This application note demonstrates how comprehensive 2D-LC can be used to resolve the complex mixture of hydrophilic phenols found in virgin olive oil and investigates differences in the phenol composition of several olive oils.

Virgin olive oil is associated with the health and nutritional benefits of the Mediterranean diet. In this respect, the presence of antioxidants, which are represented by hydrophilic phenols among others, plays an important role. Hydrophilic phenols contained in virgin olive oil include phenolic acids and alcohols, flavonoids, secoiridoids, and lignans (1,2). 

One-dimensional liquid chromatography is not able to completely resolve the complex mixture of hydrophilic phenols present in virgin olive oil (3). Due to its high separation capability, comprehensive 2D-LC can be deployed to improve the separation. 

Comprehensive 2D-LC analysis was achieved with the Agilent 1290 Infinity 2D-LC solution. The first dimension separation used an  Agilent ZORBAX RRHD Eclipse Plus Phenyl-Hexyl column (2.1 × 150 mm, 1.8-µm) with a gradient of water and methanol, each with 0.1% formic acid, at a flow rate of 0.05 mL/min. In the second dimension, an Agilent ZORBAX RRHD Eclipse Plus C18 column (3.0  × 50 mm, 1.8-µm) was used with shifted gradients of water and acetonitrile, each with 0.1% formic acid, at a flow rate of 3.0 mL/min. Modulation was realized using the Agilent 2-position/4-port duo-valve, equipped with two 60 µL loops and with a modulation time of 30 s. Detection was performed at 260 nm and by ESI–TOF–MS in negative ionization mode. Preparation of olive oil samples was carried out according to the protocol from the International Olive Council (4). 

Four different olive oil samples with high phenol content (3) purchased from Italian olive oil farms were analyzed by comprehensive 2D-LC. Figure 1 (top) exemplarily shows the 2D-LC chromatogram of one olive oil. 

MS detection showed that the main hydrophilic phenols present in the analyzed olive oils are aglycons of oleuropein, ligstroside, and their derivatives. Further, elenolic acid, luteolin, apigenin, hydroxytyrosol, and hydroxytyrosol acetate were identified in all olive oils analyzed. Compared to the olive oils A–C (from the same farm), olive oil D (from another farm) showed higher percent responses of the flavonoids apigenin and luteolin (Figure 1, bottom). 

 Figure 1: Two-dimensional-LC chromatogram of an olive oil at 260 nm (top) and visualization of differences between the olive oils analyzed (bottom); blue circles indicate higher percent responses and white circles lower percent responses of substances detected, areas indicate differences.			

The Agilent 1290 Infinity 2D-LC solution can be used to significantly improve the separation of hydrophilic phenols contained in virgin olive oil. This enables the investigation of differences between the compositions of hydrophilic phenols in olive oils. 

(3) S. Schneider, "Quality Analysis of Virgin Olive Oils – Part 6," Agilent  Application Note, publication number 5991-3801EN, (2014). 

(4) "Determination of biophenols in olive oils by HPLC," International Olive  Council: COI/T.20/DOC. 29, (2009). 

5301 Stevens Creek Blvd., Santa Clara, California 95051, USA			

Articles

Food and Beverage

Determination of Phenolic Compounds in Virgin Olive Oil Using Comprehensive 2D-LC

The physical properties of polymers and polysaccharides depend strongly on their molecular properties, the most important of which are the molecular weight and molecular weight distribution, and the molecular size and structure. Gel permeation chromatography (GPC), also called size-exclusion chromatography (SEC), is the most commonly used tool for assessing these parameters, and is enhanced by coupling it to light scattering and viscometer detectors. In particular, the concurrent measurement of Rg using multi-angle light scattering (MALS) and intrinsic viscosity offers exceptional insight into the structure of synthetic and natural polymer molecules. 

In this application note, molecular weight Rg and IV data are combined to study the structure of different polysaccharides.  

 Figure 1: Chromatogram showing RI (red), LS (90Â°) (orange), and IV (blue) detector responses for pectin.  			

Analysis was carried out using a Viscotek TDAmax system with SEC-MALS 20 detector. Viscotek A6000M columns were used and all samples were dissolved overnight in the mobile phase to a concentration of 0.5–3 mg/mL, with an injection volume of 100 µL. 

Figure 2: Overlay of SECâMALS detector signals for pectin at all angles.			

Figure 1 shows an example chromatogram of pectin including the RI, LS (90°), and IV detectors, and Figure 2 shows the angular data from the SEC-MALS 20 detector. A slight angular dependence can be seen across the different angles, allowing Rg to be measured for this sample. 

The results are also plotted on a Mark-Houwink (IV vs. MW) plot for a visual comparison. This allows comparison of different samples and is ideally suited to the study of branching and other structural changes. 

Figure 3: Mark-Houwink plot comparing pectin with other polysaccharides.			

The Mark-Houwink plot shows that pectin is the highest and therefore the least dense molecule of those under investigation. As a comparison, gum arabic, which has the highest molecular weight, is the lowest on the plot, indicating that it is the smallest or most dense of the different samples.  

In summary, the molecular weight, size, and intrinsic viscosity of pectin was measured using the Viscotek TDA and SEC-MALS 20 systems and compared with a selection of other polysaccharides. The differences in molecular structures between the different polysaccharides are very clear and easily observed using the Mark-Houwink plot. 

(1) Characterizing polysaccharide structure with SEC-MALS and intrinsic viscosity measurements, Malvern Instruments application note, 

Enigma Business Park, Grovewood Road, Malvern, UK

Pharmaceutical/Drug Discovery

Characterizing Polysaccharide Structure with SEC–MALS and Intrinsic Viscosity Measurements

Pharmaceutical and personal care products (PPCPs) are products used for personal health or cosmetic reasons. This category includes a broad group of chemical substances such as human and veterinary medicines and cosmetics. The presence of PPCPs in environmental and potable water is a widespread concern due to the potentially harmful environmental effects. Evidence suggests PPCPs are linked to some ecological damage such as delayed development in fish (1). 

To ensure the safety of water, PPCP concentrations are stringently monitored by environmental regulatory bodies, including the United States Environmental Protection Agency (US EPA) (2). Detection of PPCPs is traditionally a complicated process due to the range of substances potentially present. Here we explore a simple, more convenient method than traditional solid-phase extraction (SPE)-based methods for highly sensitive PPCP detection, using triple quadrupole liquid chromatography–mass spectrometry  (LC–MS–MS). 

Conventional methods of PPCP detection in clean water have followed the defined EPA 1694 "template" for analysis, which requires the pre-concentration of large volume water samples and tedious solid-phase extraction cleanup, followed by liquid chromatography–mass spectrometry analysis to achieve the low ng/L (ppt) level detection necessary to comply with regulations (3). 

 Figure 1: PPCPs in environmental water and nearby soil is a widespread concern.			

Bruker has explored how LC–MS–MS can be employed specifically for the analysis of PPCPs in clean water. PPCPs were detected at 1–2 ppt with a linear response up to 200 or 500 ppt. Excellent system robustness was obtained throughout the extended method development and sample analysis period. 

Figure 2: Selected MRM chromatograms for PPCPs at 2 ppt.			

Case Study: Using LC–MS–MS to Analyze PPCPs in Clean Water

The study was carried out using ultrahigh-performance liquid chromatography (UHPLC) with an integrated on-line extraction (OLE) option coupled to a triple quadrupole mass spectrometer. The OLE module enables convenient method-driven on-line sample cleanup or sample pre-concentration. 

Figure 3: Selected calibration curves.			

Several water samples were analyzed for a range of PPCP species, including tap water samples along with bottled water and creek water. Samples were analyzed targeting a wide range of PPCP species representing compounds displaying varied properties and concentrations. Tables 1a and 1b illustrate the Advance UHPLC and EVOQ instrumentation set up respectively. 

Tables 1a & 1b: Instrumentation set-up for analysis of PPCPs in clean water.			

All of the PPCPs were detected at 2 ppt or better with the injection of 0.4 mL samples with a linear response range up to 200 or 500 ppt. The fast polarity switch can analyze positive and negative PPCPs in the same analytical segment with excellent linear response for both polarities (Figures 2 and 3). The results for the analysis of tap, creek, and bottled waters are shown in Table 2. 

Table 2: Test results for selected PPCPs in real water samples.			

The Bruker Advance UHPLC with OLE coupled to EVOQ LC–MS–MS detected PPCP samples at 2 ppt or better within 0.4 mL samples. Excellent linearity, sensitivity, and robustness were achieved throughout. The technique presents a more convenient and simpler approach to PPCP analysis than traditional SPE-based methods. 

(1) K. Hirsch, "Pharmaceuticals and Personal Care Products," (2013). Available at: 

http://serc.carleton.edu/NAGTWorkshops/health/case_studies/pharmaceutical.html

(2) US EPA, "PPCPs Basic Information," (2010). Available at: 

(3) US EPA, "EPA Method 1694: Pharmaceuticals and Personal Care Products in Water, Soil, Sediment, and Biosolids by HPLC/MS/MS," (2007). 

40 Manning Road, Billerica, Massachusetts 01821, USA

Tel: (978) 663 3660  Fax: (978) 667 5993 

Environmental

Keeping Water Safe: Detecting Pharmaceutical and Personal Care Products in Water Using Liquid Chromatography–Mass Spectrometry

Low-molecular-weight heparins (LMWHs) are obtained by fractionation or depolymerization of natural heparins. They are defined as having a mass-average molecular weight of less than 8000 and for which at least 60% of the total weight has a molecular mass less than 8000. 

Size-exclusion chromatography (SEC) has been the most common way of measuring the molecular weight and molecular weight distributions of LMWHs by using the two most common detection technologies: ultraviolet (UV) coupled with refractive index (RI) detection. However, these detectors embody a relative method in order to determine molecular weights, requiring calibration standards. A newer, absolute method involves the use of multi-angle light scattering (MALS), which does not require any standards. The European Pharmacopeia (EP) monograph for LMWH specifies the use of the UV–RI detection method and provides a known calibration standard. Many laboratories around the world have adopted this method. 

 Figure 1: Examples of UV and RI traces for an LMWH sample.			

We previously developed an SEC–MALS method and found it to be very suitable for the analysis of LMWHs. We have recently adopted the UV–RI method described in the EP monograph and compared the molecular weight results generated for LMWH using each detection type. The adopted method uses an Agilent LC-1200 series HPLC, 0.2 M sodium sulphate pH 5.0 mobile phase, Tosoh TSK-gel G2000 SWxl column with Tosoh TSK-gel Guard SWxl, Waters 2487 dual wavelength UV detector, and Wyatt Optilab rEX refractive index detector. For MALS analysis, the UV detector was replaced with a Wyatt miniDAWN TREOS detector; all other method aspects remained the same. 

Figure 2: Examples of LS and RI traces for an LMWH sample.			

The results indicated that both detection types are suitable and acceptable for the analysis of LMWHs. The molecular weight and distribution results generated using each detection type are comparable. This indicates that a SEC–MALS method could be adopted in place of the SEC–UV–RI method currently required by the EP monograph, and that it would result in less time because it obviates the need for calibration standards. 

This note was graciously submitted by Lin Rao and John Beirne of Scientific Protein Laboratories LLC. 

6300 Hollister Avenue, Santa Barbara, California 93117, USA

Tel: +1 (805) 681 9009  fax: + 1 (805) 681 0123 

Medical/Biological

Molecular Weight Determination of LMWH SEC–MALS vs. SEC–UV–RI

High standards have to be met by the pharmaceutical industry when it comes to drug quality and safety. These standards are documented in pharmacopoeias as officially recognized pharmaceutical rules, and published as legal tools of customer protection by authorities such as governments and medical societies. The identification of a drug depends on sensitive, reliable instruments and methods — as does the determination of the drug's compliance with applicable regulations. 

Ion chromatography (IC) is the method of choice to determine active ingredients, excipients, and traces of impurities, as well as metabolites in the form of organic and inorganic ions or polar substances, in a number of pharmaceuticals, pharmaceutical solutions, and even body fluids. It can determine several substances within a very short time in a single analysis — and can even distinguish chemically similar analytes. The concentration of analytes can vary from ng/L up to the per cent range. The large selection of separation columns and elution systems available makes IC useful for almost any kind of analyte. Interfering effects caused by the sample matrix can easily be avoided by using the right sample preparation or choosing a suitable detection method. In-line sample preparation is a feature of many modern IC systems, as the focus of recent advances in IC has been mainly on ease of use. However, convenience is not the only advantage brought by automation of the IC process: Reducing human interference to a minimum also means reducing the chances of mistakes and contamination. 

Depending on the requirements of analyte and matrix, there is a broad range of detection methods to choose from: 					

Conductivity detection with and without suppression

Spectrophotometric detection with and without post-column derivatization (UV–vis)

Coupled detection methods such as IC–MS and IC–ICP–MS

Pharmaceutical samples come in many different forms which require different ion chromatographic approaches. What follows is an overview of frequent sample types with example analyses. 

The term "pharmaceutical solutions" denotes isotonic solutions, hemodialysis solutions, or infusion solutions. They contain anions, cations, carbohydrates, and organic acids, the concentrations of which frequently differ from one another by several orders of magnitude. Within the context of production monitoring and final quality control, an analysis method is required that can determine these ingredients with a high degree of precision. In addition, the analysis should be quick and require minimal effort. With its intelligent analytical procedure and automatic in-line sample preparation, IC fully accomplishes this task. 

Two example analyses of hemodialysis solutions are shown in Figures 1–2. Patients suffering from renal failure require hemodialysis to compensate for the loss of the kidney's blood-cleansing function. During the process, the patient's blood exchanges solutes with a hemodialysis solution through a semipermeable membrane. The exchanged solutes include, among others, waste products such as urea and phosphate, which diffuse out of the blood and into the dialysis solution along the concentration gradient. The composition of dialysis solutions is complex because the removal of solutes from the blood changes its osmotic activity; therefore, it has to take place at a controlled rate, which is achieved by the right solute concentration. A strong change in osmotic activity can cause dialysis disequilibrium syndrome where, because of the low solute concentration in blood, solutes are washed out from other body compartments. 

 Figure 1: IC measurement on a Metrosep A Supp 7 - 250/4.0 using Na2CO3 gradient elution, followed by sequential suppression and conductivity detection. (a): Anion standard including acetate and citrate; (b): acetate and citrate in hemodialysis solution.			

Figure 1 shows the simultaneous determination of citrate and acetate in diluted hemodialysis solution. In part A, an anion standard was measured; part B shows the sample determination. Citrate is added to hemodialysis solutions for its anticoagulant properties and acetate is added as a buffer substance. It is transferred to the patient's bloodstream during hemodialysis and stabilizes the blood's pH value. This is necessary because the kidneys of dialysis patients are not capable of excreting acid components – therefore, patients are often acidotic. 

Besides citrate and acetate, the chromatogram reveals the presence of a close to physiological concentration of chloride. By using physiological solute concentrations, the concentration gradient is reduced to a minimum and a dynamic equilibrium is reached between the blood and dialysis solution. The loss of certain solutes — including chloride — is thereby prevented. 

Figure 2 shows the determination of cations in hemodialysis concentrate after an automated in-line dilution step. Like chloride, the cations are present in close to physiological concentrations to avoid their drainage from patients' blood by osmosis. 

Figure 2: Cations in diluted hemodialysis concentrate using the Metrosep C 4 - 150/4.0 column and non-suppressed conductivity detection.			

Active pharmaceutical ingredients (APIs) in medicines such as gentamicin, neomycin, cefadroxil, or bethanechol chloride can be determined by IC in accordance with the regulations of the U.S. Pharmacopeia and European Pharmacopoeia. The requirements regarding precision, separation, and recovery of the analytes are described in detail in the pharmacopoeias. Figure 3 depicts the ion chromatogram of an analysis of gentamicin, an antibiotic belonging to the group of aminoglycosides. Aminoglycosides are bactericidal antibiotics that block protein biosynthesis by binding to ribosomes, thereby causing mistakes in the translation from mRNA to DNA. Gentamicin consists of several closely related compounds, namely gentamicin C

. In spite of their structural similarity, IC achieves a good separation of the different gentamicin components. 

Figure 3: IC determination of the antibiotic gentamicin by pulsed amperometric detection; column: Polymer Laboratories RP-S; eluent: 60 g/L Na2SO4, 1.75 g/L sodium octane sulphonate, 1.34 g/L NaH2PO4, 8 mL/L THF (pH = 3, H3PO4); post-column addition: 300 mmol/L NaOH.			

Apart from API analysis, it is also possible to determine impurities in pharmaceutical products by IC. Even small concentrations of an impurity can cause significant side effects. For example, in the synthesis of the antihypertensive irbesartan, azide can be detected in traces as an impurity in the product. Azide is strongly toxic to humans and its concentration in irbesartan is therefore subject to rigorous controls. The U.S. Pharmacopeia recommends ion chromatographic azide determination after direct injection according to USP<621>. In this method, a transfer solution consisting of the IC eluent and a suitable organic solvent is used to remove the API from the analytical column. However, this procedure is tedious, time-consuming, and cannot be automated.

Figure 4: Irbesartan sample spiked with 5â80 Âµg/L azide; column: Metrosep A Supp 10 - 250/4.0; eluent: 5 mmol/L Na2CO3, 5 mmol/L NaHCO3; inline matrix elimination with 70:30 (v/v) methanol/water.			

Azide determination is more selective, more sensitive, and, above all, quicker with the use of in-line matrix elimination, where the interfering pharmaceutical matrix is separated from the target analyte in the course of sample preparation. The ion chromatogram in Figure 4 shows the analysis of an irbesartan sample spiked with different concentrations of azide. The signal is recorded by a conductivity detector following sequential suppression. Table 1 lists the average recovery values of azide that were achieved over three measurements, as well as the mean conductivity measured by the detector and the relative standard deviation. 

Table 1: Precision and recovery of azide.			

The determination of azide in irbesartan with preceding matrix elimination fulfills all requirements of the regulatory authorities, which concern the selectivity of the method, its limits of detection and quantitation, precision, linearity, accuracy, and robustness. Therefore, it can be used as a quicker and more sensitive alternative to the proposed determination according to USP<621>. 

Table 2: Selection of IC applications in the pharmaceutical industry.			

Today, IC covers a diverse field of applications in the pharmaceutical industry. A selection of three out of more than 80 applications is listed in Table 2. The technique has become extremely versatile because of the large number of different columns, eluent and gradient options, sample preparation techniques, and automation possibilities that are available to the user. 

Ion Chromatography – A Versatile Tool in Pharma Analysis

Proteins have a tendency to aggregate over time and the risk for drugs in the biopharmaceutical industry is that the presence of aggregates will stimulate an immune response. Size-exclusion chromatography (SEC) is a powerful tool that is commonly used to look at the aggregation of proteins. 

SEC separates proteins by size, and is commonly used to measure molecular weight and characterize aggregation. By adding a light scattering detector to the system, the molecular weight of the protein monomer, oligomers, and aggregates in a sample can be measured independent of their elution volume. In addition, multi-angle light scattering (MALS) can also be used to measure the radius of gyration (Rg) of large aggregates that scatter light anisotropically. 

Pepsin was characterized using a Viscotek TDAmax system connected to a Viscotek SEC-MALS 20 detector. Two Viscotek protein columns were coupled together for the separation. The detectors and columns were all held at 30 °C to ensure a good separation and to maximize baseline stability of the detectors. 

 Table 1: Measured molecular weights of the different peaks of the pepsin sample.			

Figure 1 shows that the pepsin sample contains 2 main components. The molecular weight of the second peak (18.5 mL) is measured at 34.7 kDa, which is very close to the known molecular weight of pepsin (35 kDa). The larger light scattering peak at 11 mL has a much higher and more variable molecular weight, clearly identifying it as some disordered aggregates, which are unlikely to be active. The MALS plot in Figure 2 shows that these aggregates are strongly anisotropic scatterers, and therefore have a large size compared to the protein. 

Figure 1: Chromatogram of pepsin showing the refractive index (red) and SECâMALS (90Â°) (orange) detector signals.  			

As pepsin is a digestive enzyme, the broad peak at 20.9 mL is most likely to be digestion products. 

Figure 2: SEC-MALS plot showing different angular response for pepsin monomer and aggregates.			

The molecular weight and aggregate content of pepsin was successfully measured using the Viscotek SEC-MALS 20 system, and their amounts quantified. Where the aggregates are large enough, their size (Rg) can also be measured. 

(1) Measuring protein aggregation with the Viscotek SEC-MALS 20, Malvern Instruments application note, 

Enigma Business Park, Grovewood Road, Malvern, UK	

Detection and Quantification of Protein Aggregates by SEC–MALS

Using Selectivity to Enhance Separation of Analgesics

Selectivity is the most powerful tool to optimize separations in HPLC. This parameter is changed by using different bonded phases, including C18, polar embedded, phenyl bonded phases and perflorophenyl, or by changing the mobile phase. In this work, 4.6 × 50 mm Poroshell 120 columns are used to quickly evaluate method development choices for the analysis of non-steroidal anti-inflammatory drugs (NSAIDS). The short column length and high efficiency provide short analysis times and rapid equilibration, leading to fast investigations of selectivity.

Instrument:  Agilent 1260 Infinity Binary LC System 

The Agilent 1260 Infinity Binary LC System was configured as follows: 					

				 G1312B Binary Pump SL, capable of delivering up to 600 bar 			

				 G1316C Thermostatted Column Compartment (TCC) 			

				 G1376D High Performance Autosampler SL Plus 			

				 G4212A Diode Array Detector equipped with a G4212-60008   10 mm path length, 1 µL volume flow cell 

The following columns were used in this study. 					

  Agilent Poroshell 120 PFP, 4.6 ×  50 mm, 2.7-µm (p/n 699975-408) 

  Agilent Poroshell 120 EC-C18, 4.6 × 50 mm, 2.7-µm (p/n 699975-902) 

Agilent Poroshell 120 Bonus-RP, 4.6 × 50 mm, 2.7-µm (p/n 699968-901) 

  Agilent Poroshell 120 Phenyl-Hexyl, 4.6 × 50 mm, 2.7-µm  (p/n 699975-912) 

A generic gradient separation was used to evaluate these columns consisting of ammonium formate (20 mM NH

 pH 3.0) using either methanol or acetonitrile.  

Figure 1: Structures of selected analgesics.			

The analgesic materials all possess a wide variety of functional groups including fluorine (sulindac and diflunisal) and chlorine (diclofenac). The structures of the compounds examined are shown in Figure 1 and Table 1. All samples were prepared at 10 mg/mL in acetonitrile and were diluted in water to a final concentration of 0.1 mg/mL. 

 Table 1: Retention time, Log P, and pKa data for selected analgesics.			

The columns were chosen to improve selectivity in the separation. They included a highly end capped C18 column recommended as a first choice in method development (Poroshell120 EC-C18).  

Poroshell 120 Bonus-RP can be used for many of the same separations as a C18 column while avoiding some of the disadvantages of C18, such as poor wettability in high aqueous mobile phases. In addition, it is much more retentive for those molecules that can interact by hydrophobic interactions and also by H-bonding with the amide group. Compared to alkyl only phases, Bonus-RP has enhanced retention and selectivity for phenols, organic acids, and other polar solutes due to strong H-bonding between polar group (H-bond acceptor) and H-bond donors, like phenols and acids. Bonus-RP gives retention slightly less than a C18 allows, for easy column comparison without the need to change mobile phase conditions. The Bonus-RP phase gives different selectivity than C18 for polar compounds. It is also compatible with 100% water. 

Poroshell 120 Phenyl-Hexyl columns deliver unique selectivity for compounds with aromatic groups, providing superior resolution for these samples. Poroshell 120 Phenyl-Hexyl can also provide optimum separations of moderately polar compounds where typical alkyl phases (C18 and C8) do not provide adequate resolution. Acetonitrile tends to decrease the π–π interactions between aromatic and polarizable analytes and the phenyl-hexyl stationary phases, but methanol enhances those same interactions, giving both increased retention and changes in selectivity. This does not mean that acetonitrile should not be used with a phenyl bonded phase or that it might not provide an acceptable separation, but methanol is more likely to deliver the different selectivity that is desired from a phenyl phase. 

Poroshell 120 PFP columns possess a pentafluorophenyl ligand. This can provide an orthogonal separation mechanism to traditional reverse phase columns. By specifically targeting many polar retention mechanisms, PFP phases can separate analytes based on small differences in structure, substitution, and steric access to polar moieties. The resulting selectivity for positional isomers, halogenated compounds, and polar analytes is particularly useful in the analysis of complex mixtures, and small molecule pharmaceuticals  

The separation of all nine compounds was attempted on all columns surveyed. The Poroshell 120 PFP and Poroshell 120 Bonus RP columns both fully resolved all compounds in the same order, although the spacing of the peaks is more even on PFP. The Poroshell 120 Phenyl Hexyl column does not yield the same elution order as the Poroshell 120 PFP column. This means that the PFP column is not just a stronger phenyl column, other interactions beside π-π and hydrophobic interactions are in play. All four columns elute acetominophen (APAP) and phenacetin first. The Poroshell 120 EC-C18 column did not fully separate three compounds (Tolmetin, Ketoprofen, and Sulindac).  

Figure 2: Separation of analgesics using Agilent Poroshell 120 columns using methanol.			

Figure 3 shows the separation on all four columns using acetonitrile. In this case, only Poroshell 120 PFP resolves all compounds, and Poroshell 120 EC-C18 and Poroshell Phenyl Hexyl columns elute all compounds in the same order. Typically π-π interactions with Phenyl Hexyl columns are overwhelmed in acetonitrile. Again, the PFP and Bonus RP columns have very similar elution orders (with the exception of the last two peaks).   

Figure 3: Separation of analgesics using Agilent Poroshell 120 columns with acetonitrile.			

Since the Poroshell 120 PFP phase almost separates all nine compounds when using methanol or acetonitrile, it provides the best method development option for further development.  

Figure 4: Poroshell 120 EC-C18 retention time vs. log P values.			

Table 1 lists the retention time of all nine analytes on the four columns using both methanol and acetonitrile. Log P and pKa data are also listed. The Log P refers to the equilibrium distribution of a single substance between two solvent phases separated by a boundary. It was discovered that the narcotic action of many simple organic solutes was reflected rather closely by their oil-water partition coefficients. The oil was later replaced by octanol (5). In Figure 4, Log P values correspond to retention time on the Poroshell 120 EC-C18 column using methanol or acetonitrile. Figure 5 shows the correlation of Poroshell EC-C18 and PFP retention time data in methanol and acetonitrile for the last seven analgesics. The first two analgesics, acetominophen and phenacetin, remain in the same elution order in all solvent column combinations. With these compounds removed, the correlation between EC-C18 and PFP retention in methanol (5a) and acetonitrile (5b) is poor and highly indicative of orthogonality. 

Figure 5: Poroshell 120 EC-C18 retention time vs. Poroshell 120 PFP retention time.			

Analysis problems can be quickly resolved by including survey methods with generic gradients as part of the method development scheme. This work demonstrates how different chemistries and organic modifiers such as acetonitrile and methanol can develop different selectivity and can be used to optimize the separation. In this case, using an alternative selectivity column such as Poroshell 120 PFP yielded better results than a more commonly used C18 chemistry. Fluorinated stationary phases are useful because of their enhanced interaction with halogens, and conjugated compounds. 

(1) A. Streitweiser Jr. and C.H. Heathcock, 

http://en.wikipedia.org/wiki/Non-steroidal_anti-inflammatory_drug

(3) J. Sangster, "Logkow: A Database of Evaluated Octanol/Water Partition Coefficents," (Log P), 

http://logkow.cisti.nrc.ca/logkow/index.jsp

2850 Centerville Road, Wilmington, Delaware 19808, USA 		

Fast Screening Methods for Analgesics and Non-Steroidal Anti-Inflammatory (NSAIDS) Drugs by HPLC with Agilent Poroshell 120 Columns

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). The DAR describes the degree of drug addition that directly impacts both potency and potential toxicity of the therapeutic, and can have significant effects on 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. Calculations based on UV absorption are often complicated by similarities in extinction coefficients of the antibody and small molecule. Mass spectrometry, though a powerful tool for M

 determination, depends on uniform ionization and recovery between compounds — which is not always the case for ADCs. 

Here we present a method for DAR determination based on SEC–MALS in conjunction with UV absorption and differential refractive index detection. Figure 1 shows UV traces for two model ADCs; molecular weights of the entire ADC complexes are determined directly from light scattering data. 

Component analysis is automated within the ASTRA 6 software package by using the differential refractive index increments (dn/dc) and extinction coefficients, which are empirically determined for each species or mined from the literature, to calculate the molar mass of the entire complex as well as for each component of the complex. 

 Figure 1: Molar masses for two distinct ADC formulations are determined using SECâMALS analysis.			

In this example an antibody has been alkylated with a compound having a nominal molecular weight of 1250 Da (Figure 2). Molar masses of the antibody fractions are similar, which indicates that the overall differences between the two formulations reflect distinct average DARs that 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 uniform throughout the population eluting in that peak. 

Figure 2: Molar masses for the antibody and total appended drug are calculated in the ASTRA software package based on prior knowledge of each component’s extinction coefficent and dn/dc, allowing determination of DAR based on a nominal Mw of 1250 Da for an individual drug.			

Tel: +1 (805) 681 9009  Fax: +1 (805) 681 0123 

Antibody Drug Conjugate (ADC) Analysis with SEC–MALS

Soil is a complex matrix consisting of organic and inorganic material. It possesses many active sites that can retain pesticides and other residues. Compared to other matrices, soil samples are more difficult to extract and often require longer extraction times. Certain compounds may be covalently bound to the soil. These can only be removed using acid or base hydrolysis. If a hydrolysis step is employed, this may have a detrimental effect on pH sensitive analytes. This technique was not evaluated in the course of this study. 

The aim of this study was to evaluate the effectiveness of the QuEChERS extraction and cleanup approach for pesticides in soil. A group of 21 LC–MS–MS amenable pesticides, comprising various chemical properties, were used for the study. 

O content) into a 50 mL centrifuge tube. Alternatively, weigh 3 g air-dried soil sample into a 50 mL tube and add 7 mL H

O, vortex briefly, and allow to hydrate for 30 min. 

2. Add 10 mL of acetonitrile to each sample. 

3. Shake (manually or mechanically) or vortex samples for 5 min to extract pesticides. (In this study a Spex SamplePrep Geno/Grinder 2010 operated at 1500 rpm was used). 

 (citrate salts) Mylar pouch to each centrifuge tube. 

5. Immediately shake samples for at least 2 min.  

6. Centrifuge for 5 min at ≥ 3000 rcf. 

7. Transfer a 1 mL aliquot of supernatant to a 2 mL CUMPSC18CT (MgSO

9. Centrifuge for 2 min at high rcf (e.g. ≥ 5000). 

10. Filter purified supernatant through a 0.2 μm syringe filter directly into a sample vial. 

In the dSPE step, a combination of PSA/C18 gives cleaner extracts than PSA alone. In this study, no major variation in results was observed between PSA and PSA/C18. In fact, the PSA/C18 gave slightly better results overall. 

Thiabendazole gave low, though reproducible recovery throughout the study. Thiabendazole is a planar pesticide and could potentially be retained by strong hydrophobic interactions on the soil. In addition, it is a basic compound that is positively charged at low pH and is capable of being retained on the soil through ionic interactions, particularly by humic/fulvic acids. 							

2731 Bartram Road, Bristol, Pennsylvania 19007, USA  

The Application Notebook-07-02-2014