromatography (RPLC) and RPLC–MS are presented. Cysteine is pre-derivatised with purified 2-chloro-1-methylquinolinium tetrafluoroborate (CMQT) and separated from other derivatisation products on a narrow-bore 50 mm × 2 mm I.D. monolithic C₁₈ column with UV detection at 355 nm. For reversed-phase LC (RPLC) the separation is carried out isocratically using a mobile phase of 50 mM trichloroacetic acid (TCA) adjusted to pH 2.5 with lithium hydroxide (LiOH) and acetonitrile (83:14) pumped at 1.5 mL/min with an elevated column temperature. For RPLC–MS an ammonium acetate and acetonitrile gradient method was developed with a reduced flow rate of 0.3 mL/min. The treatment of the samples consisted of dividing them into two aliquots, the first aliquot is analysed for cysteine and the second aliquot is analysed for cystine after its quantitative reduction to cysteine using tris(2-carboxyethyl)phosphine (TCEP). Both methods are linear, with R² > 0.999 for 0.25–500 μM for cysteine and 0.25–250 μM for cystine using the LC–UV method, sensitive, with detection limit of 36 nM for cysteine, and precise, with ≤1.1% RSD for both retention time and peak area (n = 6). Samples (n = 31) of an industry standard and supplied chemically defined media feedstock were analysed, finding cysteine ranging from 1.56 to 2.26 μg/mL and cystine from 1062.02 to 1348.13 μg/mL.     

A simple approach to reduce dimensionality from comprehensive two-dimensional liquid chromatography coupled with a multichannel detector

Comprehensive two-dimensional liquid chromatographic (LC × LC) systems play an ever increasing role in separation and characterization of complex samples. When coupled with multichannel detectors, such as the diode array detector, these LC × LC systems become especially useful for non-target analysis and identification of patterns based on the information extracted from those complex samples. Nevertheless, due to the large amount of data generated by these systems, the extraction of useful information for the identification of patterns still is one of the major drawbacks for a wider application of this technique. As a preliminary step in data treatment, we have developed a simple and fast way to deal with this large amount of multi-dimensional data by identifying the three-dimensional (3D) regional maxima of each chromatographic peak generated in a LC × LC–DAD system: retention times at the peak maximum in the first- and second-dimensions and the wavelength of the maximum UV absorption. This dataset is then used to build a 3D fingerprinting of the given sample, which alongside the 3D fingerprinting of other samples, can be used to identify different patterns associated with the specific properties of every sample under study. The applicability of the developed methodology was further assessed by performing a non-target LC × LC–DAD analysis of four Portuguese red wine samples.     

Fast high performance liquid chromatography analysis in lipidomics: Separation of radiolabelled fatty acids and phosphatidylcholine molecular species using a monolithic C18 silica column

HPLC procedures using conventional C₁₈ columns are usually used to separate simple and complex lipid mixtures but these methods of separation remain often laborious and very slow. Here, monolithic columns were successfully applied to separate lipids - radiolabelled fatty acid mixtures and individual phosphatidylcholine (PC) molecular species. For that, isocratic elution was performed using two Chromolith™ Performance RP-18e columns connected in series. Detection was achieved by online measurement of radioactivity for radiolabelled fatty acids and by UV absorbance at 205 nm for PC molecular species. The performances of such silica rods were compared to conventional reverse-phase silica columns. Monolithic stationary phase separated radiolabelled fatty acids and PC molecular species two times and four times faster, respectively. In each analysis, monolithic columns allowed better separation efficiency per unit of time, with lower inlet pressure. The main advantages of this method for lipid separation are that, under isocratic conditions, it is simpler and much faster, while remaining accurate and selective when compared to conventional methods. Therefore, monolithic columns may represent a powerful tool for the near future in the field of lipidomics.     

Separation of intact proteins on porous layer open tubular (PLOT) columns

Porous layer open tubular (PLOT) polystyrene divinylbenzene columns have been used for separating intact proteins with gradient elution. The 10 μm I.D. × 3 m columns were easily coupled to standard liquid chromatography–mass spectrometry (LC–MS) instrumentation with commercially available fittings. Standard proteins separated on PLOT columns appeared as narrow and symmetrical peaks with good resolution. Average peak width increased linearly with gradient time (t_G) from 0.14 to 0.33 min (t_G 20 and 120 min, respectively) using a 3 m column. With shorter columns, peak widths were larger and increased more steeply with gradient time. Theoretical peak capacity (n_c) increased with column length (tested up to 3 m). The n_c increased with t_G until a plateau was reached. The highest peak capacity achieved (n_c = 185) was obtained with a 3 m column, where a plateau was reached with t_G 90 min. The within- and between column retention time repeatabilities were below 0.6% and below 2.5% (relative standard deviation, RSD), respectively. The carry-over following injection of 0.5 ng per protein was less than 1.1%. The retention time dependence on column temperature was investigated in the range 20–50 °C. Proteins in a skimmed milk sample were separated using the method.     

High-temperature two-dimensional gas chromatography of hydrocarbons up to nC60 for analysis of vacuum gas oils

In a tense energetic context, the characterization of heavy petroleum fractions becomes essential. Conventional comprehensive two-dimensional gas chromatography (2D-GC or GC × GC) is widely used for middle distillates analysis, but only a few applications are devoted to these heavier fractions. In this paper, it is shown how the optimization of GC × GC separation allowed the determination of suitable high-temperature (HT) conditions, adjusting column properties and operating conditions. 2D separations were evaluated using 2D separation criteria and a new concept of 2D asymmetry (As_2D). New HT conditions allowed the extension of GC × GC range of applications to heavier hydrocarbons, up to nC₆₀. A first application of high-temperature two-dimensional gas chromatography (HT-2D-GC) to a full vacuum gas oil (VGO) feed stock is described. Comparisons with other standardized methods illustrate the high potential of HT-2D-GC for heavy fractions analysis.     

A vacuum assisted dynamic evaporation interface for two-dimensional normal phase/reverse phase liquid chromatography

A vacuum assisted dynamic solvent evaporation interface for coupling of two-dimensional normal phase/reverse phase liquid chromatography was developed and evaluated. A normal-phase liquid chromatographic (NPLC) column of a 250 mm × 4.6 mm I.D. 5 μm CN phase was used as the first dimension, and a reversed-phase liquid chromatographic (RPLC) column of 250 mm × 4.6 mm I.D. 5 μm C₁₈ phase was used as the second dimension. The eluent from the first dimension flowed into a fraction loop, and the solvent in the eluent was dynamically evaporated and removed by vacuum as it was entering the fraction loop of the interface. The non-evaporable analytes was retained and enriched in about 5–25 μL solution within the loop. Up to 1 mL/min of mobile phase from the first dimension can be evaporated and removed dynamically by the interface. The mobile phase from the second dimension then entered the loop, and dissolved the concentrated analytes retained inside the loop, and carried them onto the second dimension column for further separation. The operation conditions of the two dimensions were independent from each other, and both dimensions were operated at their optimal chromatographic conditions. We evaluated the interface by controlling the loop temperature in a water bath at normal temperature, and investigated the sample losses by using standard samples with different boiling points. It was found that the sample loss due to evaporation in the interface was negligible for non-volatile samples or for components with boiling point above 340 °C. The interface realizes fast solvent removal of mL volume of fraction and concentration of the fraction into tenth of μL volume, and injection of the concentrated fraction on the secondary column. The chromatographic performance of the two-dimensional LC system was enhanced without compromise of separation efficiency and selectivity on each dimension.     

Determination of mercury species by the diffusive gradient in thin film technique and liquid chromatography – atomic fluorescence spectrometry after microwave extraction

A diffusive gradient in thin films technique (DGT) was combined with liquid chromatography (LC) and cold vapor atomic fluorescence spectrometry (CV-AFS) for the simultaneous quantification of four mercury species (Hg²⁺, CH₃Hg⁺, C₂H₅Hg⁺, and C₆H₅Hg⁺). After diffusion through an agarose diffusive layer, the mercury species were accumulated in resin gels containing thiol-functionalized ion-exchange resins (Duolite GT73, and Ambersep GT74). A microwave-assisted extraction (MAE) in the presence of 6 M HCl and 5 M HCl (55 °C, 15 min) was used for isolation of mercury species from Ambersep and Duolite resin gels, respectively. The extraction efficiency was higher than 95.0% (RSD 3.5%). The mercury species were separated with a mobile phase containing 6.2% methanol + 0.05% 2-mercaptoethanol + 0.02 M ammonium acetate with a stepwise increase of methanol content up to 80% in the 16th min on a Zorbax C18 reverse phase column. The LODs of DGT–MAE–LC–CV-AFS method were 38 ng L⁻¹ for CH₃Hg⁺, 13 ng L⁻¹ for Hg²⁺, 34 ng L⁻¹ for C₂H₅Hg⁺ and 30 ng L⁻¹ for C₆H₅Hg⁺ for 24 h DGT accumulation at 25 °C.     

A study of the precision and accuracy of peak quantification in comprehensive two-dimensional liquid chromatography in time

Simulated chromatographic data were used to determine the precision and accuracy in the estimation of peak volumes (i.e., peak sizes) in comprehensive two-dimensional liquid chromatography in time (LC × LC). Peak volumes were determined both by summing the areas in the second dimension chromatograms and by fitting the second dimension areas to a Gaussian peak. The Gaussian method is better at predicting the peak volume than the moments method provided there are at least three second dimension injections above the limit of detection (LOD). However, when only two of the second dimension signals are substantially above baseline, the accuracy and precision of the Gaussian fit method become quite poor because the results from the fitting algorithm become indeterminate. Based on simulations in which the modulation ratio (M_R = 4¹σ/t_s) and sampling phase (?) were varied, we conclude for well-resolved peaks that the optimum precision in peak volumes in 2D separations will be obtained when the M_R is between two and five, such that there are typically four to ten second dimension peaks recorded over the eight σ width of the first dimension peak. This sampling rate is similar to that suggested by the Murphy–Schure–Foley criterion. This provides an RSD of approximately 2% for the signal-to-noise ratio used in the present simulations. The precision of the peak volume of experimental data was also assessed, and RSD values were in the range of 4–5%. We conclude that the poorer precision found in the LC × LC experimental data as compared to LC may be due to experimental imprecision in sampling the effluent from the first dimension column.     

Data analysis programs for comprehensive two-dimensional chromatography

User-friendly and easy-to-use laboratory-written programs for visualisation and interpretation of comprehensive two-dimensional chromatographic data were developed. The programs that are not tied to any particular commercial instrument, and data obtained either by comprehensive two-dimensional liquid (LC × LC) or gas (GC × GC) chromatography can be analysed. Operations of the programs allow visualisation of 2D and 3D plots, comparison of two 2D plots at a time, as well as determination of retention times and peak heights and volumes.     

Fast method for monitoring phospholipase A2 activity by liquid chromatography–electrospray ionization mass spectrometry

A new liquid chromatography–electrospray ionization mass spectrometry (LC–ESI-MS) method for the fast determination of phospholipase A₂ (PLA₂) activity has been developed. For the first time, the method allows the parallel detection of glycerophosphatidylcholine (GroPCho) as PLA₂ substrate as well as of its products fatty acid (FA) and lyso-GroPCho. ESI-MS was carried out in negative ion mode, detecting the FA as [M − H]⁻ ions and the lyso-GroPCho and GroPCho as acetate adducts [M + Ac]⁻. Utilizing a fast gradient on a short C₅-modified silica gel column with 3 μm particles, five GroPChos, five FAs and six lyso-GroPChos could be separated according to their chain length in less than 3 min. A very high average chromatographic efficiency of 41,200 theoretical plates (plate height 0.5 μm) was achieved for the separation of the GroPChos. The method was applied for monitoring the release of arachidonic acid (20:4 FA) and 1-stearoyl-lyso-sn-GroPCho (18:0 GroPCho) from unilamellar vesicles of 1-stearoyl-2-arachidonoyl-sn-GroPCho (18:0/20:4 GroPCho). With a limit of detection of 0.5 pmol (total amount injected on column) for the FAs and lyso-GroPChos and 1.5 pmol for the GroPChos as well as a linear range of 1.5 decades, the method has proven to be suitable for the monitoring of different secretory PLA₂ (sPLA₂) conversions. Furthermore, it was applied to screen a small library of PLA₂ inhibitors for their activity towards sPLA₂ type V and snake venom of Bothrops moojeni. In both cases, active samples could be directly identified. With its short analysis time, its high chromatographic efficiency and the parallel detection of substrate and all products, the developed LC–ESI-MS method is well suited for the analysis of PLA₂ activity.     

Ring-opening metathesis polymerization-derived monolithic anion exchangers for the fast separation of double-stranded DNA fragments

Ring-opening metathesis polymerization- (ROMP) derived monoliths were prepared from 5-norborn-2-enemethyl bromide (NBE-CH₂Br) and tris(5-norborn-2-enemethoxy)methylsilane ((NBE-CH₂O)₃SiCH₃) within the confines of surface-silanized borosilicate columns (100 × 3 mm I.D.), applying Grubbs’ first generation benzylidene-type catalyst [RuCl₂(PCy₃)₂(CHPh)]. Monoliths were converted into weak anion exchangers via reaction with diethyl amine. The resulting monolithic anion exchangers demonstrated a very good potential for the anion-exchange separation of nucleic acids applying a phosphate buffer (0.05 mol/L, pH 7) and NaCl (1.0 mol/L) as a gradient former. Fast and efficient separations, indicated by sharp and highly symmetric analyte peaks, were established. Except for the 267 and 298 base pair fragments, the eleven fragments of a ds-pUC18 DNA Hae III digest were baseline separated within ∼8 min. Nineteen fragments of a ds-pBR322 Hae III digest were separated within ∼12 min. There, only the 192 and 213 base pair fragments and the 458, 504 and 540 base pair fragments coeluted. A ds-pUC18 DNA Hae III digest was used as a control analyte in evaluating the influence of organic additives on the mobile phase such as methanol and acetonitrile on nucleic acid separation. Methanol, and even better, acetonitrile improved the separation efficiency and shortened the analysis time.     

Development and validation of a liquid chromatographic method for the analysis of capreomycin sulfate and its related substances

A gradient LC method for the analysis of capreomycin sulfate and its related substances was developed. The chromatographic conditions include the use of a Hypersil base deactivated C₁₈ (250 mm × 4.6 mm, 5 μm) column maintained at 25 °C, a mobile phase containing acetonitrile, phosphate buffer pH 2.3 and 0.025 M hexanesulfonate at a flow rate of 1.0 mL/min and UV detection performed at 268 nm. Good separation of the four active components of capreomycin and eleven unknown impurities was achieved. A system suitability test to check the quality of the separation is specified. The method shows good repeatability, linearity and robustness.     

Large-scale pesticide testing in olives by liquid chromatography–electrospray tandem mass spectrometry using two sample preparation methods based on matrix solid-phase dispersion and QuEChERS

In this work we have evaluated the performance of two sample preparation methodologies for the large-scale multiresidue analysis of pesticides in olives using liquid chromatography–electrospray tandem mass spectrometry (LC–MS/MS). The tested sample treatment methodologies were: (1) liquid–liquid partitioning with acetonitrile followed by dispersive solid-phase extraction clean-up using GCB, PSA and C₁₈ sorbents (QuEChERS method – modified for fatty vegetables) and (2) matrix solid-phase dispersion (MSPD) using aminopropyl as sorbent material and a final clean-up performed in the elution step using Florisil. An LC–MS/MS method covering 104 multiclass pesticides was developed to examine the performance of these two protocols. The separation of the compounds from the olive extracts was achieved using a short C₁₈ column (50 mm × 4.6 mm i.d.) with 1.8 μm particle size. The identification and confirmation of the compounds was based on retention time matching along with the presence (and ratio) of two typical MRM transitions. Limits of detection obtained were lower than 10 μg kg⁻¹ for 89% analytes using both sample treatment protocols. Recoveries studies performed on olives samples spiked at two concentration levels (10 and 100 μg kg⁻¹) yielded average recoveries in the range 70–120% for most analytes when QuEChERS procedure is employed. When MSPD was the choice for sample extraction, recoveries obtained were in the range 50–70% for most of target compounds. The proposed methods were successfully applied to the analysis of real olives samples, revealing the presence of some of the target species in the μg kg⁻¹ range. Besides the evaluation of the sample preparation approaches, we also discuss the use of advanced software features associated to MRM method development that overcome several limitations and drawbacks associated to MS/MS methods (time segments boundaries, tedious method development/manual scheduling and acquisition limitations). This software feature recently offered by different vendors is based on an algorithm that associates retention time data for each individual MS/MS transition, so that the number of simultaneously traced transitions throughout the entire chromatographic run (dwell times and sensitivity) is maximized.     

Comprehensive two-dimensional liquid chromatography separations of pharmaceutical samples using dual Fused-Core columns in the 2nd dimension

This paper focuses on the application of RPLC × RPLC to pharmaceutical analysis and addresses the specific problem of separating co-eluting impurities/degradation products that maybe “hidden” within the peak envelope of the active pharmaceutical ingredient (API) and thus may escape detection by conventional methods. A comprehensive two-dimensional liquid chromatograph (LC × LC) was constructed from commercially available HPLC equipment. This system utilizes two independently configurable 2nd dimension binary pumping systems to deliver independent flow rates, gradient profiles and mobile phase compositions to dual Fused-Core secondary columns. Very fast gradient separations (30 s total cycle time) were achieved at ambient temperature without excessive backpressure and without compromising optimal 1st dimension sampling rates. The operation of the interface is demonstrated for the analysis of a 1 mg/ml standard mixture containing 0.05% of a minor component. The practicality of using RPLC × RPLC for the analysis of actual co-eluting pharmaceutical degradation products, by exploiting pH-induced changes in selectivity, is also demonstrated using a three component mixture. This mixture (an API, an oxidation product of the API at 1.0%, w/w, and a photo degradant of the API at 0.5%, w/w) was used to assess the stability indicating nature of an established LC method for analysis of the API.     

Silica-based ionic liquid coating for 96-blade system for extraction of aminoacids from complex matrixes

1-Vinyl-3-octadecylimidazolium bromide ionic liquid [C₁₈VIm]Br was prepared and used for the modification of mercaptopropyl-functionalized silica (Si-MPS) through surface radical chain-transfer addition. The synthesized octadecylimidazolium-modified silica (SiImC₁₈) was characterized by thermogravimetric analysis (TGA), infrared spectroscopy (IR), ¹³C NMR and ²⁹Si NMR spectroscopy and used as an extraction phase for the automated 96-blade solid phase microextraction (SPME) system with thin-film geometry using polyacrylonitrile (PAN) glue. The new proposed extraction phase was applied for extraction of aminoacids from grape pulp, and LC–MS–MS method was developed for separation of model compounds. Extraction efficiency, reusability, linearity, limit of detection, limit of quantitation and matrix effect were evaluated. The whole process of sample preparation for the proposed method requires 270 min for 96 samples simultaneously (60 min preconditioning, 90 min extraction, 60 min desorption and 60 min for carryover step) using 96-blade SPME system.     

Direct aqueous supercritical fluid extraction coupled on-line with liquid chromatography–tandem mass spectrometry for the analysis of polyether ionophore antibiotics in water

A direct aqueous SFE system designed to extract water samples contained in vials has been coupled on-line with a reverse phase LC–MS–MS system using a single 10-port valve. An SFE trap system using C₁ stationary phase connected to a C₁₈ analytical HPLC column enabled the SFE–LC–MS–MS analysis of three polyether ionophore antibiotics in water using a step gradient. A quantitative SFE–LC–MS–MS method has been developed whereby the progress of SFE can be monitored directly on-line such that ionophore recovery profile data from a single water sample can be obtained. Using a continuous direct aqueous SFE period of 75 min, the SFE–LC–MS–MS recoveries of the ionophores were: monensin 76.2% with RSD 4.1%, lasalocid 84.6% with RSD 3.8% and narasin 91.2% with RSD 3.2%. With positive ion electrospray ionization, the SFE–LC–MS–MS system using a 4 mL water sample provided multiple reaction monitoring (MRM) limits of detection for monensin and lasalocid each equivalent to 90 ng/L whereas 30 ng/L for narasin. A two-way valve controlling carbon dioxide distribution to the SFE vessel has provided a means for the initial investigation of the recovery of ionophore sodium salts from water using static SFE.     

Simultaneous determination of sex hormones in egg products by ZnCl2 depositing lipid, solid-phase extraction and ultra performance liquid chromatography/electrospray ionization tandem mass spectrometry

A method was developed for simultaneous determination of residues of 17 sex hormones in egg products. Target compounds were extracted from samples with methanol in an ultrasonic bath, effectively separated from lipids in the extracts by ZnCl₂ depositing filtration and purified using a C₁₈ solid-phase extraction (SPE) and followed by NH₂ SPE cartridge. The analytes were quantified by liquid chromatography using a BEH C₁₈ column coupled to an electrospray ionization tandem mass spectrometer (LC-ESI-MS/MS) operating in negative mode for estrogens and in positive multiple reaction monitoring mode for androgens. The parameters of the mass spectrometer and the composition of mobile phase and additives were also optimized to enhance detection sensitivity. Average recoveries of the target compounds varied from 70.0% to 121.0% with relative standard deviations ranging from 2.3% to 11.2% at two fortification levels. The limits of detection (LOD) of the method were from 0.002 μg kg⁻¹ to 0.23 μg kg⁻¹ and the limits of quantification (LOQ) were in the range of 0.007-0.76 μg kg⁻¹.