High-throughput screening for enzyme inhibitors using frontal affinity chromatography with liquid chromatography and mass spectrometry

This work presents new frontal affinity chromatography (FAC) methodologies for high-throughput screening of compound libraries, designed to increase screening rates and improve sensitivity and ruggedness in performance. A FAC column constructed around the enzyme N-acetylglucosaminyltransferase V (GnT-V) was implemented in the identification of potential enzyme inhibitors from two libraries of trisaccharides. Effluent from the FAC column was fractionated, sequentially processed via LC/MS, and referenced to a similar analysis through a control FAC column lacking the enzyme. The resulting multidimensional data sets were compared across corresponding sample and control fractions to identify binders, in a semiautomated approach. A strong binder in the protonated form at m/z 795 was identified from the first library of 81 compounds, exhibiting an estimated Kd value of 0.3 microM. Other binders yielded Kd values ranging from 0.35 to 3.35 microM. To demonstrate the improvement in performance of this FAC-LC/MS approach over the conventional online FAC/MS approach, 15 compounds from this library were blended with a second library of 1000 synthetic trisaccharides and screened against GnT-V. All ligands in the 15-compound set were identified in this larger screen, and no ligands of greater affinity than compound 1 were found. Our results show that FAC-LC/MS is a reliable method for screening large compound libraries directly and useful for large-scale ligand discovery initiatives.     

Global protein identification and quantification technology using two-dimensional liquid chromatography nanospray mass spectrometry

A simple and reliable method is described here for the identification and relative quantification of proteins in complex mixtures using two-dimensional liquid chromatography/tandem mass spectrometry. The method is based on the classical proteomic analysis where proteins are digested with trypsin and the resulting peptides are separated by multidimensional liquid chromatography. The separated peptides are analyzed by tandem mass spectrometry and identified via a database search algorithm such as SEQUEST. The peak areas (integrated ion counts over the peptide elution time) of all identified peptides are calculated, and the relative concentration of each protein is determined by comparing the peak areas of all peptides from that protein in one sample versus those from the other. Using this strategy, we compared the relative level of protein expression of A431 cells (an epidermal cell line) grown in the presence or absence of epidermal growth factor (EGF). Our results are consistent with the published observations of the transient effects of EGF. In addition, the difference in the concentrations of several phosphopeptides determined in our studies suggests the possibility of several new targets involved in the EGF cell-signaling pathway. This global protein identification and quantification technology should prove to be a valuable means for comparing proteomes in biological samples subjected to differential treatments.     

Simultaneous quantification of homocysteine and folate in human serum or plasma using liquid chromatography/tandem mass spectrometry

A unified extraction and quantification procedure based on stable isotope-dilution liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been developed for the simultaneous determination of total homocysteine and folate (5-methyltetrahydrofolic acid and folic acid) levels in human serum and plasma. This is the first report documenting the simultaneous extraction and quantification of these structurally dissimilar analytes. Analytes are quantitatively isolated from samples (500 microL) prior to LC/MS/MS analysis using a two-step stabilization process combined with C18 solid-phase extraction. The method exhibits excellent linearity over 4 orders of magnitude for each analyte. Measurement repeatability (RSD, N = 2) ranged from 0.3% to 3% for all analytes over 1 day of analysis. Total method variability (RSD, N = 6) ranged from 0.7% to 10% for all analytes over three independent days of analysis. The accuracy and practical applicability of the method were demonstrated by applying the method to the quantitative determination of each analyte in a new NIST serum Standard Reference Material (NIST SRM 1955 Homocysteine and Folate in Frozen Human Serum) and in a small subset of normal donor plasma samples.     

An identification method for altered proteins in tissues utilizing fluorescence derivatization, liquid chromatography, tandem mass spectrometry, and a database-searching algorithm

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is now widely used as a tool for proteomic studies. For the sensitive determination of proteins in 2D-PAGE, fluorescence derivatization of primary amino moieties of proteins with cyanine dyes was recently developed. However, precipitation of the proteins could occur if completely derivatized because of the lower solubility of the resultant derivatives owing to the hydrophobicity of the reagents and the loss of the hydrophilic primary amino moieties. Thus, in this paper, a water-soluble and thiol-specific fluorogenic reagent, ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate, was adopted for the derivatization of proteins in tissues either with and without stimulation. Then, the method follows a separation of the derivatives by liquid chromatography with fluorescence detection, an isolation of only the altered proteins, an enzymatic digestion of the isolated proteins, and an identification of the proteins by liquid chromatography/MS/MS with the database-searching algorithm. By using this method, we identified the altered expressions of five increased proteins (e.g., pancreatic polypeptide) as well as three decreased proteins (e.g., insulin 2) in the islets of Langerhans in Wistar rats 2 days after they were subcutaneously administered with dexamethasone.     

Development and evaluation of a candidate reference method for the determination of total cortisol in human serum using isotope dilution liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry

Cortisol is an important diagnostic marker for the production of steroid hormones, and accurate measurements of serum cortisol are necessary for proper diagnosis of adrenal function. A candidate reference method involving isotope dilution coupled with liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been developed and critically evaluated. An isotopically labeled internal standard, cortisol-d(3), was added to serum, followed by equilibration and solid-phase and ethyl acetate extractions to prepare samples for liquid chromatography/mass spectrometry electrospray ionization (LC/MS-ESI) and liquid chromatography/tandem mass spectrometry electrospray ionization (LC/MS/MS-ESI) analyses. (M + H)(+) ions at m/z 363 and 366 for cortisol and its labeled internal standard were monitored for LC/MS. The transitions of (M + H)(+) --> [(M + H)(+) - 2H(2)O] at m/z 363 --> 327 and 366 --> 330 were monitored for LC/MS/MS. The accuracy of the measurement was evaluated by a comparison of results of this candidate reference method on lyophilized human serum reference materials for cortisol [Certified Reference Materials 192 and 193] with the certified values determined by gas chromatography/mass spectrometry reference methods and by a recovery study for the added cortisol. The results of this method for total cortisol agreed with the certified values within 1.1%. The recovery of the added cortisol ranged from 99.8% to 101.0%. This method was applied to the determination of cortisol in samples of frozen serum pools. Excellent precision was obtained with within-set CVs of 0.3%-1.5% and between-set CVs of 0.04%-0.4% for both LC/MS and LC/MS/MS analyses. The correlation coefficients of all linear regression lines ranged from 0.998 to 1.000. The detection limits (at a signal-to-noise ratio of approximately 3-5) were 10 and 15 pg for LC/MS and LC/MS/MS, respectively. This method, which demonstrates good accuracy and precision, and is free from interferences from structural analogues, qualifies as a candidate reference method and can be used as an alternative reference method to provide an accuracy base to which the routine methods can be compared.     

A liquid chromatography--tandem mass spectrometry multiresidue method for quantification of specific metabolites of organophosphorus pesticides, synthetic pyrethroids, selected herbicides, and deet in human urine

The ability to estimate low-dose human exposure to commonly used pesticides often is requested in epidemiologic studies. Therefore, fast and robust methods are necessary that can measure many analytes in the same sample. We have developed a method for high-throughput analysis of 19 markers of commonly used pesticides in human urine. The analytes were seven specific metabolites of organophosphorus pesticides, five metabolites of synthetic pyrethroids, six herbicides or their metabolites, and one insect repellant. Human urine (2 mL) was spiked with stable isotopically labeled analogues of the analytes, enzymatically hydrolyzed, extracted using solid-phase extraction, concentrated, and analyzed using high-performance liquid chromatography-tandem mass spectrometry. The sample was divided into two portions and analyzed on two different mass spectrometers, one using atmospheric pressure chemical ionization (APCI) and the other using turbo ion spray atmospheric pressure ionization (TIS). All analytes except the pyrethroid metabolites were analyzed using APCI. The detection limits for all analytes ranged from 0.1 to 1.5 ng/mL of urine, with the majority (17) below 0.5 ng/mL. The analytical precision for the different analytes, estimated as both the within-day and between-day variation, was 3-14 and 4-19%, respectively. The extraction recoveries of the analytes ranged from 68 to 114%. The throughput, including calibration standards and quality control samples, is approximately 50 samples a day. However, the analysis time with the TIS application is much shorter, and if only pyrethroid metabolite data are of interest, the throughput can be increased to 100-150 samples/day.     

Accurate mass liquid chromatography/mass spectrometry on orthogonal acceleration time-of-flight mass analyzers using switching between separate sample and reference sprays. 1. Proof of concept

This paper describes the use of two separate electrosprays for introducing sample and reference for accurate mass liquid chromatography/mass spectrometry (LC/MS) on an orthogonal acceleration time-of-flight mass analyzer. This is carried out using an adaptation of the multiplexed electrospray ion source in which only two of the sprays are utilized. Results are shown for the positive ion detection of trace-level components in complex matrixes and good mass accuracies are obtained, even for very low level components. An example of accurate mass measurements obtained using negative ion LC/MS is also shown. To obtain additional structural information, an example of cone voltage fragmentation is included and shows that good mass accuracy can be obtained for both precursor and fragment ions.     

Ultra-sensitive quantification of corticosteroids in plasma samples using selective solid-phase extraction and reversed-phase capillary high-performance liquid chromatography/tandem mass spectrometry

Low-dose corticosteroids may provide a favorable benefit/risk ratio for many therapeutic applications. However, the extremely low plasma drug concentrations achieved, in conjunction with the insufficient sensitivity/ selectivity of current analytical methods, renders the evaluation of corticosteroid pharmacokinetics (PK) a significant challenge under such conditions. Furthermore, targeted therapeutic strategies involving administration by inhalation or intraocular injection could result in very low but sustained systemic corticosteroid concentrations, which must be quantified to determine potential side effects. Here we describe a robust method for the ultrasensitive quantification of corticosteroids in plasma samples. This was achieved by the combination of a selective solid-phase extraction (SPE) with a highly sensitive capillary LC (microLC)-MS/MS analysis. SPE washing and elution conditions were optimized so that target drugs are selectively extracted from plasma. By eliminating most undesirable compounds from the sample matrix, this selective SPE procedure enabled a high sample loading volume on the microLC column without compromising chromatographic performance and operational robustness and helped to achieve ultralow detection limits for the corticosteroids in plasma. The effect of microLC separation on the signal-to-noise ratio of corticosteroid peaks in plasma samples was investigated. It was found that with sufficient microLC separation, sensitivity was improved because of a decrease in matrix effects and the removal of endogenous interferences. Detection limits of four clinically important corticosteroids (budesonide, dexamethasone, triamcinolone acetonide, and dexamethasone acetate) ranged from 0.2 to 1 pg/mL in plasma, and linearity was good for all drugs in the range of 5-5000 pg/mL. Accuracy was 88-107% and the variation (CV%) was 2.3-11.1%. A limit of quantification (LOQ) of 5 pg/mL was validated for all four compounds. We applied this method to quantify the low levels of triamcinolone acetonide (TACA) in porcine plasma following suprachoroidal administration, which is necessary to estimate systemic drug exposure resulting from this novel clinical approach for treating inflammatory diseases of the eye. TACA in plasma could be quantified at low pg/mL levels for up to 90 days posttreatment. To our knowledge, this is the first practical analytical approach that can monitor plasma corticosteroids after intraocular administration, given the ultralow plasma concentrations achieved. In summary, this strategy enables PK analysis of corticosteroids in treatment regimens that result in extremely low systemic concentrations, and the approach can be extended for the sensitive quantification of other drugs.     

Accurate mass liquid chromatography/mass spectrometry on quadrupole orthogonal acceleration time-of-flight mass analyzers using switching between separate sample and reference sprays. 2. Applications using the dual-electrospray ion source

A new electrospray dual sprayer, LockSpray, was developed for accurate mass measurements on a quadrupole orthogonal acceleration time-of-flight mass spectrometer (oa-Q-ToF). With the dual-sprayer ion source, both sprays are orthogonal to each other. A mechanism similar to the one employed on the multiplexed electrospray source (MUX) allows switching between reference and sample sprayer. The reference sprayer is optimized for low flow rates, whereas the sample sprayer is a conventional Z-spray type sprayer. Earlier work using a modified MUX ion source on an orthogonal acceleration time-of-flight instrument showed promising results. In this paper, examples obtained with the LockSpray, specifically designed for accurate mass measurements on an oa-Q-ToF, are presented. The examples include results obtained for the identification of impurities in drug substances such as cimetidine and rosiglitazone, using accurate mass tandem mass spectrometry in both positive and negative ion electrospray modes. Good mass accuracies, i.e., within 2 mDa of the theoretical value, were obtained in MS and MS/MS operation.     

Towards absolute quantification of therapeutic monoclonal antibody in serum by LC-MS/MS using isotope-labeled antibody standard and protein cleavage isotope dilution mass spectrometry

Although LC-MS methods are increasingly used for the absolute quantification of proteins, the lack of appropriate internal standard (IS) hinders the development of rapid and standardized analytical methods for both in vitro and in vivo studies. Here, we have developed a novel method for the absolute quantification of a therapeutic protein, which is monoclonal antibody (mAb). The method combines liquid chromatography tandem mass spectrometry (LC-MS/MS) and protein cleavage isotope dilution mass spectrometry with the isotope-labeled mAb as IS. The latter was identical to the analyzed mAb with the exception that each threonine contains four (13)C atoms and one (15)N atom. Serum samples were spiked with IS prior to the overnight trypsin digestion and subsequent sample cleanup. Sample extracts were analyzed on a C18 ACE column (150 mm x 4.6 mm) using an LC gradient time of 11 min. Endogenous mAb concentrations were determined by calculating the peak height ratio of its signature peptide to the corresponding isotope-labeled peptide. The linear dynamic range was established between 5.00 and 1000 microg/mL mAb with accuracy and precision within +/-15% at all concentrations and below +/-20% at the LLOQ (lower limit of quantification). The overall method recovery in terms of mAb was 14%. The losses due to sample preparation (digestion and purification) were 72% from which about 32% was due to the first step of the method, the sample digestion. This huge loss during sample preparation strongly emphasizes the necessity to employ an IS right from the beginning. Our method was successfully applied to the mAb quantification in marmoset serum study samples, and the precision obtained on duplicate samples was, in most cases, below 20%. The comparison with enzyme-linked immunosorbent assay (ELISA) showed higher exposure in terms of AUC and Cmax with the LC-MS/MS method. Possible reasons for this discrepancy are discussed in this study. The results of this study indicate that our LC-MS/MS method is a simple, rapid, and precise approach for the therapeutic mAb quantification to support preclinical and clinical studies.     

Analysis of endocrine disruptors, pharmaceuticals, and personal care products in water using liquid chromatography/tandem mass spectrometry

A method has been developed for the trace analysis of 27 compounds from a diverse group of pharmaceuticals, steroids, pesticides, and personal care products. The method employs solid-phase extraction (SPE) and liquid chromatography/tandem mass spectrometry (LC/MS/MS), using electrospray ionization (ESI) in both positive and negative modes and atmospheric pressure chemical ionization in positive mode. Unlike many previous methods, a single SPE procedure using 1 L of water coupled to a simple LC method is used for all ionization modes. Instrument detection limits for most compounds were below 1.0 pg on column with reporting limits of 1.0 ng/L in water. Recoveries for most compounds in deionized water were greater than 80%. Sulfuric acid was found to be the preferred sample preservative, and structures of all MS/MS product ions are proposed. Matrix effects from waters with a high content of treated municipal effluent were observed in both ESI modes and are discussed in the paper.     

Expediting the method development and quality control of reversed-phase liquid chromatography electrospray ionization mass spectrometry for pharmaceutical analysis by using an LC/MS performance test mix

Mass spectrometry combined with liquid chromatography (LC/MS) has become an important analytical methodology in both pharmaceutical and biomolecule analyses. LC/MS, especially with reversed-phase HPLC (RP-LC), is extensively used in the separation and structural identification of pharmaceutical samples. However, many parameters have to be considered when a new LC/MS method is developed for either separation and structural analysis of unknown mixtures or quantitative analysis of a set of known compounds in an assay. The optimization of a new LC/MS method can be a time-consuming process. A novel kit-LC/MS performance test mix-composed of aspartame, cortisone, reserpine, and dioctyl phthalate has been developed to accelerate the process of establishing a new RP-LC/MS method. The LC/MS mix makes the evaluation and validation of an LC/MS method more efficient and easier. It also simplifies the quality control procedure for an LC/MS method in use.     

Study of the hydrolysis of a sulfonylurea herbicide using liquid chromatography with diode array detection and mass spectrometry by three-way multivariate curve resolution-alternating least squares

This research is focused on the development of a novel, automated chemometric method for obtaining relevant chemical information from time-course measurements of an evolving chemical system. This paper describes an investigation of the hydrolysis of Ally, which is a sulfonylurea herbicide. The hydrolysis of this compound is observed at different pHs and temperatures by reversed-phase liquid chromatography using a diode array detector. The data are analyzed using a three-way, multivariate curve resolution technique. Of special interest was the application of a closure constraint in the kinetic dimension followed by the determination of the rate constants for each step of the pathway by using a differential equation solver and nonlinear fitting of the data.     

Trace analysis of trimethoprim and sulfonamide, macrolide, quinolone, and tetracycline antibiotics in chlorinated drinking water using liquid chromatography electrospray tandem mass spectrometry

A multirun analytical method has been developed and validated for trace determination of 24 antibiotics including 7 sulfonamides, 3 macrolides, 7 quinolones, 6 tetracyclines, and trimethoprim in chlorine-disinfected drinking water using a single solid-phase extraction method coupled to liquid chromatography with positive electrospray tandem mass spectrometry detection. The analytes were extracted by a hydrophilic-lipophilic balanced resin and eluted with acidified methanol (0.1% formic acid), resulting in analyte recoveries generally above 90%. The limits of quantitation were mostly below 10 ng/L in drinking water. Since the concentrated sample matrix typically caused ion suppression during electrospray ionization, the method of standard addition was used for quantitation. Chlorine residuals in drinking water can react with some antibiotics, but ascorbic acid was found to be an effective chlorine quenching agent without affecting the analysis and stability of the antibiotics in water. A preliminary occurrence study using this method revealed the presence of some antibiotics in drinking waters, including sulfamethoxazole (3.0-3.4 ng/L), macrolides (1.4-4.9 ng/L), and quinolones (1.2-4.0 ng/L).     

Isotope dilution high-performance liquid chromatography/tandem mass spectrometry method for quantifying urinary metabolites of atrazine, malathion, and 2,4-dichlorophenoxyacetic acid.

We have developed an isotope dilution high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) method for quantifying the urinary metabolites of the pesticides atrazine, malathion, and 2,4-dichlorophenoxyacetic acid (2,4-D). Urine samples are extracted with an organic solvent, and the organic fraction is concentrated. The concentrate is then analyzed using HPLC/MS/MS. The limits of detection for the metabolites are less than 0.5 microgram/L (parts per billion) in 10 mL of urine, with a high degree of accuracy and precision.     

Determination of suitable column geometries by means of van Deemter and kinetic plots for isothermal and isocratic method development in high-temperature liquid chromatography isotope ratio mass spectrometry

The method of high-temperature liquid chromatography isotope ratio mass spectrometry (HTLC-IRMS) is used to determine the origin or authenticity of compounds. Currently, the drawback of this hyphenation is the interface which causes pronounced band broadening due to a large extra-column volume. Therefore, the aim of this study is to determine suitable column geometries and particle sizes at different temperature and to study the effect of extra-column band broadening. The tools to assess the efficiency of columns are van Deemter and kinetic plots. By comparison of different column geometries and particle sizes, it could be shown that 3.0 mm ID columns achieve a higher performance than 2.1 mm ID columns and a particle size of 1.7 μm is advantageous over 3.5 and 5.0 μm particles when the injection volume is adjusted to 2 μL and the temperature is higher than 60 °C. Because water was the mobile phase, the retention factor could not be kept constant at different column temperatures. The lower retention factor at elevated temperatures leads to a decrease of the plate number, because of the relatively larger contribution to extra-column band broadening at lower retention factors. This is the reason why 3.0 mm ID columns should be preferred for the HTLC-IRMS hyphenation when the separation is carried out under isothermal and isocratic conditions.     

Trace determination of macrolide and sulfonamide antimicrobials, a human sulfonamide metabolite, and trimethoprim in wastewater using liquid chromatography coupled to electrospray tandem mass spectrometry

An analytical method has been developed and validated for the simultaneous trace determination of four macrolide antibiotics, six sulfonamides, the human metabolite N4-acetylsulfamethoxazole, and trimethoprim in wastewater. The method was validated for tertiary, secondary, and-unlike in previously published methods-also for primary effluents of municipal wastewater treatment plants. This wide range of application is necessary to thoroughly investigate the occurrence and fate of chemicals in wastewater treatment. Wastewater samples were enriched by solid-phase extraction, followed by reversed-phase liquid chromatography coupled to tandem mass spectrometry using positive electrospray ionization. Recoveries from all sample matrixes were generally above 80%, and the combined measurement uncertainty varied between 2 and 18%. Concentrations measured in tertiary effluents ranged between 10 ng/L for roxithromycin and 423 ng/L for sulfamethoxazole. Corresponding levels in primary effluents varied from 22 to 1450 ng/L, respectively. Trace amounts of these emerging contaminants reach ambient waters, since all analytes were not fully eliminated during conventional activated sludge treatment followed by sand filtration. In the case of sulfamethoxazole, the amount present as human metabolite N4-acetylsulfamethoxazole had to be taken into account in order to correctly assess the fate of sulfamethoxazole in wastewater treatment.     

High-throughput sequential injection method for simultaneous determination of plutonium and neptunium in environmental solids using macroporous anion-exchange chromatography, followed by inductively coupled plasma mass spectrometric detection

This paper reports an automated analytical method for rapid and simultaneous determination of plutonium and neptunium in soil, sediment, and seaweed, with detection via inductively coupled plasma mass spectrometry (ICP-MS). A chromatographic column packed with a macroporous anion exchanger (AG MP-1 M) was incorporated in a sequential injection (SI) system for the efficient retrieval of plutonium, along with neptunium, from matrix elements and potential interfering nuclides. The sorption and elution behavior of plutonium and neptunium onto AG MP-1 M resin was compared with a commonly utilized AG 1-gel-type anion exchanger. Experimental results reveal that the pore structure of the anion exchanger plays a pivotal role in ensuring similar separation behavior of plutonium and neptunium along the separation protocol. It is proven that plutonium-242 ((242)Pu) performs well as a tracer for monitoring the chemical yield of neptunium when using AG MP-1 M resin, whereby the difficulties in obtaining a reliable and practicable isotopic neptunium tracer are overcome. An important asset of the SI setup is the feasibility of processing up to 100 g of solid substrates using a small-sized (ca. 2 mL) column with chemical yields of neptunium and plutonium being ≥79%. Analytical results of three certified/standard reference materials and two solid samples from intercomparison exercises are in good agreement with the reference values at the 0.05 significance level. The overall on-column separation can be completed within 3.5 h for 10 g of soil samples. Most importantly, the anion-exchange mini-column suffices to be reused up to 10-fold with satisfactory chemical yields (>70%), as demanded in environmental monitoring and emergency scenarios, making the proposed automated assembly well-suited for unattended and high-throughput analysis.     

Measurement of selected polybrominated diphenyl ethers, polybrominated and polychlorinated biphenyls, and organochlorine pesticides in human serum and milk using comprehensive two-dimensional gas chromatography isotope dilution time-of-flight mass spectrometry

A new method using comprehensive two-dimensional gas chromatography and isotope dilution time-of-flight mass spectrometry (GCxGC-IDTOFMS) for the simultaneous measurement of selected polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs), and brominated flame retardants is presented. In contrast to the reference methods based on classical GC/MS, a single injection of the extract containing all compounds of interest results in accurate identification and quantification. Using GCxGC ensures the chromatographic separation of most compounds, and TOFMS allows mass spectral deconvolution of coeluting compounds as well as the use of (13)C-labeled internal standards for quantification. Isotope ratio measurements of the most intense ions for both native and labels ensure the required specificity. The use of this new method with an automated sample preparation procedure developed at the Centers for Disease Control and Prevention (CDC) for the analysis of human serum and milk compared favorably to conventional isotope-dilution one-dimensional gas chromatography-high-resolution mass spectrometry (GC-IDHRMS) for the different human serum and milk pools tested. The instrumental detection limits ranged between 0.5 pg/microL and 10 pg/microL and the method detection limits ranged between 1 and 15 pg/microL (N = 59 analytes). The reproducibility of the method was almost as good as with GC-IDHRMS, the relative standard deviations ranging between 1 and 11% for OCPs measured in human serum. OCP, PBDE, and PCB levels measured using the two methods were highly correlated, and the deviations between the two methods were below 20% for most analytes with concentrations above 1 ng/g milk lipids.     

Combining drop-to-drop solvent microextraction with gas chromatography/mass spectrometry using electronic ionization and self-ion/molecule reaction method to determine methoxyacetophenone isomers in one drop of water

A novel analytical technique termed drop-to-drop solvent microextraction (DDSME) was developed to determine three methoxyacetophenone isomers in one drop of water, which were then detected by gas chromatography/mass spectrometry using electronic ionization mass spectrometry for quantification analysis and self-ion/molecule reaction/tandem mass spectrometry for isomer differentiation. The best optimum parameters for the DDSME technique were as follows: extraction time, 5 min; using toluene as the extraction solvent; volume of extraction solvent, 0.5 microL and no salt addition. The advantages of this method are rapidity, convenience, ease of operation, simplicity of the device, and extremely little solvent and sample consumption. The limit of detection (LOD) for this technique was 1 ng/mL. The relative standard deviation was less than 2.6% (n = 5). The linear range of the calibration curve of DDSME is from 0.01 to 5 microg/mL with correlation coefficient (r2) of >0.954. In the comparison of the LOD of DDSME with other sample pretreatment methods including liquid/liquid extraction (LLE), single-drop microextraction (SDME), solid-phase microextraction (SPME), and liquid-phase microextraction (LPME) using a dual gauge microsyringe with hollow fiber methods, this method shows much better in sensitivity than the LLE (25 ng/mL) and it is compatible with SDME (0.5 ng/mL), SPME (0.5 ng/mL), and LPME using a dual gauge microsyringe with a hollow fiber (1 ng/mL). However, DDSME was more convenient than the LPME using a dual gauge microsyringe with a hollow fiber method and much lower cost than the SPME technique.