	Quality and consistency in reagents is critical to successful drug discovery and development. When targeting a particular protein of interest, in vitro experiments should be performed with proteins of biological properties similar to those for in vivo tests. It is important that molecularity, purity, shape, and degree of heterogeneity remain the same when any alterations are made to the model protein or the formulation buffer. Multi-angle light scattering (MALS) combined with size-exclusion chromatography (SEC-MALS) is a very useful technique to monitor the solution properties of the protein as changes to reagents are made.

	As an example, crystallization studies typically have a higher rate of success when the proteins involved are simplified, for example, truncated or expressed in bacteria to minimize post-translational modifications. However, these forms may not be amenable to traditional characterization such as plate-based assays and certain biophysical techniques, where native protein is preferred. Affinity tags or changes to buffer excipients may also be required. All of these differences increase the likelihood of altering solution properties.

	The example in this note shows how light scattering data obtained with a DAWN® MALS detector and analyzed by ASTRA® software (both from Wyatt Technology, Santa Barbara, CA) were used to used to elucidate the solution properties of protein expressed from two different constructs. Construct A is the shorter of the two, designed for crystallization efforts.

	Presented with data from Figure 1, combined with knowledge that the enzyme showed activity and was the predominant species in mass spectrometry, one might not question the suitability of this reagent. SEC-MALS data (Figure 2), however, show that this enzyme is in fact quite heterogeneous.

	Construct B addressed this; it is the full-length enzyme. MALS data show that the protein is in its biologically active form (dimer)and is highly homogeneous. This information gave the project team confidence to move forward with this construct for crystallography, NMR, and high-throughput screening (HTS) efforts.

	This note graciously submitted by Mark Tardie and George Karam; Pfizer Central Research, Groton, CT.

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Articles

Medical/Biological

Ensuring Protein Regent Quality by SEC-MALS

Issue PDF

Click the title above to open the LCGC North America February 2017 Application Notebook Supplement, Vol 35 No s2, in an interactive PDF format.

Vol 35 No 2 LCGC North America February 2017 Application Notebook PDF

	Antibodies are Y-shaped proteins predominantly produced by plasma cells and used by the immune system to neutralize pathogens. Antibodies glycoproteins part of the immunoglobulin superfamily, constituting most of the gamma globulin fraction of the blood proteins. Although the general structure of all antibodies are very similar, a small region at the tip of the protein drastically varies, allowing millions of antibodies with slightly different tip structures to exist.

	The study of antibodies began in the late 1800s and has been continuing through today. Recently, there has been an increase of antibody research, causing a growing need for better antibody analysis. HPLC and Size Exclusion Chromatography (SEC) are ideal for determining molecular weight and size of the antibodies.

	Shodex introduces the LW-803 column with a durable silica based packing material for antibody SEC analysis. This column has an adopted pore size suitable for analyzing proteins with a molecular weight of several hundreds of thousands.It also corresponds to the USP L20, L33, and L59. Shodex LW-803 is appropriate for proteins and specifically antibody analysis, and has demonstrated the determination of molecular weight for seven proteins.

	The analysis of seven proteins (Thyroglobulin, ?-Globulin, BSA, Ovalbumin, Myoglobin, Ribonuclease A, and Aprotinin) was accomplished with Shodex PROTEIN LW-803 (8.0 mm ID ? 300 mm, 3 µm), a silica-based column for aqueous SEC antibody analysis. Column temperature was kept at room temperature and flow rate was 1.0 mL/min. Eluent conditions were 50 mM sodium phosphate buffer (pH 7.0) with 0.3 M NaCl. The HPLC system was coupled with a UV detector set to 280 nm.

	Seven proteins with molecular weights ranging from 6,500 to 670,000 Da were analyzed on the PROTEIN LW-803 (Figure 1). From the seven proteins, a calibration curve could be formed. From this curve, unknown antibody proteins can be analyzed to determine the molecular weight.

	Shodex PROTEIN LW-803, a Size Exclusion Chromatography (SEC) chromatography column is suitable for antibody protein molecular weight analysis.

	Column: Shodex PROTEIN LW-803 (8.0 mm I.D. ? 300 mm)
	Eluent: 50 mM Sodium phosphate buffer (pH 7.0) + 0.3 M NaCl
	Flow rate: 1.0 mL/min
	Detector: UV (280 nm)
	Column temp.: Room temperature


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A New Silica-Based Aqueous SEC Column for Analysis of Antibody Proteins

The C3 epimers of 25-hydroxyvitamin D have lower bioactivity than the primary metabolites and, unless they are chromatographically separated, can cause clinical vitamin D levels to be overestimated. Raptor FluoroPhenyl columns provide baseline resolution of all key compounds, and the method established here allows accurate quantification in a fast, 5-min analysis time (7-min total cycle time). This method is recommended for laboratories interested in reporting C3 epimer concentrations separately in order to obtain more accurate results for the clinical diagnosis of vitamin D status.

	Vitamin D analysis has increased dramatically in clinical practice, due to its association with multiple human diseases and the prevalence of vitamin D deficiency worldwide. Vitamin D exists in two forms, vitamin D2 and vitamin D3. These parent compounds undergo metabolism to form 25-hydroxyvitamin D2 [25(OH)D2] and 25-hydroxyvitamin D3 [25(OH)D3], which are used as biomarkers for the assessment of vitamin D status in patients. However, C3 epimeric forms of 25-hydroxyvitamin D-3-epi-25-hydroxyvitamin D2 [3-epi-25(OH)D2] and 3-epi-25-hydroxyvitamin D3 [3-epi-25(OH)D3]-have been identified and may contribute a large portion of the total 25-hydroxyvitamin D concentration, particularly in infant populations. Structurally, the C3 epimeric forms differ from the primary forms only in the configuration of the hydroxyl group in the third carbon position. Studies have shown that the C3 epimers of 25-hydroxyvitamin D have much lower bioactivity than the primary metabolites; therefore, specific quantitation of the C3 epimers is necessary for proper clinical assessment of vitamin D status. Since the C3 epimers are isobaric to the primary metabolites (Figure 1), chromatographic separation is necessary for accurate quantitation. In this study, the Raptor FluoroPhenyl column was used for the chromatographic separation of the primary 25-hydroxyvitamin metabolites from their C3 epimers. As established here, the chromatographic method was able to quickly and accurately quantify both C3 epimers of 25-hydroxyvitamin D2 and D3 and the primary metabolites in fortified serum.

Calibration Standards and Serum Control Samples

	Synthetic human serum [SeraFlx LCMSMS (Cerilliant)] was fortified with four analytes-25-hydroxyvitamin D2, 25-hydroxyvitamin D3, 3-epi-25-hydroxyvitamin D2, and 3-epi-25-hydroxyvitamin D3-to prepare calibration standards and QC samples. The concentrations of the calibration standards ranged from 1 to 100 ng/mL and the QC samples were prepared at 4, 20, and 80 ng/mL for all analytes. In addition, beagle serum was fortified with all four analytes at 8 ng/mL. All samples were subjected to the sample preparation procedure detailed below. All samples, including the beagle serum samples, were quantified using the calibration standards prepared in synthetic human serum.

	Serum (400 µL) was mixed with 15 µL of internal standard solution (1 µg/mL of d6-25-hydroxyvitamin D3 in methanol), 0.2 M ZnSO

 (400 µL), and methanol (800 µL) in a 4 mL glass vial. A 2 mL aliquot of hexane was added and the samples were mixed for 90 s. Then, the samples were centrifuged for 10 min at 4,300 rpm. The hexane layer was removed and evaporated to dryness under nitrogen at 55 °C. The dried extract was reconstituted with 100 µL of a water:methanol solution (50:50) and injected (10 µL) for analysis.

	LC analysis was performed on a Shimadzu Nexera XR LC coupled with a Sciex API 4000 mass spectrometer. Instrument conditions were as follows and analyte transitions are provided in Table I.

	It was determined that a 1/x weighted linear regression was the best fit for the standard curves of all four analytes (Figure 2). Standard deviations were ≤10% (except for the lowest concentration, which was ≤20%) and individual R-squared values ranged from 0.996 to 0.999. Examples of typical calibration curves are shown in Figure 2. The LOD for all compounds was 0.3 ng/mL, as estimated from the signal-to-noise value of the 1 ng/mL standard injection.

	Three independent analyses were conducted on multiple days and three replicates of the QC samples were included in each analysis. Quantitative analysis of three QC levels of fortified synthetic human serum samples showed acceptable method accuracy with percent recoveries within 10% of the nominal concentration for all QC levels. Individual %RSD values ranged from 0.9–6.6% and 2.2–5.3% for intraday and interday analyses, respectively, indicating that an acceptable level of method precision was obtained (summary data presented in Table II). Accurate quantitative analysis was possible because the Raptor FluoroPhenyl column provided baseline resolution of both C3 epimers of 25-hydroxyvitamin D from the primary metabolites (Figure 3). These compounds cannot be separated on standard C18 columns, but the unique selectivity of the Raptor FluoroPhenyl column allowed complete separation in a fast, 5-min analysis time (7 min total cycle time).

	Once validated, the method was used to analyze the blank and 8 ng/mL fortified beagle serum samples (Figures 4 and 5). As shown in Table III, acceptable accuracy and precision results were achieved. For most compounds, the average percent recoveries of three sets of analyses were within 5% and the %RSD values ranged from 2.3–2.9%. However, because the beagle serum contained a high endogenous concentration of 25-hydroxyvitamin D3, accuracy and precision for this compound could not be assessed at the 8 ng/mL fortification level.

	The unique selectivity of the Raptor FluoroPhenyl column allows fast quantitative analysis of the C3 epimers of 25-hydroxyvitamin D and the primary metabolites in serum. The analytical method established here is applicable to the clinical analysis of total 25-hydroxyvitamin D concentration. Because all key compounds are chromatographically separated, this method provides analytical labs with the option to report C3 epimer concentrations separately, providing more accurate results for the clinical diagnosis of vitamin D status.


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	tel. (800)356-1688, fax (814) 353-1300
	Website: 

Rapid Analysis of C3 Epimers of 25-Hydroxyvitamin D in Serum by LC–MS/MS

	Poly(lactic-co-glycolic acid) (PLGA) is a copolymer based on glycolic acid and lactic acid. The two monomer units are linked together by ester linkages and form linear polyester chains. The final product is biodegradable and biocompatible, and is approved by the U.S. Food and Drug Administration (FDA) for production of various therapeutic devices as well as for drug delivery applications. The properties of PLGA can be tuned by the ratio of the two monomers and by the overall molar mass distribution.

	The characterization of PLGA by means of conventional size exclusion chromatography (SEC) is problematic because of the lack of suitable calibration standards. In addition, the linear polyester structure can be modified by the addition of small amounts of polyfunctional monomer to obtain branched chains of differing degrees of branching. The degree of branching becomes an additional parameter that can be used to adjust PLGA properties-all of which renders conventional column calibration an inadequate analytical technique.

	In this application note, two commercially available samples were analyzed in the ASTRA

 software by SEC coupled to a Multi-Angle Light Scattering (MALS) detector (DAWN

), and a differential viscosity detector (ViscoStar

). The MALS and RI signals were combined to determine absolute molecular weight, independently of elution time or column calibration. The ViscoStar was used in order to measure intrinsic viscosity (IV) and uncover additional information about the molecular structure of the analyzed polymers. In addition to molar mass distributions, the SEC-MALS-IV system yields the relationship between intrinsic viscosity and molar mass (Mark-Houwink-Sakurada plot) that can provide deep insight into the molecular structure of the polymers being analyzed.

	In Figure 1, the molar mass distributions are given as differential distribution plots. As seen from the plots, the two samples span markedly different molar mass ranges. The Mark-Houwink-Sakurada plots of the two samples are shown in Figure 2 together with the plot of linear polystyrene that is shown simply for the sake of comparison. The slope of the Mark-Houwink-Sakurada plot of the linear polystyrene is 0.71, a typical value for linear random coils in thermodynamically good solvents. The slope of the red sample roughly corresponds to a linear structure as well. However, there is a slight indication of deviation from linearity at the region of high molar masses that may indicate the presence of branched molecules. The Mark-Houwink-Sakurada plot of the blue sample is curved. Curvature of the Mark-Houwink-Sakurada plot generally reveals branching. In addition, the slope of the higher molar mass portion of the Mark-Houwink-Sakurada plot of 0.48 suggests significant branching.

	SEC-MALS-IV is an excellent method for the characterization of PLGA polyesters as it has the ability to determine not only the absolute molar mass distribution, but to reveal subtle differences in PLGA's molecular structure.


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	tel. +1 (805) 681-9009
	Website: 

Characterization of PLGA Using SEC-MALS-IV

	Organic arsenic compounds added to feed stocks of chicken and poultry pose serious environmental and ecological threats. National and worldwide health organizations, such as the U.S. Environmental Protection Agency and the U.S. Food and Drug Administration, have recently implemented more stringent concentration limits for arsenic species in drinking water and foodstuffs.

	Analysis of arsenic species is made challenging due to diverse sample matrices. To circumvent these problems, an HPLC ICP-MS method has been developed. Resolution of arsenic compounds is achieved by ion exchange chromatography on a Hamilton PRP-X100 column, a 55% cross-linked polystyrene-divinylbenzene copolymer functionalized with quaternary ammonium anion-exchanger group. Detection is accomplished by inductively coupled plasma-mass spectrometry.


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	tel. (775) 858-3000, (800) 648-5950
	Website: 

Food & Beverage

Arsenic Speciation on Hamilton PRP-X100

	This application note outlines a simple, fast, and cost-effective QuEChERS-based method for the determination of limonin in citrus juice. Limonin is extracted from a variety of juice samples using acetonitrile and citrate-buffered salts. The sample extract undergoes cleanup by dispersive-SPE (dSPE) using primary-secondary amine (PSA), C18, and graphitized carbon black (GCB) to remove unwanted matrix components, including sugars, acids, and pigments, and yield a clear sample extract. Analysis is performed by LC–MS/MS using a Selectra® C18 HPLC column (although HPLC-UV can also be used).

	a) Allow citrus juice to come to room temperature and thoroughly mix the sample before analysis.

	b) Transfer 10 mL of juice into a 50 mL polypropylene centrifuge tube.

 Mylar pouch and vortex or shake (manually or mechanically) for 5 min.

		For this work a SPEX® SamplePrep® 2010 Geno/Grinder® was used (1500 rpm).

	e) Centrifuge the samples at ≥3000 rcf for 5 min.

	c) Centrifuge the samples at ≥3000 rcf for 5 min.

	d) Transfer 500-600 µL of purified supernatant into an autosampler vial for analysis.

	Samples were fortified with limonin at 10 µg/mL (50 µL of a 2 mg/mL acetonitrile standard). A six-point matrix-matched calibration curves were prepared at 1, 2, 5, 10, 15, and 20 µg/mL. Non-fortified juice samples were extracted according to the QuEChERS procedure described above and 1 mL of purified extracts were fortified with 5, 10, 25, 50, 75, and 100 µL of a 200 µg/mL acetonitrile standard.

	* One outlier was omitted from the final results.

	This application note outlines a simple, fast, and cost-effective QuEChERS-based method for the determination of limonin in citrus juice. Citrus juice samples are extracted with citrate-buffered QuEChERS salts followed by dSPE cleanup of the supernatant using PSA, C18, and GCB, yielding a clear extract. Analysis is performed by LC–MS/MS using a Selectra® C18 HPLC column. Various citrus juices (lemon, lime, orange, and grapefruit) were evaluated for recovery and repeatability using fortified samples. Excellent recovery (88 to 97%) and repeatability (≤ 7.5%) were obtained using the outlined method.


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Analysis of Limonin in Citrus Juice Using QuEChERS and LC–MS/MS

-resveratrol in wine is complicated by two factors: relatively low levels and interferences from matrix components. Here, we present two useful approaches to overcoming these issues depending on the instrumentation available. For high performance liquid chromatography (HPLC) analyses with ultraviolet (UV) detection, matrix peaks can be removed by microextraction using a packed sorbent while simultaneously concentrating the 

-resveratrol peak by a factor of two. For liquid chromatography–mass spectrometry (LC–MS), the extracted ion chromatogram for the [M + H]

 analyte ion can be used to obtain specificity without prior extraction procedures.

	Resveratrol is a naturally occurring stilbenoid compound found in certain plant components, such as grape skins, peanuts, and berries. Although the practical extent of its therapeutic effects is debated, resveratrol has been variously reported to exhibit cardio-protective (1), anticancer (2), and neuroprotective (3) properties. A common point of contention with findings regarding the purported health benefits of resveratrol is that many such studies are performed 

 or with animal subjects (4). Hence, the conclusions may not necessarily translate to 

 human clinical trials, which are still relatively scarce at present (5). For example, the low bioavailability of resveratrol may not allow for the relatively high concentrations used to achieve significant efficacy in some 

	Nevertheless, the amount of resveratrol in food and beverage products is of ongoing interest to producers and consumers. Red wine, a common food source of resveratrol, is therefore pertinent to analytical study in furtherance to these investigations. The content of resveratrol can vary widely among red wine samples depending on factors such as region of cultivation and grape variety, but reported values have ranged up to 14.3 mg/L (7). With ultraviolet (UV) detection in high performance liquid chromatography (HPLC) analyses, the lower levels found in some samples may be somewhat difficult to adequately quantitate. Furthermore, peaks from other components in the wine extracts may interfere with resveratrol detection. Liquid chromatography coupled to mass spectrometry (LC–MS) has been shown to be a powerful tool capable of detecting 

resveratrol down to 0.3 pmol (8) and can also differentiate based on the mass-to-charge ratio (

), but many laboratories may lack this more sophisticated instrumentation. For analyses with UV-based instruments, a sample cleanup strategy is generally required before HPLC analysis.

	In recent years, miniaturized solid-phase extraction (SPE) techniques have become an advantageous alternative to traditional sample cleanup or preconcentration procedures (9). Using conventional liquid–liquid extraction (LLE), relatively large amounts of solvent are typically required for a single sample, which increases cost for both solvent usage and disposal. The process is also not amenable to automation, taking up valuable analyst time. SPE greatly reduces solvent consumption compared to LLE, but can be attenuated further with more modern microextraction techniques. In addition to solvent savings, miniaturized SPE approaches require very low amounts of sample (on the order of microliters versus milliliters for SPE), which can be beneficial if the sample is expensive or scarce.

	Microextraction by packed sorbent can be thought of as a type of miniaturized SPE technique where the solid phase is a small packed bed (~1–2 mg) incorporated into the barrel of a pipet needle. Carryover from previous samples using the same cartridge can be reduced or eliminated compared to traditional SPE because of the smaller sorbent bed and lower wash volumes involved (10). The process is also generally faster and more readily automated than SPE or LLE, since the packed sorbent bed is part of the syringe (11).

	The extraction technique entails the same general principles as chromatography, with a few notable caveats. Because the process must be quantitative to be of use, extremes in retention or elution behavior are preferable. A sample is loaded onto the cartridge under strongly retaining conditions such that the analyte of interest will be quantitatively adsorbed onto the sorbent. Strongly eluting conditions are used to then completely remove the analyte from the packed bed and into the sample vial for analysis. Since the sorbent is smaller in microextraction by packed sorbent than in traditional SPE, greater attention must be paid to ensure the analyte loading capacity of the packed bed is not exceeded.

	There are two primary reasons to incorporate a microextraction by packed sorbent procedure into sample preparation. First, compounds that are not retained on the sorbent are removed from the final sample, which might otherwise show up as interfering peaks in the subsequent HPLC chromatograms. Hence, the technique can improve the specificity of the analysis. Second, if the volume of liquid used in the elution step is lower than that of the loading step, an increase in analyte concentration is obtained. This can be performed by using one large volume in the loading step or through a series of extract–discard cycles of equal volume to the elution step. If an analyte is generally found in low levels in a particular sample, this microextraction by packed sorbent preconcentration technique may be an effective strategy to adequately detect and quantitate it. The resulting concentration increase is then accounted for in the analytical calculations, allowing for the quantitation of analytes present at levels below the linear range of the calibration curve.

	In the present work, microextraction by packed sorbent approaches were investigated as a means to increase the concentration of resveratrol in red wine samples while simultaneously removing undesirable compounds from the extracts. A silica hydride–based C18 material was used as the sorbent in the cartridges and compared to an analogous C18 phase based on conventional silica. To our knowledge, this is the first instance of silica hydride sorbents used for a microextraction sample preconcentration application. Previous work on the utility of these materials as analytical HPLC columns in food and beverage analyses has been demonstrated for limonin in orange juice (12) and histamine in foodstuffs (13). Silica hydride–based sorbents have also been used in traditional SPE in an application analyzing components of dark chocolate (14).

	One notable study (15) reported the use of microextraction by packed sorbent in analysis of 

resveratrol in wine samples, but in that case a preconcentration factor of the extract was not used in the final method. Preconcentration had been investigated through the use of multiple extract–discard cycles of sample aliquots on the cartridges, and increases in peak areas were observed, but the results were not quantitative (for example, the use of five extraction cycles in the microextraction by packed sorbent preconcentration step in sample preparation yielded significantly less than a fivefold increase in peak area in the HPLC data, relative to that of the single extraction cycle). If the increase in concentration is to be accurately accounted for in the calculations, the peak area must be directly proportional to the preconcentration factor. Hence, another aspect of this study was to investigate the preconcentration aspect of the microextraction by packed sorbent technique more thoroughly.

	Because of the inherent sensitivity and specificity capabilities of LC–MS, its use was investigated for analysis of 

-resveratrol in red wine as an alternative strategy to the microextraction by packed sorbent approach. This may be pertinent to researchers investigating resveratrol at very low levels or in more complex samples such as plasma extracts. Analysts can isolate the extracted ion chromatogram (EIC) corresponding to the [M + H]+ ion for resveratrol, leading to superior specificity compared to UV-based analyses. As such, extraction-based sample cleanup procedures may be unnecessary with the use of this more sophisticated instrumentation.

	Resveratrol capsules and red wine (Merlot, 2012, cultivated in California) were purchased from local supermarkets. Formic acid and a 

-resveratrol reference standard were from Sigma–Aldrich. Acetonitrile, isopropanol, methanol, and deionized water (HPLC grades) were obtained from GFS Chemicals, Inc.

	A Hewlett-Packard 1100 HPLC system consisting of an autosampler, degasser, binary pump, and variable wavelength UV detector (304 nm) was used. The system was interfaced with Agilent Chemstation software and a Syscon REX-C100 air temperature controller (RKC) with a temperature maintained at 25 °C. The 50 mm × 2.1 mm analytical column was packed with a Bidentate C18 stationary phase (BDC18, MicroSolv Tech. Corp.), which had a particle size of 4 µm and a pore size of 100 Å. The final mobile phase obtained after method development used a gradient: Solvent A was premixed 90% deionized water–10% acetonitrile–0.1% formic acid (v/v) and solvent B was acetonitrile with 0.1% formic acid (v/v). The gradient program held at 100% A for 2 min, decreased linearly to 50% A in 7 min, and increased linearly back to 100% A in 1 min. The post time was 5 min. The flow rate was 0.3 mL/min and the injection volume was 2.0 µL.

	An Agilent 1200SL Series LC system, including degasser, binary pump, temperature-controlled autosampler, and temperature-controlled column compartment was used. The mass spectrometer system was an Agilent Model 6220 MSD TOF with a dual sprayer electrospray source (ESI). The 75 mm × 4.6 mm analytical column was packed with a BDC18 stationary phase that had a particle diameter of 4 µm and a pore size of 100 Å. The final mobile phase obtained after method development used a gradient: Solvent A was deionized water with 0.1% formic acid (v/v) and solvent B was acetonitrile with 0.1% formic acid (v/v). The gradient program held at 90% A for 2 min, decreased linearly to 40% A in 16 min, and increased linearly back to 90% A in 2 min. The flow rate was 0.4 mL/min and the injection volume was 1.0 µL. Using the positive ionization mode, 

-resveratrol was detected as the [M + H]+ ion (

-resveratrol was prepared by weighing 50.0 mg of the reference standard on a Sartorius Handy H51 analytical balance. The material was quantitatively transferred to a 50-mL amber volumetric flask containing a portion of 50% deionized water–50% acetonitrile. It was sonicated for 10 min using a VWR Symphony ultrasonic bath and then diluted to mark. After mixing, aliquots were transferred to amber volumetric flasks using a calibrated SGE eVol automatic delivery pipet and diluted to mark to obtain concentrations of 0.5, 1.0, 2.0, 5.0, and 10.0 ppm with a diluent of 95% deionized water–5% acetonitrile. Aliquots of the standard solutions were then transferred to 2 mL amber glass autosampler vials (MicroSolv Tech. Corp.). These solutions were then used to produce calibration curve data from HPLC–UV analyses.

	The contents of a capsule containing 100 mg of 

-resveratrol were transferred to a 100-mL amber volumetric flask, and a portion of 50% deionized water–50% acetonitrile diluent was added. The flask was sonicated for 10 min and then diluted to the mark. After mixing, a portion was filtered with a 0.22-µm nylon syringe filter (MicroSolv Tech. Corp.). The theoretical concentration of 

-resveratrol in the stock solution filtrate was 1000 ppm. A 1.00-mL aliquot of the filtrate was transferred to a 100-mL amber volumetric flask and was then diluted to the mark with 95% deionized water–5% acetonitrile and mixed thoroughly. This diluted solution had a 

	A syringe needle with a C18 sorbent cartridge, either silica hydride based (50 µm, 60 Å, synthesized in house) or type B silica based (45 µm, 60 Å), was attached to the automatic delivery pipet described and conditioned with 100 µL of isopropanol, 100 µL of methanol (two times), and then 100 µL of deionized water. A 100-µL aliquot of the 10.0 ppm 

-resveratrol solution was loaded onto the sorbent and the liquid was discarded. The extract–discard step was repeated zero to three times depending on the preconcentration factor used (see Table I for total aliquot load volumes, final concentrations, and analyte mass load). The sample was eluted from the sorbent into a glass amber autosampler vial (with a glass small volume insert) using a 100-µL aliquot of methanol in each case. The samples were analyzed by HPLC–UV and the peak areas were compared to the calibration curve linear model to obtain percent recovery values.

	A portion of red wine was filtered with a 0.22-µm nylon syringe filter. A syringe needle with a C18 sorbent cartridge (either silica hydride based or type B silica based) was attached to the automatic delivery pipet described previously and was conditioned with 100 µL of isopropanol, 100 µL of methanol (two times), and then 100 µL of deionized water. A 100-µL aliquot of the filtrate was loaded onto the sorbent and the liquid was discarded. The process was repeated for a twofold increase in concentration. The sample was eluted from the sorbent into a glass amber autosampler vial (with a glass small volume insert) using a 100-µL aliquot of methanol. This sample was then analyzed by HPLC–UV and the peak area was compared to the calibration curve line of best fit to obtain an estimate of concentration and then divided by two to take into account the 2× factor from the preconcentration.

-resveratrol stock solution, 1.00 mg of resveratrol was dissolved in 1.00 mL of a solution containing 50% deionized water–50% acetonitrile–0.1% formic acid. The sample for LC–MS analysis was diluted 1:100 with 50% deionized water–50% acetonitrile–0.1% formic acid (v/v).

	A red wine sample was prepared by filtering through a 0.45-µm nylon filter (MicroSolv Technology Corp.) and diluting 1:5 with 50% deionized water–50% acetonitrile–0.1% formic acid (v/v). This sample was analyzed by LC–MS as well as a red wine sample spiked with the reference standard solution.

	It was important to use amber flasks and vials in this investigation because 

-resveratrol has been reported to degrade upon exposure to sunlight (16). The formation of a degradant means that some of the 

-resveratrol has been used up, skewing quantitative accuracy. Indeed, the effect of amber versus clear vials was studied during method development and degradation was attenuated using the former. Figure 1 shows inter-day overlays of the same 10 ppm 

-resveratrol standard solution, with one portion stored in an amber autosampler vial (Figure 1a) and another in a clear autosampler vial (Figure 1b). A second peak began to appear in both samples, but the amber vial showed less degradation in the studied timescale. The extra peak was significantly more prominent using the clear vial after three days.

	Using reference standard solutions, a calibration curve for 

-resveratrol could be constructed on which to base subsequent percent recovery and sample quantitation studies. To optimize sensitivity in terms of the detector, 

-resveratrol's maximum absorption wavelength of 304 nm was chosen. The data showed good linearity in the studied range of 0.50–10.0 ppm 

 = 0.99992), with a line of best fit equation 

 – 2.12. Estimates for limit of detection (LOD) and limit of quantitation (LOQ) were also obtained using the signal-to-noise ratio (S/N) of the analyte peak from the 0.5 ppm calibration curve solution (S/N = 91.3). With S/N definitions of 3 for LOD and 10 for LOQ, the values were then estimated at 0.017 ppm and 0.055 ppm, respectively.

	To ensure quantitative recovery in the microextraction by packed sorbent samples, various aspects of the technique should be considered and optimized. The loading solvent is an example of one such parameter. Because the retention of resveratrol on the sorbent proceeds via a reversed-phase mechanism, the water content should ideally be as high as possible. In practice, the low solubility of resveratrol in water (24) will require that some acetonitrile be present to allow for complete dissolution. As such, a diluent of 95% deionized water–5% acetonitrile was chosen. If the water content is too low, quantitative adsorption of resveratrol onto the sorbent may not be achieved, leading to lower recovery.

	The choice of the eluting solvent is equally important to ensure that the analyte is quantitatively desorbed from the cartridge and into the autosampler vial. For this reason, the composition of the eluting solvent should consist of at least 60% strong solvent and ideally 100% (25). In reversed-phase, the strong solvent is generally acetonitrile or methanol and the weak solvent is water. Various eluting solvents were studied (see Table II), and the results were consistent with theory. The 100% deionized water solvent showed the lowest recovery (3.6%) since 

-resveratrol is strongly retained on the cartridge under these conditions. When 15% acetonitrile was present, a significant increase to 55.0% recovery was observed. The use of 50% acetonitrile or greater resulted in nearly complete recovery (94.6%), since these are strongly eluting conditions in reversed phase. Of all the solvents investigated, neat methanol produced the highest recovery at 99.8%. Neat acetonitrile was investigated as well, but it resulted in a distortion of peak shape in the HPLC chromatograms on some occasions (data not shown).

	A review of the microextraction by packed sorbent technique (17) recommends a washing step between the loading and elution steps to help remove weakly adsorbed sample contaminants, with a highly or completely aqueous solvent being the ideal choice for reversed phase applications. However, this step was omitted in the present investigation for two reasons. First, the sample diluent was 95% aqueous with standard solutions or 100% aqueous with wine samples, so aspirating and discarding an extra aliquot of water was deemed unnecessary; removal of contaminants was already accomplished by discarding the sample aliquot. Second, the data from Table II described earlier demonstrated that 3.6% of the 

-resveratrol was leached from the sorbent using deionized water as the eluting solvent. Hence, a washing step would be expected to lower analyte recovery and was therefore detrimental to accurate quantitation.

	Resveratrol is a moderately hydrophobic compound. As such, it can be retained in microextraction by packed sorbent with a C18 sorbent and reversed-phase conditions. An experimental silica hydride–based C18 material was compared to a C18 sorbent based on conventional type B silica. Silica hydride is a unique material that is virtually free of residual silanols (>95% of Si–OH moieties are replaced by Si–H groups) (18). These silanol groups are known to produce undesirable effects in HPLC and potentially in microextraction by packed sorbent as well. The Si–H group is also relatively nonpolar, which may impart differences in hydrophobicity among the two materials. These types of differences could translate into advantages in extraction efficiency or ability to remove sample contaminants. Subsequent studies comparing the two sorbents could determine if either of these were the case.

	Because the microextraction by packed sorbent cartridge is smaller than the cartridge in an SPE format, the possibility of sample overload should be considered more carefully. If the loading capacity of the sorbent is exceeded, the process may no longer be quantitative, which would defeat the purpose of the technique. Percent recovery studies at increasing concentration factors can help empirically determine whether the technique is quantitative at a given concentration factor. To this end, various 

-resveratrol preconcentration samples were investigated (1–4×) using both sorbents and the percent recoveries were calculated and compared (see Figure 2). The preconcentration can be accomplished by multiple sample load–discard cycles of a given volume followed by an elution of the same volume. The analyst then records the resulting peak areas at each preconcentration level and calculates the percent recoveries. The data demonstrated that only the 2× concentration increase was quantitative while unacceptable deviation from linearity occurred at higher preconcentration levels. In comparing the two types of C18 sorbents, both performed well up to 2×, but neither was able to provide accurate results at the 3× or 4× level. The results suggest that, among other possible explanations, there may be a cumulative effect of a small amount of 

-resveratrol leaching from the sorbent bed in multiple extractions, along the same lines as discussed earlier in Table II. The effect would become more pronounced with increasing numbers of extract–discard cycles, leading to more significant deviation from ideal recovery. This data may also help explain the findings of Gonçalves and Câmara (15), who observed nonquantitative increases in peak area using five and 10 extraction cycles relative to one cycle. For this reason, only the 2× preconcentration factor was used with the subsequent red wine sample.

	Thus far, the performances of the two sorbents were mostly comparable, with the silica hydride-based sorbent providing slightly higher recovery. However, in the red wine extracts, significant advantages were observed using the silica hydride–based C18 sorbent compared to both the C18 sorbent based on conventional silica and a filtration-only protocol (

 without microextraction by packed sorbent). Figure 3 compares the chromatograms that were obtained in each case. In Figure 3a, many peaks are present in the wine sample, especially near the solvent front and in the region where 

-resveratrol is eluted. Because of coelution phenomena from these latter interferences, no 

-resveratrol peak was detected. Use of a type B silica–based C18 sorbent was effective in removing some of these peaks (Figure 3b), but still no 

-resveratrol peak was observed. Only with the use of the silica hydride–based sorbent (Figure 3c) were enough matrix interferences removed from the extract to detect the analyte peak.

-resveratrol from the 2× red wine microextraction by packed sorbent extract using the C18 silica hydride sorbent could then be correlated with the calibration curve to obtain a concentration of 0.64 ppm in the final sample. Accounting for the twofold increase in concentration via microextraction by packed sorbent, the original concentration of 

-resveratrol in the wine sample was then calculated to be 0.32 ppm. Hence, even though the calibration curve only went down to 0.50 ppm, the analyte could still be made to fall within the studied linear range using the preconcentration strategy. This concentration is within the general range of 

-resveratrol in red wine samples reported by Stervbo and colleagues (7).

	For investigations requiring more sophisticated detection methods, LC–MS was also studied in its application in resveratrol analysis. Figure 4 shows unspiked and spiked red wine extracts using LC–MS without microextraction by packed sorbent treatment. Because of the specificity of MS detection through the use of EICs, a 

-resveratrol peak could be detected using only filtration and dilution in the sample preparation (Figure 4a), a feat which could not be achieved in the HPLC–UV investigations. The spiked sample showed that the correct peak assignment was made (Figure 4b). The data demonstrate that LC–MS may be an alternative approach to microextraction by packed sorbent in addressing the issue of interfering matrix components in red wine samples.

	Using quantitative preconcentration microextraction by packed sorbent approaches with a silica hydride–based C18 sorbent, 

-resveratrol could be adequately detected and correlated to a linear calibration curve model. The same technique was ineffective when a C18 sorbent based on conventional silica was studied because of the insufficient removal of matrix peaks that were coeluted with 

-resveratrol. Likewise, only using filtration without microextraction by packed sorbent was also shown to be impractical with UV detection. However, 

-resveratrol was detected with LC–MS without microextraction by packed sorbent because of the added specificity and sensitivity afforded by MS. Hence, two viable approaches can be used in the analysis of 

	The authors would like to acknowledge Microsolv Technology Corporation for donation of the analytical columns used in this study and SGE Analytical for preparing the microextraction by packed sorbent cartridges.

	(2) M. Jang, L. Cai, G.O. Udeani, K.V. Slowing, C.F. Thomas, W.W. Beecher, H.H.S. Fong, N.R. Farnsworth, A.D. Kinghorn, R.G. Mehta, R.C. Moon, and J.M. Pezzuto, 

	(3) E. Tellone, A. Galtieri, A. Russo, B. Giardina, and S. Ficarra, 

	(4) J. Tomé-Carneiro, M. Larrosa, A. González-Sarrías, F.A. Tomás-Barberán, M.T. García-Conesa, and J.C. Espín, 

	(5) C. Andres-Lacueva, M. Urpi-Sarda, R. Zamora-Ros, and R.M. Lamuela-Raventos, in 

Plant Phenolics and Human Health: Biochemistry, Nutrition, and Pharmacology,

 C.G. Fraga, Ed. (John Wiley & Sons, New York, 2010), chapter 13, pp. 265–297.

	(7) U. Stervbo, O. Vang, and C. Bonnesen, 

	(8) L. Mark, M.S.P. Nikfardjam, P. Avar, and R. Ohmacht, 

	(9) C. Silva, C. Cavaco, R. Perestrelo, J. Pereira, and J.S. Câmara, 

	(10) E. Candish, A. Gooley, H.-J. Wirth, P.A. Dawes, R.A. Shellie, and E.F. Hilder, 

	(12) J.E. Young, J.J. Pesek, and M.T. Matyska, 

	(13) A. Dang, J.J. Pesek, and M.T. Matyska, 

	(14) Y. Nolvachai, C. Kulsing, R.I. Boysen, M.T. Matyska, J.J. Pesek, P.J. Marriott, and M.T.W. Hearn, 

	(16) T.S. Figueiras, M.T. Neves-Petersen, and S.B. Petersen,

 is with MicroSolv Technology Corporation in Eatontown, New Jersey. 

Michael V. Lim, Joshua Topete, Hao Hang, Marion Gahol, Joseph J. Pesek, 

are with San José State University in San José, California. Direct correspondence to: 

Improved Sensitivity and Specificity for trans-Resveratrol in Red Wine Analysis with HPLC–UV and LC–MS

A novel method for the retention and quantification of polar neurotransmitters is presented. A reversed phase method using Scherzo SS-C18 column is compared to a HILIC method using a Polyhydroxyethyl column. 

	When analyzing highly polar compounds by reversed phase HPLC there exist several challenges, the most obvious of which is that polar compounds are difficult to retain using non-polar stationary phases like C18. A common solution is the use of aqueous normal phase or HILIC methods with stationary phases that can be wetted and that retain this aqueous layer on the surface of the particle. The difficulty in using HILIC methods is that the wetting process can often be quite lengthy, and maintaining a consistent aqueous surface is difficult.

	In the example shown here we seek to provide an alternative to HILIC methods by using a novel multi-mode column called the Scherzo SS-C18. This stationary phase has both reversed-phase (C18) and polar components with the addition of a high density of strong anion and cation exchange ligands. This strategy for increasing polar retention is much more predictable run to run and does not require lengthy column pre-treatments.

	MRM transitions and running conditions are within chromatograms.

	Experimental data courtesy of Dr. D. Koop, Oregon Health and Sciences University, Portland, OR.

	Chromatograms of the neurotransmitter acetylcholine using Scherzo SS-C18 and HILIC columns are shown in Figure 1 and Figure 2, respectively.

	Both columns shown in Figure 1 and in Figure 2 retain the neurotransmitter acetylcholine well. Two instant observations are peak shape and detection limits. In particular, despite longer retention (~ 20% more), the peak shape on Scherzo SS-C18 is better as evident by peak width at half height which is about 50% of the HILIC column. This observation indicates overall significantly better performance of a Scherzo SS-C18 column and higher peak capacity that leads to better separations. This also contributes to a roughly 2.5X increase in sensitivity with the Scherzo SS-C18 column compared to the HILIC column (125 pg vs. 300 pg).

	At approximately 3 min of retention, both methods can be justified for high throughout applications, however, considering the long and kinetically-slow conditioning on the HILIC column, we feel that Scherzo SS C-18 is a better choice. In contrast, the HILIC column, while it has comparable run times, requires over 2.5 h conditioning in this application, practically disqualifying this HILIC column for high throughput use.

	The multi-mode Scherzo SS-C18 column shows great promise in successful retention of small, polar molecules (e.g., neurotransmitters). The column retains these types of analytes as well or better than a HILIC column, it has excellent sensitivity, eluents are MS-friendly, and, most importantly, it does not need lengthy conditioning. This feature alone significantly reduces overall separation times and operation costs, which potentially makes this column an attractive alternative for separation of polar analytes.


	1104 NW Overton St., Portland, OR 97209
	tel. (888) 456-HPLC, (215) 665-8902, fax (501) 646-3497
	Website: 

General

Comparison of Multi-Mode Scherzo SS-C18 and HILIC Mode Column in the Retention

	Organochlorine pesticides are among the more notorious organic pollutants. Gaining wide spread attention into the latter part of the 20th century, and ultimately leading to their ban, OCPs remain with us as a legacy contaminant in the environment. In water samples, OCPs are monitored by the U.S. Environmental Protection Agency by various methods including methods EPA 508, EPA 608, and EPA 8081.

	The use of solid phase extraction (SPE) can rapidly increase both extraction efficiency and reduce lab solvent usage. By implementing the FMS TurboTrace® ABN system with its dual cartridge functionality, an in-line florisil cartridge can be added to perform automated extract clean-up during the elution step.

		n-hexane, pesticide grade or equivalent

		Anhydrous sodium sulfate, ACS grade or equivalent

 SPE cartridge with 20 mL 10% acetone in hexane

	2. Condition C18 SPE cartridge with 10 mL acetone

	3. Condition C18 SPE cartridges with 20 mL H

	4. Load samples across C18 SPE cartridges via vacuum

	6. Automatic solvent rinse of sample bottle with 10% acetone in hexane

	7. Bottle rinse loaded across both cartridges and collected in SuperVap concentrator

	8. Additional 10 mL elution solvent passed C18 cartridges

	9. 10 mL elution solvent passed across Florisil cartridge and nitrogen purged to SuperVap

	2. Evap mode: 10 psi nitrogen with sensor

	Results of IPR study performed on Turbo Trace ABN system with in-line Florisil.

	Extraction of the water samples performed on the TurboTrace ABN system enabled a fully automated extraction process using its dual cartridge capabilities. Samples were loaded across the C18 cartridges, then eluted through the Florisil cartridges in succession. The addition of Na

 to the clean-up cartridge enabled residual water removal from the extract without a separate, manual step. Analytical results yielded spiked recoveries of Organochlorine pesticides to all be between 70–130%, with low RSDs.


	580 Pleasant Street, Watertown, MA 02472
	tel. (617) 393-2396, fax (617) 393-0194
	Website: 

Environmental

Automated Extraction Of Organochlorine Pesticides with In-Line Florisil Cleanup

	Perfluorinated chemicals (PFCs), also known as PFAAs, can be found in commercial and industrial uses such as firefighting foams, package material coatings, nonstick cookware, waterproofing, and stain-proof fabrics. PFCs are all manmade and have unique properties such as repelling water, oil, and heat. They are very difficult to break down and persist in the environment. Small amounts of PFCs can dissolve in water and is a cause for concern for human exposure, especially in drinking water sources such as large public water systems and private wells. A recent health advisory has been issued by U.S. Environmental Protection Agency (EPA). Water utilities should notify customers if greater than 70 ppt (0.07 µg/L) PFOS or PFOA or a total for the two combined are detected in the water supply.

	The United States is not the only country concerned about exposure to PFCs. In Europe, the twelfth meeting of the United Nations Persistent Organic Pollutants Review Committee was held in Rome in September 2016 to move the consideration of PFCs forward for further regulatory consideration. Rules developed under this framework will have global impact. This work will demonstrate the capability of the SmartPrep® Cartridge Extraction II System for the extraction of six PFCs in compliance with US EPA 537 or ISO 25101:2009 (1).

	Solid phase extraction (SPE) is used to remove and concentrate the PFCs from water samples. In U.S. EPA method 537, a 0.5 g, 6-mL SPE cartridge containing styrene divinylbenzene (SDVB) sorbent phase is specified. The analysis is done using HPLC–MS/MS for a sensitive detection step.

	After demonstrating that the system does not contribute background to the extraction step, the Method Detection Limits (MDL) were determined from seven samples run on the SmartPrep over a three day period. The MDL values for this method with this sample size and equipment are extremely low (Table I). They will be able to measure water samples at the U.S. Health Advisory level of 0.07 µg/L for PFOS or PFOA with MDLs of 0.004 and 0.002 respectively. The limit of quantitation (LOQ) is 0.013 µg/L for PFOS and 0.0076 µg/L for PFOA, which is acceptable for very low level determinations.

	PFC compounds are becoming an increasingly important concern as research into health effects uncovers other related illnesses. As countries around the world move to regulate exposure through drinking water, reliable, and accurate measurement methods become critical.

	Methods US EPA 537 and ISO 25101:2009 specify extraction and concentration with SPE and analysis using HPLC–MS/MS. Automation of the cartridge SPE step is important to ensure the best reproducibility with the least chance of contamination. It also ensures the efficient use of technician time through walk-away operation.

	(1) The Extraction of Perfluorinated Alkyl Acids (PFAAs) in Drinking Water Using Automated Cartridge SPE, AN1131610_01, available from 


	16 Northwestern Drive, Salem, NH 03079
	tel. (603) 893-3663
	Website: 

The Extraction of Perfluorinated Alkyl Acids (PFAAs) in Drinking Water Using Automated Cartridge SPE

Microwave-assisted extraction technology offers multiple benefits over traditional Soxhlet extraction and other non-conventional systems. ETHOS X with its new FastEX-24 rotor and disposable glass vials offers reliable extraction of contaminants from soil in compliance with EPA 3546 along with easy handling and high extraction efficiency.

	The extraction of contaminants, such as PCBs, semivolatile organic compounds, and PAHs, from soils requires solvent extraction. Microwave-assisted extraction (MAE) overcomes the limitations of Soxhlet extraction, resulting in rapid sample preparation with reduced amounts of solvents while working at higher temperatures and pressures. The process is a partitioning of the compounds of interest from the sample matrix into the solvent within a closed vessel. EPA 3546 method provides guidelines to work with the MAE technology, thus improving the quality and the productivity of environmental laboratories.

	Milestone's new ETHOS X microwave extraction system can extract organic target compounds from soils, in full compliance with EPA 3546 (100–115 °C and 50–150 psi). Disposable glass vials and contactless temperature control in all positions makes the Milestone ETHOS X with the FastEX-24 rotor a unique and innovative solution for the extraction of contaminants from soils, providing unmatched ease of use and low running costs. The ETHOS X is capable of processing up to 30 g of sample per vessel (up to 24 samples simultaneously), thereby improving the limit of quantitation (LOQ) for analysis. The handling is very easy as the sample is weighed directly into the disposable glass vial, hexane/acetone or CH

/acetone (1:1) is added, and the vessel is loaded into the FastEX rotor. After 10–20 min of microwave heating, the sample is ready to be filtered and analysed by gas chromatography.

	The ETHOS X enables simultaneous solvent extraction of up to 24 samples (from weighing to filtration) in only 40 min. This results in the capacity to extract over 200 samples in an 8-hour workday. Contamination, memory effects, and cleaning are eliminated due to the use of disposable glass vials. The use of contactless temperature control ensures high reproducibility and full recovery of the target analytes for full compliance with EPA 3546. The ETHOS X with all its features, fully addresses the needs of environmental laboratories in terms of productivity, ease of use, running costs, and extraction quality. The ETHOS X equipped with the FastEX-24 rotor provides superior extraction capability for easier analysis.


	25 Controls Drive, Shelton, CT 06484
	tel: (866) 995-5100, fax (203) 925-4241
	Website: 

Improving the Quality and Productivity of Environmental Extraction

	Glyphosate, the active ingredient in Roundup®, is the most widely used herbicide in the world. In the United States, the Environmental Protection Agency (EPA) regulates Glyphosate and sets maximum amount of herbicide allowed to be present in assorted crops as well as drinking water. Recent research, however, has raised concerns about Glyphosate safety and its prevalence in the environment. In response to the evidence of increased human exposure to this herbicide, the U.S. Food and Drug Administration (FDA) announced it will begin testing Glyphosate in foods including soybeans, corn, milk, and eggs.

	Pickering Laboratories has over 30 years of experience manufacturing and selling instruments and reagents for Glyphosate analysis in accordance to the US-EPA Method 547 for Glyphosate Analysis in Drinking Water as well as the AOAC Method 991.08 for Glyphosate Analysis in Environmental Waters. These HPLC methods are based on post-column derivatization technology with florescence detection. The AOAC Official Method 2000.05 for Analysis of Glyphosate in Crops describes an easy clean-up procedure using cation-exchange cartridges that was successfully combined with Pickering Laboratories' post-column derivatization for analysis of Glyphosate in crops such as soy, corn, alfalfa, and sunflower seeds as well as vegetables such as tomatoes and broccoli (1,2). The downside of this sample preparation technique is the fairly high volume of the water-based solution used to elute Glyphosate from the clean-up cartridge and consequently long evaporation times.

	The presented post-column derivatization method for Glyphosate analysis utilizes an accelerated sample preparation procedure to quickly and efficiently analyze Glyphosate in a wide range of foods. The analytical method is sensitive and selective, and it can be easily implemented in any laboratory.

	To 25 g of homogenized sample, add enough water (after estimating moisture content) such that the total volume of water is 125 mL. Blend at high speed for 3–5 min and centrifuge for 10 min. Transfer 20 mL of the aqueous extract into a centrifuge tube and add 15 mL of Methylene Chloride. Shake for 2–3 min and centrifuge for 10 min. Use the upper layer for SPE clean-up.

	1) For samples that absorb large amounts of water, reduce test portion to 12.5 g while keeping water volume the same.

	2) For samples with high fat content, perform the Methylene Chloride partitioning twice.

	Condition the SAX cartridge with 5 mL of Methanol followed by 5 mL of D.I. water, draining the liquid to the top of the sorbent bed.Transfer 1 mL of extract into cartridge and elute to the top of the sorbent bed. Wash with 5 mL of Methanol. Elute Glyphosate with 5 mL of Elution Solution into collection vessel for evaporation. Evaporate to dryness at 55 °C under Nitrogen. Dissolve the residue in 1.5 mL of a 10% RESTORE™ in water solution. Filter through a 0.45 µm syringe filter and inject onto the HPLC column.

	The simple and robust post-column method was shown to be suitable for analysis of Glyphosate on sub-ppm levels in many types of foods. Accelerated sample preparation improves throughput of samples and reduces cost of testing for busy laboratories. The sensitivity of the method allows for detection of Glyphosate well below residue tolerances set by regulatory agencies.

The Application Notebook-02-01-2017