	This application note describes the determination of the triphenylmethane dyes Malachite Green and Crystal Violet and their metabolites from the aquaculture samples brown trout, shrimp, and tuna using dispersive SPE (dSPE) with CHROMABOND® QuEChERS mixes for sample clean-up. The eluates from dSPE cleaning are finally analyzed by HPLC–MS/MS using the biphenyl phase NUCLEODUR® π

	Motivated by various potential health benefits, human feeding behaviour is changing, leading to an increasing demand for aquatic products. Farming aquatic species is becoming an important way of producing large amounts of aquatic products all over the world. One challenge of farming aquatic species is the control of infectious diseases.

	Triphenylmethane dyes (TPM) are organic compounds originally used as textile and paper dyes and have been found to be effective as antibacterial, antifungal, and antiparasitic agents in fisheries. They accumulate in fish and metabolize to the equivalent, colourless leuco-forms, which are also known as mutagenic (1). To protect human health, triphenylmethane dyes have been banned in many countries according to the recommendations of national and international related agencies. For example, the European Union has implemented a minimum required performance limit (MRPL) for the sum of Malachite Green and Leucomalachite Green of 2 μg/kg (2).

	QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe) methodology is known as a common sample preparation technique in modern analysis of pesticides in food, and offers a quick and cost-efficient determination of different analytes in strongly matrixâcontaminated samples by GC–MS and LC–MS (3).

	Biphenyl-modified HPLC phases feature a better retention of aromatic analytes compared to classical C

 phases due to additional π-π interactions.

		Weigh out 5 g of homogenized sample into a 50 mL brown centrifuge tube

		Add 5 μL of internal standard solution (β = 1 μg/mL Malachite Green-d5 and Leucomalachite Green-d5 each in acetonitrile)

		Add 25 μL of standard solution (β = 200 ng/mL Malachite Green, Leucomalachite Green, Crystal Violet, Leucocrystal Violet each in acetonitrile) for determining recovery rate

		Add 10 mL 1% acetic acid in acetonitrile and shake

		Add the CHROMABOND® QuEChERS extraction Mix II (MachereyâNagel REF 730971)

		Shake vigorously for 30 s and cool the mixture down in an ice bath

		Centrifuge the mixture at 4500 rpm for 5 min at 4 °C

		Put 5 mL acetonitrile supernatant in a 15 mL brown centrifuge tube

		Add the CHROMABOND® QuEChERS clean-up Mix III (MachereyâNagel REF 730972) (for samples with high fat content add 1 mL hexane)

		Dilute the extract 1:1 with an aqueous solution of 5 mmol/L ammonium acetate + 1 mL/L formic acid and filter through a syringe filter (CHROMAFIL® Xtra PET-20/13, Macherey-Nagel REF 729208)

 5 mmol/L ammonium acetate + 0.1% formic acid in water
	

20–85% B in 10 min, 85% B for 5 min, 85–20% B in 1 min, 20% B for 5 min
	

 LCMS 8050 (Shimadzu); ion source: ESI, positive ionization mode; scan type: MRM; interface heater on; interface current: 4000 V; interface temperature: 300 °C; DL temperature: 250 °C; nebulizing gas flow: 3.00 L/min; heating gas on; heating gas flow: 10.00 L/min; heat block: 400 °C; drying gas on; drying gas flow: 10.00 L/min

	The identification and quantification of TPM dyes in the cleaned sample extracts of brown trout, shrimp, and tuna were successfully performed by ESI mass spectrometry. Calibration curves were in the concentration range between 0.5 ng/mL and 50 ng/mL for each analyte. Figure 1 shows the chromatogram of QuEChERS extract from the tuna sample spiked with 1 µg/kg with high signal response and less matrix interferences.

	By using the QuEChERS methodology it was possible to recover more than 70% of TPM dyes from the different matrices (see Figure 2). The use of brown centrifuge tubes protects the analytes from lightâinduced degradation and leads to better sensitivities and recovery rates.

	In summary, the presented application describes a quick and convenient method for the determination of TPM dyes from aquatic samples.

		BfR Expert Opinion No. 007/2008, 24 August 2007.

		Official Journal of the European Communities L 221, 17 August 2002, 8–36.	

		M. Anastassiades, S.J. Lehotay, D. Stajnbaher, and F.J. Schenck, J. AOAC Int. 86, 412–431 (2003).	

		SPE Application Nos. 306560, 306570 and 306580, MACHEREY-NAGEL, available from 

		HPLC Application No. 128430, MACHEREY-NAGEL, available from 

	Neumann-Neander-Str. 6 – 8, 52355 Düren, Germany 

Food and Beverage

This application note describes the determination of the triphenylmethane dyes Malachite Green and Crystal Violet and their metabolites from the aquaculture samples brown trout, shrimp, and tuna using dispersive SPE (dSPE) with CHROMABOND QuEChERS mixes for sample clean-up.

Determination of Triphenylmethane Dyes from Aquaculture Samples

	Glycoproteomics aims at the concomitant identification of not only the composition of the glycan but also the sites of glycosylation and the determination of the protein attached with glycans. However, glycopeptide analysis is challenging because their microheterogeneity results in the reduced concentration of each individual glycopeptide molecule compared to unmodified peptides, even if they are obtained from the very same digest sample (1). Also, glycopeptides exhibit poor ionization efficiency compared to their nonglycosylated counterparts (2). Therefore, it is essential to perform selective and efficient glycopeptide enrichment by solid-phase extraction (SPE) prior to detection and identification by mass spectrometry (MS) analysis (3–10). Hydrophilic interaction liquid chromatography (HILIC) SPE has been extensively applied in the past decade because of its low bias towards different glycan types (9,10). In contrast to normalâphase liquid chromatography, the HILIC retention mechanism is mainly based on the hydrophilic partitioning of the analyte to the enriched superficial water layer surrounding the surface of the polar stationary phase (11). Ionic interaction and hydrogen bonding may also be involved in the separation depending on the sample properties and the character of HILIC stationary phase. Here, we demonstrate the glycopeptide enrichment efficiency of iSPE®-HILIC material for glycoproteomics applications using a well characterized glycoprotein standard.

	Sample Preparation and HILIC SPE Enrichment: Three micrograms of standard glycoprotein (human IgG) were subjected to trypsin digestion. Glycopeptides were enriched using 25 mg iSPE®âHILIC material (50 μm, 60 Å, HILICON) by a drop-HILIC approach as described earlier (9). The dried samples, which contained about an aliquot of 30 ng enriched glycopeptides in each sample, were reconstituted with 100 μL of 0.1% TFA and used for the MALDIâTOF-MS analyses. Equal volumes of sample and matrix (20 mg/mL 2,5-dihydroxybenzoic acid [DHB] in 30% acetonitrile/0.1% TFA) were spotted onto a MTP 384 target plate ground steel (Bruker Daltonics).

	MS System: MALDI-TOF-MS analysis was performed on a spectrometer equipped with Smartbeam™ 3D laser optics running at 5000 Hz and controlled by FlexControl 4.0 software (Bruker Daltonics). MS analysis was performed in reflectorâpositive mode, and the spectra were acquired within the mass range of m/z 1000 to 4000. In total, 20,000 shots were accumulated per spot, baseline corrected, and smoothed using Gauss algorithm with 

	The efficiency and selectivity of the iSPE®-HILIC material for glycopeptide enrichment were evaluated using a human Immunoglobulin G (IgG) mix. The tryptic (glyco)peptides mixture was analyzed using MALDI-TOF-MS before and after HILIC enrichment. As shown in Figure 1(a) with non-enriched samples, no signals corresponding to the IgG glycopeptides could be detected. Nevertheless, after glycopeptide enrichment using iSPE®-HILIC material, a total of 14 individual glycopeptides were identified from 30 ng of glycoprotein by MALDI-TOF-MS, shown in Figure 1(b). Next to these glycopeptides, a few unmodified polar peptides (in the lower m/z region as shown in Figure 1[b]) were also coenriched, but they did not interfere with glycopeptide detection. Considering the fact that HILIC enriches glycopeptides based on their hydrophilicity, it does not come as a surprise other hydrophilic compounds are coenriched (10). Such an unavoidable coenrichment of smaller hydrophilic peptides usually does not interfere with the ionization and detection of the target glycopeptides in the higher m/z range. For detailed information on the detected peptides, please contact us to retrieve the list of identified peptides corresponding to the different IgG subclass by MALDI-TOF-MS analysis in the nonenriched sample and the list of tryptic IgG Fc N-glycopeptides detected in the samples after iSPE®-HILIC enrichment.

	In summary, iSPE®-HILIC offers a robust, reliable, and easily implemented solution for effective glycopeptides enrichment in glycoproteomics. In combination with optimized sample digestion protocols (for example, salt removal), iSPE®-HILICâbased drop-HILIC glycopeptides enrichment opens a wide range of opportunities for site-specific and in-depth glycoproteomics research.

		K. Stavenhagen, P.H. Jensen, F. Altmann, and N.H. Packer, 

		Y. Pasing, A. Sickmann, and U. Lewandrowski, 

		B.A. Budnik, R.S. Lee, and J.A. Steen, 

		H.J. An, J.W. Froehlich, and C.B. Lebrilla, 

		Q. Li, Y. Xie, M. Wong, and C.B. Lebrilla, 

		K. Alagesan, S.K. Khilji, and D. Kolarich, 

		M. Thaysen-Andersen, S. Mysling, and P. Højrup, 

Medical/Biological

Demonstratation of the glycopeptide enrichment efficiency of iSPE®-HILIC material for glycoproteomics applications using a well characterized glycoprotein standard.

Effective Enrichment of Glycopeptides in Drop‑HILIC Approach Using iSPE®-HILIC Material

Coffee is one of the most widely consumed beverages in the world, partly due to the stimulating effect of its caffeine content. Like most crops, the application of pesticides in coffee cultivation is a common practice to increase production yields. This application note details an optimized method for the extraction and cleanup of pesticide residues from coffee using a QuEChERS extraction procedure followed by a silica gel SPE cleanup.

	a) Add 10 mL brewed coffee (pH adjusted to about 8 with 1 N NaOH) and 10 mL acetonitrile (MeCN) to a 50-mL centrifuge tube.

	b) Add the QuEChERS extraction salts from the Mylar pouch (ECMSSC50CT-MP) to the 50-mL tube, and shake vigorously for 1 min manually or using a Spex 2010 Geno-Grinder at 1000 strokes/min.

	d) Transfer 5 mL supernatant to a clean test tube, add 1.5 mL toluene, and evaporate to about 1 mL.

	a) Add about ½ inch of anhydrous sodium sulfate to a silica gel SPE cartridge (CUSIL156), and attach the SPE cartridge to a glass block or positive pressure manifold.

	b) Wash the SPE cartridge with 6 mL dichloromethane, soak for 1 min, drain to waste, and dry the SPE cartridge for 1 min under full vacuum or pressure.

	c) Condition the SPE cartridge with 2 × 6 mL hexane by gravity.

	d) Insert glass collection container into the manifold, load the 1 mL concentrated sample onto the SPE cartridge, rinse the test tube with 6 mL of 15% acetone in n-hexane and apply the rinsate to the SPE cartridge, and collect.

	e) Continue to elute with 3 × 6 mL of 15% acetone in n-hexane by gravity.

	f) Add 1.5 mL ethyl acetate to the eluate container and evaporate to 1 mL.

	g) Add internal standard, vortex for 30 s, and inject 1 μL into the GC–MS system for analysis.

 Agilent 6890N GC coupled to a 5975C MSD
	

 Restek Rxi®-5Sil MS (30 m × 0.25 mm, 0.25-µm)
	

 60 °C for 1 min, 10 °C/min to 310 °C, hold for 2 min; 28 min total
	

UCT, LLC
	2731 Bartram Rd, Bristol, Pennsylvania 19007, USA
	

This application note details an optimized method for the extraction and cleanup of pesticide residues from coffee using a QuEChERS extraction procedure followed by a silica gel SPE cleanup.

Determination of Pesticides in Coffee with QuEChERS Extraction and Silica Gel SPE Cleanup

	Nowadays, tattoos have become increasingly popular with both men and women, and particularly with younger people. However, an increase in popularity has meant that more and more adverse effects, such as infections and allergies, have been reported. This is due to the fact that tattoo inks can be complex in their formulation, containing several out of more than 100 pigments and additives, and also a significant quantity of impurities. The pigments are normally of particularly low purity and a majority of the ingredients are not even approved for application in cosmetic products in the European Union. Despite this, harmonized and well-established analytical methods for the comprehensive characterization of tattoo ink ingredients are still lacking (1).

	A powerful technology for the investigation of the particulate content in tattoo inks is asymmetrical flow field-flow fractionation (AF4) coupled with multi-angle light scattering (MALS) and inductively-coupled plasma mass spectrometry (ICP-MS) (2). In this study, AF4-MALS-ICP-MS was applied to characterize four commercial tattoo inks: outlining black, purple deep violet, green grasshopper, and blue ice.

Sizing of Particulate Tattoo Ink Ingredients Using AF4âMALS

	Using AF4-MALS, the particle size distribution in all four investigated tattoo inks could be assessed and all were found to have a significant nanoparticulate content. While outlining black, green grasshopper, and blue ice revealed monomodal particle size distributions ranging from 30–350 nm, 35–550 nm, and 25–450 nm radii of gyration (Rg), respectively, deep purple violet showed a bimodal distribution with particle sizes ranging from
	20–550 nm Rg (Figure 1).

Chemical Identification of Particulate Tattoo Ink Ingredients Using AF4-ICP-MS

	By coupling to ICP-MS, AF4 enabled size-resolved chemical identification of particulate tattoo ink ingredients. By this means, it could be demonstrated that outlining black contains particulate titanium (Ti) and copper (Cu), but no aluminium (Al), while green grasshopper and blue ice contain all three elements in particulate form. Purple deep violet, however, contains particulate Ti and Al, but no Cu (Figure 2). All investigated metals were most likely present as pigments in their oxidic state as TiO2, Al2O3, and CuO.

Comparison of AF4-ICP-MS with Batch ICP-MS Results

	By comparing the results obtained from AF4-ICP-MS with batch ICP-MS measurements, a quantitative differentiation between particulate and dissolved metals in the investigated tattoo inks could be achieved. While Al was predominantly dissolved, most of the Ti and Cu was present in particulate form (Table 1).

	This study demonstrates the excellent suitability of AF4-MALS-ICP-MS for the comprehensive characterization of tattoo inks. Besides the particle size distributions obtained from the MALS data, ICP-MS also provides statements on the elemental distributions of various metals as a function of the respective particle sizes, offering valuable insight into the composition of the inks. This can not only facilitate the reliable identification and quantification of possible allergenic ingredients (and thus a better risk assessment) but may also help tattoo ink manufacturers to improve their formulations, eventually leading to safer products.

		P. Piccinini, S. Pakalin, L. Contor, I. Bianchi, and C. Senaldi, 

 (Final report, EUR 27947 EN), doi: 10.2788/011817.	

		B. Bocca, E. Sabbioni, I. Micetic, A. Alimonti, and F. Petrucci, 

Journal of Analytical Atomic Spectrometry


	Max-Planck-Strasse 14, 86899 Landsberg, Germany
	Tel.: +49 8191 9856880  Fax: +49 8191 98568899
	

In this study, AF4-MALS-ICP-MS was applied to characterize four commercial tattoo inks: outlining black, purple deep violet, green grasshopper, and blue ice.

Characterization of Metal-Containing Nanoparticles in Tattoo Ink Using Asymmetrical Flow Field-Flow Fractionation Coupled with Multi-Angle Light Scattering and ICP-MS

Abuse of synthetic opioid prescription painkillers such as fentanyl, along with a rapidly growing list of illicit analogues, is a significant public health problem. In this study, we developed a simple dilute-andâshoot method that provides a fast 3.5-min analysis of fentanyl and related compounds (norfentanyl, acetyl fentanyl, alfentanil, butyryl fentanyl, carfentanil, remifentanil, and sufentanil) in human urine by LC–MS/MS using a Raptor Biphenyl column.

	In recent years, the illicit use of synthetic opioids has skyrocketed, and communities worldwide are now dealing with an ongoing epidemic. Of the thousands of synthetic opioid overdose deaths per year, most are related to fentanyl and its analogues. With their very high analgesic properties, synthetic opioid drugs such as fentanyl, alfentanil, remifentanil, and sufentanil are potent painkillers that have valid medical applications; however, they are also extremely addictive and are targets for abuse. In addition to abuse of these prescription drugs, the current opioid crisis is fueled by a growing number of illicit analogues, such as acetyl fentanyl and butyryl fentanyl, which have been designed specifically to evade prosecution by drug enforcement agencies.

	As the number of opioid drugs and deaths increases, so does the need for a fast, accurate method for the simultaneous analysis of fentanyl and its analogues. Therefore, we developed this LC–MS/MS method for measuring fentanyl, six analogues, and one metabolite (norfentanyl) in human urine. A simple diluteâandâshoot sample preparation procedure was coupled with a fast (3.5 min) chromatographic analysis using a Raptor Biphenyl column. This method provides accurate, precise identification and quantitation of fentanyl and related compounds, making it suitable for a variety of testing applications, including clinical toxicology, forensic analysis, workplace drug testing, and pharmaceutical research.

 The analytes were fortified into pooled human urine. An 80 µL urine aliquot was mixed with 320 µL of 70:30 water–methanol solution (fivefold dilution) and 10 µL of internal standard (40 ng/mL in methanol) in a Thomson SINGLE StEP filter vial (Restek cat. #25895). After filtering through the 0.2 µm PVDF membrane, 5 µL was injected into the LC–MS/MS.

Calibration Standards and Quality Control Samples:

 The calibration standards were prepared in pooled human urine at 0.05, 0.10, 0.25, 0.50, 1.00, 2.50, 5.00, 10.0, 25.0, and 50.0 ng/mL. Three levels of QC samples (0.75, 4.0, and 20 ng/mL) were prepared in urine for testing accuracy and precision with established calibration standard curves. Recovery analyses were performed on three different days. All standards and QC samples were subjected to the sample preparation procedure described.

	LC–MS/MS analysis of fentanyl and its analogues was performed on an ACQUITY UPLC instrument coupled with a Waters Xevo TQ-S mass spectrometer. Instrument conditions were as follows, and analyte transitions are provided in Table 1.

Analytical column:                

Raptor Biphenyl (5 µm, 50 mm× 2.1 mm; cat. #9309552)
	

                      Raptor Biphenyl EXP guard column cartridge, (5 µm, 5 mm × 2.1 mm; cat. #930950252)
	

                  0.1% Formic acid in water
	

                   0.1% Formic acid in methanol

	                                    0.00                  30

	                                    2.50                  70

	                                    2.51                  30

	                                    3.50                  30

                         0.4 mL/min
	

                         Positive ESI

 All eight analytes were well separated within a 2.5-min gradient elution (3.5-min total analysis time) on a Raptor Biphenyl column (Figure 1). No significant matrix interference was observed to negatively affect quantification of the fivefold diluted urine samples. The 5-µm particle Raptor Biphenyl column used here is a superficially porous particle (SPP) column. It was selected for this method in part because it provides similar performance to a smaller particle size fully porous particle (FPP) column, but it generates less system back pressure.

 Linear responses were obtained for all compounds and the calibration ranges encompassed typical concentration levels monitored for both research and abuse. Using 1/x weighted linear regression (1/x

 for butyryl fentanyl), calibration linearity ranged from 0.05 to 50 ng/mL for fentanyl, alfentanil, acetyl fentanyl, butyryl fentanyl, and sufentanil; from 0.10 to 50 ng/mL for remifentanil; and from 0.25 to 50 ng/mL for norfentanyl and carfentanil. All analytes showed acceptable linearity with r

 values of 0.996 or greater and deviations of <12% (<20% for the lowest concentrated standard).

 Based on three independent experiments conducted on multiple days, method accuracy for the analysis of fentanyl and its analogues was demonstrated by the %recovery values, which were within 10% of the nominal concentration for all compounds at all QC levels. The %RSD range was 0.5–8.3% and 3.4–8.4% for intraday and interday comparisons, respectively, indicating acceptable method precision (Table 2).

	A simple dilute-and-shoot method was developed for the quantitative analysis of fentanyl and its analogues in human urine. The analytical method was demonstrated to be fast, rugged, and sensitive with acceptable accuracy and precision for urine sample analysis. The Raptor Biphenyl column is well suited for the analysis of these synthetic opioid compounds and this method can be applied to clinical toxicology, forensic analysis, workplace drug testing, and pharmaceutical research.


	110 Benner Circle, Bellefonte, Pennsylvania 16823, USA
	Tel. 1 (814) 353 1300
	

 In this study, we developed a simple dilute-and‑shoot method that provides a fast 3.5-min analysis of fentanyl and related compounds (norfentanyl, acetyl fentanyl, alfentanil, butyryl fentanyl, carfentanil, remifentanil, and sufentanil) in human urine by LC–MS/MS using a Raptor Biphenyl column.

Analysis of Fentanyl and Its Analogues in Human Urine by LC–MS/MS

	Sphingolipids are major components forming biological membranes, and they serve as intracellular signalling molecules. It is important to measure the amount of these molecules in biological samples because they have a massive influence on various metabolic diseases such as obesity, diabetes, and Alzheimer’s disease. However, in LC analyses of sphingophospholipids, such as sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P), a phosphate group in these molecules causes significant peak tailing, and therefore loss of sensitivity and reproducibility. Recently, an improved method using a YMC-Triart C18 metal-free column was reported by Dr. Gowda 

Adsorption of Compounds with Phosphate Groups

	In LC analysis, compounds containing phosphate groups tend to be adsorbed on the metallic surfaces in the flow path of the LC system. This results in peak tailing, carryover, and insufficient sensitivity.

	It is important to use a column packed with a packing material containing less metal impurities to prevent these problems. Material used for the column hardware is also important. Stainless steel-free column hardware is effective to improve peak shapes, especially for highly sensitive analyses such as LC–MS.

	YMC-Triart C18 metal-free columns are ideal for highly sensitive analysis of coordination compounds because their hardware consists of a PEEK-lined stainless steel tube and PEEK frits.

	Significant peak tailing was observed for the conventional stainless steel column.

	On the other hand, peak shape and intensity were improved using the YMC-Triart C18 metal-free column.


	Schöttmannshof 19 · 46539 Dinslaken
	

Sphingolipids are major components forming biological membranes, and they serve as intracellular signalling molecules. It is important to measure the amount of these molecules in biological samples because they have a massive influence on various metabolic diseases such as obesity, diabetes, and Alzheimer’s disease. Recently, an improved method using a YMC-Triart C18 metal-free column was reported by Dr. Gowda et al.

LC–MS/MS Analysis of Sphingophospholipids Using a Metal-Free Column

	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 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®), a refractive index detector (Optilab®), 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 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 a 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, 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 because it has the ability to determine not only the absolute molar mass distribution but also to reveal subtle differences in PLGA’s molecular structure.


	6330 Hollister Ave, Santa Barbara, California 93110, USA
	

Pharmaceutical/Drug Discovery

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®), a refractive index detector (Optilab®), and a differential viscosity detector (ViscoStar®).

The Application Notebook-09-01-2019