	Supplements containing amino acids are widely used as part of the treatment of a number of medical conditions, including ALS, some brain conditions caused by liver disease, muscle degeneration and movement disorders. Amino acids supplements are also used by athletes to improve performance and prevent muscle breakdown. These supplements are available in variety of forms and dosages, and they often contain other ingredients like vitamins, minerals, herbal extracts, and flavorings.

	Manufacturers are responsible for supporting label claims. The testing of dietary ingredients as well as finished products for active components is an important part of any quality assurance program.


	Figure 2: Chromatogram of powdered drink mix.

	Analysis of amino acids using cation-exchange columns and Ninhydrin post-column reagent is a well-established methodology that is recommended by the European Pharmacopeia. Pickering Laboratories developed and validated a post-column method for amino acids analysis of supplements that is sensitive, selective and can be used without modifications to analyze capsules, tablets, drinks, and other samples. Accelerated methods are available for samples with a limited number of amino acids.


	Figure 3: Chromatogram of encapsulated amino acids supplement.

	Column: High-efficiency Lithium cation-exchange column, 4.6 × 75 mm, Catalog Number 0354675T
	Flow rate: 0.55 mL/min
	Mobile Phase: See method in Table I

	Post-Column System: Pinnacle PCX
	Reactor volume: 0.5 mL
	Reactor Temperature: 130 °C
	Flow rate: 0.3 mL/min
	Detection: UV-vis 570 nm for primary amino acids, 440 nm for secondary amino acids
	Injection Volume: 10–50 µL

	Place 100 mg of homogenized sample into a 25 mL volumetric flask. Bring to volume with Li220 Diluent. Sonicate for 30 min and filter through a glass microfiber filter if the solution is cloudy. Dilute the sample to fit within calibration curve as necessary. More than one dilution may be needed if concentrations of amino acids differ by more than 10 times. Filter the final solution though 0.45 µm Nylon filter before injecting.


	1280 Space Park Way, Mountain View, CA 04043
	tel. (800) 654-3330, (650) 694-6700
	

Articles

Medical/Biological

Pickering Laboratories - Analysis of Amino Acids in Dietary Ingredients and Supplements

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-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.

	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 dilute-and-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 I.

	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.


	Figure 1: The Raptor Biphenyl column effectively separated all target compounds in urine with no observed matrix interferences. Peak elution order: norfentanyl-D

, norfentanyl, remifentanil, acetyl fentanyl-

, acetyl fentanyl, alfentanil, fentanyl-D

, carfentanil, butyryl fentanyl, sufentanil-D

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

 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 

 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 II).


	Table II: Accuracy and precision results for fentanyl and related compounds in urine QC samples

	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, PA 16823
	tel. 1 (814) 353-1300
	

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

Intrinsically-disordered proteins cannot be analyzed by SEC column calibration. SEC-MALS provides accurate molecular masses of these proteins and of their complexes with other proteins.

	The S100 family is a group of dimeric, calcium-binding proteins which have been found to be overexpressed in several types of cancer. S100 proteins bind to the C-terminal region (293-393) of p53, the most critical tumor suppressor involved in apoptosis, cell cycle arrest and DNA repair. Since S100 is dimeric and the C-terminus of p53 encompasses a tetramerization domain, the potential range of complexes incorporating p53 and S100 is large.

	The p53 variants have a large fraction of intrinsically disordered structure and do not elute according to globular protein standards in size-exclusion chromatography (SEC). However, multi-angle light scattering determines molecular mass from first principles. SEC-MALS determines the molecular weight of the complexes independently of protein standards. We utilized SEC-MALS to investigate how S100 proteins bind to p53 in its different oligomeric states, using monomeric (L344P) and dimeric (L344A) p53 mutants.

	The experimental system consisted of an HPLC and SEC column plumbed in series with a DAWN MALS detector (Wyatt Technology, Santa Barbara) and an Optilab differential refractive index (dRI) detector (Wyatt Technology).

	The p53 mutants and S100 proteins were first characterized separately by SEC-MALS to determine their molecular masses and homogeneity. They were then incubated at various stoichiometric ratios-4:1, 2:1, 1:1, 1:2, and 1:4-and analyzed on the SEC-MALS system.

	Molar mass results of the individual p53 proteins are overlaid with the light scattering (solid lines) and dRI chromatograms (dotted lines) in Figure 1. The molar masses correspond to a monomer of 11.2 kDa for the L344P variant, a dimer for the L344A variant, and a tetramer for p53 wild-type.


	Figure 1: Analytical SEC-MALS of p53(293-393) variants. Overlay of the individual SEC-MALS elution profiles of p53 (293-393)L344P (red), p53 (293-393)L344A (green), and p53 (293-393) wild type.

	Chromatograms and molar masses for the pre-incubated mixtures are presented in Figure 2. Monomeric p53 (293-393)L344P (11.2 kDa) and dimeric S100B (21.4 kDa) both eluted with a retention time of ~17.5 min. The complex molar mass of ~32 kDa at ~16 min. is independent of the relative ratio of the two proteins, indicating that one dimer of S100B binds only one monomer of p53. No complexes consisting of a dimer of S100B and two monomers of p53 (~43 kDa) could be detected. In addition, S100B was able to disrupt the dimeric p53(293-393)L344A mutant and form a complex with the monomer.


	Figure 2: SEC-MALS of S100B and p53(293-393)L344P in different concentration ratios.

	Interestingly, SEC-MALS revealed that S100B was also able to form a stable complex with tetrameric p53 (293-393) consisting of 4 dimers of S100B bound to a tetramer of p53.

	SEC-MALS helped understand how S100 proteins influence the oligomerization of p53. Based on these results and in vivo studies, we established a binding model where S100 proteins can activate tetrameric p53 but inhibit p53 activity binding to the monomer by shifting the tetramerization equilibrium. SEC-MALS is a valuable tool in structural biology for studying the stoichiometry and formation of many types of protein-protein complexes.


	6330 Hollister Avenue, Santa Barbara, CA 93117
	tel. +1 (805) 861-9009
	

Wyatt - Stoichiometry of Intrinsically- Disordered Protein Complexes

Semi-volatile organic compounds (SVOCs) are a subgroup of volatile organic compounds (VOCs) that have a high molecular weight and high boiling points. They are primarily composed of pesticides and herbicides. Prolonged exposure to these compounds, especially indoors, raises a public health concern as many of these compounds have been listed by the US EPA as hazardous air pollutants (HAPs). This classification applies to pollutants that can cause serious health effects such as: allergies, asthma, endocrine and thyroid disruption, reproductive toxicity, fetal and child development delays, and cancer.

	Due to their persistent nature, SVOCs continue to accumulate and concentrate in our environment. To ensure our safety, these compounds must be extracted, identified, and quantified. EPA 3545 is a method for the extraction of water-insoluble or slightly water-soluble volatile and semi-volatile compounds in soils, clays, sediments, sludges, and waste solids. It is typically used for SVOC analysis. The EDGE™ Extraction System from CEM meets the requirements of EPA 3545 while yielding an efficient extract that is filtered, cooled, and ready for analysis in under 5 min.

	The EDGE combines the process of pressurized fluid extraction and dispersive solid-phase extraction in one instrument, yielding rapid and efficient extracts every time. The easy-to-assemble Q-Cup™ sample holder offers a unique open cell concept that creates a dispersive effect to promote rapid extraction and filtration. Soil samples from 1 g to 30 g can be extracted and NaSO

 (or other additives) can be included with the sample as needed. The EDGE will use only 40 mL of solvent per extraction, including solvent for diffusive extraction, sample rinse, and cleaning of the system.

	Ten g of sand, loam, or clay samples spiked with 250 µL of spike solution were weighed into an assembled Q-Cup containing a Q-Disc™. The Q-Cups were placed in the EDGE removable rack with collection vials and the rack was positioned in the EDGE. The pre-programmed One Touch Method™ for EPA 3545 was used. Upon extraction completion, the extracts were injected into the Agilent 7890A with a 5975C MSD for analysis adhering to EPA 8270. A Phenomenex ZB-5MSplus 30 m, 0.25 mm column was used.

	Sand, loam, and clay purchased from Sigma Aldrich were spiked with SPEX CertiPrep TCLP Base/Neutral/Acid Extractable Spike Solution in Methylene Chloride (Part#: TCLP-BNA). All samples were extracted via the EDGE and Soxhlet. A 50:50 mixture of acetone-hexane was used as the extraction and rinse solvent. The system was washed with hexane and acetone.


	Table I: Percent recovery data as compared to Soxhlet for spiked sand, loam, and clay

	The EDGE extracted sand, loam, and clay samples in under 5 min, including filtration, cooling, and system washing. No post clean up, solvent exchange, or concentration was necessary. The extracts were directly injected into the GC-MS for analysis. The recoveries for all three types of spiked soil were comparable to Soxhlet. Table I shows the % recoveries as compared to Soxhlet from spiked soil of some difficult to extract semi-volatile organic compounds. The high % recoveries of EDGE extractions shows that EDGE is a suitable tool for SVOC extraction per EPA 3545.


	P.O. Box 200, Matthews, NC 28106
	tel. (704) 821-7015, (800) 726-3331, fax (704) 821-7894
	

Environmental

CEM Corporation - Rapid Extraction of Semi-Volatile Organic Compounds from Soil in Accordance with EPA 3545

	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.


	Figure 1: Borrowed and adapted (with permission) from P. Alava et al., Biomed. Chromatogr. 26, 524–533 (2012).

	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.


	4970 Energy Way, Reno, NV 89502
	tel. (775) 858-3000, (800) 648-5950
	

Food & Beverage

Hamilton - Arsenic Speciation on Hamilton PRP-X100

Phosphorylated compounds readily adsorb to stainless steel surfaces present within the flow path of an HPLC system. This process can lead to poor peak shape, low recovery of sample, and reduction in sensitivity. Here we show that our metal-free column housing technology can improve analysis of these compounds.

	HPLC columns are commonly made with stainless steel 316, which is a combination of iron, carbon, manganese, and silicon, with high amounts of nickel, chromium, and molybdenum. The corrosion resistance of stainless steel is caused by the formation of a passive layer of chromium and nickel oxides on the surface, which prevent penetration of corrosive elements. These oxide layers have a high affinity to phosphorylated compounds, particularly those that have accessible phosphate groups.

	An illustration of this interaction is shown in Figure 1. As the analyte band travels through the column, it will lose sample through adsorption to the metal surface, resulting in low recovery and a reduction in sensitivity. Adsorption also interferes with uniform sample flow causing peak tailing which can be observed in the resulting chromatogram.


	Figure 1: A simplified schematic diagram of analyte loss caused by adsorption of phosphorylated compounds to stainless steel HPLC column housing.

	We recently developed metal-free housing to prevent such unwanted interactions. Our design utilizes stainless steel hardware, in order to preserve high pressure capabilities, with internal PEEK coatings (tubing, end fittings, inlet and outlet frits). This removes the largest contribution of metal surfaces within the HPLC sample flow path.

	Figure 2 shows several triphosphates under identical conditions in both stainless steel and metal-free housings.


	Figure 2: Triphosphates on stainless steel versus metal-free columns.

	All triphosphates tested show evidence of adsorption and poor peak shapes with a metal column, which is not seen on our metal-free column. Marked improvements in peak shape and sample recovery, were observed as evidenced by the noticeably larger area under each curve.

	When troubleshooting issues with poor peak shape and recovery, contributions of interaction with the column housing are often overlooked. Here we show that there can be considerable interaction of certain analytes with metal surfaces within the column, which can be remedied with the use of a metal-free column. We propose that in any trouble shooting scenario where peak shape cannot be improved through modification of the mobile phase, the use of a metal-free column should be considered.


	2892 NW Upshur St., Portland, OR 97210
	tel. (888) 456-HPLC, (215) 665-8902, fax, (501) 646-3497
	Website: 

General

Metal-Free Columns Significantly Improve Peak Shape, Recovery, and Sensitivity for Phosphorylated Compounds

Drug-antibody ratio (DAR) is a critical attribute of antibody-drug conjugates (ADCs). DAR may often be determined quickly and effectively by combining SEC with three online detectors: MALS, UV, and dRI.

	There has been a significant resurgence in the development of antibody-drug conjugates (ADC) as target-directed therapeutic agents for cancer treatment. Among the factors critical to effective ADC design is the drug-antibody ratio (DAR), which describes the degree of drug appended to the antibody and directly impacts both potency and potential toxicity of the therapeutic. In addition to therapeutic/physiological effects, DAR can also affect drug product properties such as stability and aggregation. Determination of DAR is, therefore, of critical importance in the development of novel ADC therapeutics.

	DAR is typically assessed by mass spectrometry (MALDI-TOF or ESI-MS) or UV spectroscopy, both of which are subject to certain limitations: 1) calculations based on UV absorption are often complicated by similarities in extinction coefficients of the antibody and small molecule; 2) mass spectrometry, though a powerful tool for molecular weight determination, depends on uniform ionization and recovery between compounds-which is not always the case for ADCs. We present here a method for DAR determination based on SEC-MALS in conjunction with UV absorption and differential refractive index (dRI) detection, which overcomes these limitations for many ADCs.

	Two model ADCs based on the same monoclonal antibody and drug-linker system, but different conjugation processes, were analyzed. In this example the antibody has been alkylated with a compound having a nominal molar mass of 1250 g/mol.

	Each ADC was passed through size-exclusion chromatography (SEC) followed by the HPLC's UV detector, a DAWN multi-angle light-scattering (MALS) detector (Wyatt Technology, Santa Barbara) and an Optilab differential refractive index (dRI) detector (Wyatt Technology). Protein molar mass (

) of the ADC were determined at each elution volume by analysis of signals from the three detectors.

	Data collection and analysis were performed in the ASTRA software (Wyatt Technology) using ASTRA's Protein Conjugate Analysis method. Conjugate analysis is automated within ASTRA using the differential refractive index increments (dn/dc) and UV extinction coefficients, which are determined empirically for each species or mined from the literature.

	Figure 1 shows UV traces for the two model ADCs along with the calculated values of 

of each. As expected, the molar masses of the antibody components are quite similar; but 

 differs considerably. Despite the drug constituting just a small fraction of overall molar mass, this highly sensitive system shows robust 

 values which are well-distinguished from each other and vary little over the peak. The inset presents just 

, clearly showing the sensitivity of the system which differentiates the two molecules quite well.


	Figure 1: UV chromatograms of ADC1 and ADC2, overlaid with the molar masses of the complex as well as protein and drug components determined by MALS-UV-dRI analysis. Inset shows just the total complex masses of the two molecules. Peak-average results and final DAR are indicated in the table.

	Final DAR is calculated from the ratio of 

 to the molar mass of a single drug + linker compound, shown in the table. The results are consistent with values obtained by orthogonal techniques. Note that the molar mass traces for the conjugated moiety represent the total amount of attached pendant groups; the horizontal trends indicate that modification is nearly uniform throughout the population eluting in that peak.

	This analysis was successful thanks to the relatively large total appended mass, about 5% (ADC2) to 8% (ADC1) of the total molar mass of the complex. In general, for accurate SEC-MALS-UV-dRI protein conjugate analysis, the modifier mass should be at least 3% of the total. Hence a lower degree of conjugation might not have been amenable to analysis by this method.

	SEC-MALS-UV-dRI provides an easy, accurate means to determine DAR of ADCs in the course of product development and quality control. It is suitable for relatively high DAR values where the total drug-linker mass is at least 3% of the overall complex mass.


	6330 Hollister Avenue, Santa Barbara, CA 93117
	tel. +1 (805) 681-9009
	

Pharma/Drug Discovery

Drug-Antibody Ratio of an Antibody-Drug Conjugate by SEC-MALS

	- Separated five standard proteins at 3.0 mL/min

	- Maintained the same separationat 1.5 mL/min flow rate

	- Analysis can be completed in under 1 min

	SP-FT for Rapid Analysis using conventional HPLC


	420 Lexington Avenue, Suite 2335A, New York, NY 10170
	tel. (212) 370-0033, fax (212) 370-4566
	

SP-FT for Rapid Analysis Using Conventional HPLC

	UCT's Refine™ Ultra-Filtration technology allows for sample precipitation and filtration to occur simultaneously in the individual column/well. A novel, hydrophobically treated, submicron frit combination facilitates the removal of sample proteins without a complicated extraction.

	a) Place a collection plate under the Refine™ Ultra-Filtration Plate.

	b) Add 250 µL of urine and 250 µL Abalonase Ultra Enzyme working solution.

	d) Further dilute sample by adding 500 µL D.I. H

	e) Mix sample. This can be executed via vortexing at maximum speed or multiple pipette aspirations and dispenses.

	f) Filter the sample using one of the following techniques:

	1. Centrifuge: For 5 min at 500 g or until filtrate is collected.

	2. Vacuum: Apply vacuum at ~20" of Hg for up to 5 min or until filtrate is collected.

	3. Positive Pressure: Apply 2–5 psi using positive pressure for up to 5 min or until filtrate is collected.

 Agilent™ 1200 HPLC and AB Sciex™ 4000 Q Trap (MS/MS)

 UCT Selectra® DA HPLC column 100 × 2.1 mm, 3 µm

 UCT Selectra® DA guard column 10 × 2.1 mm, 3 µm

	Table I: Percent reduction in analyte suppression following use of Refine™ plate compared to dilute and shoot preparation for hydrolyzed urine samples.

	Using the above procedure, the Refine™ Ultra-Filtration technology allowed for a reduction in instrumental back pressure and removal of unwanted matrix components in urine samples. Overall, this allows for an end user to have enhanced analyte selectivity and an increase in HPLC/UPLC column life.


	Figure 1: Back pressure per sample injection of Refine™ Ultra Filtration technology versus a dilute and shoot preparation for hydrolyzed urine samples.


	2731 Bartram Rd, Bristol, PA 19007
	tel: (800) 385-3153
	

The Application Notebook-06-01-2018