Hyaluronic acid (HA) is an ubiquitous, very high molar mass polysaccharide that has been of particular importance in opthalmic surgery. HA acts as a molecular "shock-absorber" and stabilizer for cells and its visco-elastic properties are valuable for separating tissue and maintaining shape. It is a critical component in tissue lubrication and is believed to play a leading role in wound repair. Finally, HA's property of non-pyrogenicity makes it an ideal sheath for implants, whose presence might cause the body to suffer an immune response.

Among the many benefits high molecular weight HA hold are											

the maintenance of tissue space for surgery

HA's therapeutic effectiveness depends critically on molecular weight: the higher the molecular weight, the longer its benefit. However, because of its visco-elastic properties, standards-based GPC analysis is inappropriate for characterizing HA. There exist no standards identical to HA and the desirability of altering experimental conditions renders conventional GPC/SEC impractical. 

Combining a DAWN with HPLC separation, however, provides an ideal platform for absolute characterization, since the light-scattering measurements do not depend on pump speed, polymer standards or molecular conformation. 

A DAWN was connected to a GPC/SEC line (100 mM NaN03 buffer, TSK-Gel G6000PW column, Optilab DSP refractometer, Waters 510 pump) and generated the data required to determine not only absolute molar mass, but also molecular size, for a variety of HA products. 

 Figure 1: From the molar mass versus time plot, subtle differences can be seen among the samples.			

Figure 1 shows the molar mass versus time (with the 90° lightscattering chromatograms in the background) for the three samples, ranging from approximately 2 million to less than 200K Daltons. Figure 2 illustrates how profoundly different the samples are by revealing their differential molar mass distributions. These indicate that the samples will behave in different ways when used medically, depending on the content of their high molecular weight HA. 

Figure 2: The differential molar mass distributions calculated by ASTRA immediately confirm the large differences among the three HA samples.			

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Articles

Hyaluronic Acid (Polysaccharides)

Heterocyclic amines (HCAs) are a class of compound that are formed when amino acids from meat proteins react with glucose and creatine under high-temperatures, as is associated with frying or grilling over an open flame (1–3). Studies have correlated the consumption of high levels of HCAs with an increased risk of some forms of cancer. Though this has only been demonstrated in animal models, investigations into the possible correlation between cooked meat consumption and elevated cancer risks will continue in both Europe and the United States (1). 

Analysis of HCAs in food products can be challenging due to a number of factors including: (1) their relatively low level in food products, necessitating the use of highly sensitive techniques such as LC–MSMS, (2) the complex nature of the sample matrix, which can compromise HPLC column lifetime and confound analysis and (3) the basic nature of these analytes, which can lead to peak tailing issues. Herein, we present a sensitive method for the analysis of HCAs which utilizes solidphase extraction (SPE) for cleanup and a sub-2µm core-shell UHPLC column for the subsequent LC–MS-MS analysis. 

 Figure 1: Representative chromatogram of 12 eterocyclic amines analysed using a Kinetex 1.7Âµm C18 core-shell UHPLC column.			

Sample Preparation and Solid-phase Extraction:

For detailed sample preparation and SPE conditions, please contact Phenomenex. In brief, salmon tissue meat purchased from a grocery store was pulverized and then spiked with a mixture of HCAs and appropriate internal standards at a final concentration of 25 ng/g of tissue. This mixture was then subject to liquid-liquid extraction using hexane and acetonitrile. The aqueous acetonitrile layer was recovered for subsequent solid-phase extraction using a polymer-based strong cation-exchange cartridge (Strata™ -X-C 60 mg/3 mL). 

Figure 2: A representative extracted ion chromatogram for 7,8- and 4,8-dimethyl IQx, isomeric HCAs that must be baseline-resolved to enable accurate quantitation using LCâMS-MS. 			

 Kinetex® 1.7µm C18 with SecurityGuard™  ULTRA C18 

 A: 30 mM Ammonium formate; B: Acetonitrile 

 (80:20) A/B to (40:60) A/B in 3 min, then step to 100% B and hold for 1 min, then re-equilibrate at (80:20) A/B for 1min 

http://www.cancer.gov/cancertopics/factsheet/Risk/cooked-meats

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Food and Beverage

Rapid LC–MS–MS Analysis of Heterocyclic Amines in Salmon

This application demonstrates that stringent sensitivity requirements for the detection of potentially harmful primary aromatic amines can be fulfilled when using an Agilent 1290 Infinity LC system equipped with the 1290 large volume injection kit.

Primary aromatic amines (PAAs) can originate from printing azodyes and azo-pigments. PAAs are potentially harmful and suspected to cause cancer; they have to be detected and determined. 

Regulation (EC) 10/2011 sets a limit of 10 ppb for the sum of the released PAAs from plastic food contactmaterials. In the field of paper and board for food contact a similar limit is mentioned within BfR recommendation XXXVI. Moreover, the upcoming German regulation for printing inks used for food contact materials (Druckfarben V) negates the already introduced limit of 10 ppb for the sum of the released PAAs to all materials. Recently, problems have been reported within industry and enforcement authorities regarding the release of carcinogenic PAAs from heavily printed paper bags and napkins. 

Since the legal limits apply to the sum of all PAAs, these compounds have to be detected down to a level of at least 1 ppb (1 ng/mL) for an individual PAA. 

To meet these requirements, two modifications were made to the standard configuration of the Agilent 1290 Infinity LC System. A 60-mm Max-Light high sensitivity flow cell was used with the 1290 Infinity Diode Array Detector. A 40-µL loop including the 1290 large volume injection kit to inject volumes up to 120 µL was used with the 1290 Infinity Autosampler. 

The method that was finally applied was developed by using a mixture of six primary aromatic amines at 100 ng/mL: 1) aniline, 2) 

-toluidine, 4) 2-methyl-5-nitroanilin, 5) 2, 4-dimethylanilin and 6) 2, 4-dichloranilin (Figure 1). 

 Figure 1: Primary aromatic amines at 100 ng/mL: 1) Aniline: 3.314 min, 2) o-Anisidine: 4.882 min, 3) o-Toluidine: 5.159 min, 4) 2-Methyl-5-nitroanilin: 6.488 min, 5) 2,4-dimethylanilin: 7.908 min and 6) 2,4-dichloranilin: 11.373 min.			

The method starts with an enrichment step for the first minute followed by a steep increase to the starting conditions at 20% methanol. The gradient separation was done in the following 13 min up to 70% methanol on an Agilent ZORBAX Eclipse Plus C18, RRHT, 3.0 × 100 mm, 1.8 µm column. 

With the final method, a calibration was done for all PAAs from 100  ng/mL down to a level of 2 ng/mL (2 ppb). All linearity correlation coefficients were above 0.99983. The concentration level at 2 ng/mL was defined as the limit of quantification (LOQ signaltonoise ratio around 10). The limit of detection (LOD) was 0.5 ng/mL (signaltonoise ratio around 3). 

A statistical evaluation was done on the 10 ng/mL level. The relative standard deviation (RSD) of the retention times was between 0.039% and 0.057%, the area RSDs were between 0.5% and 2.7%. Relative standard deviation of the compound response factors were between 0.004 and 0.017%. 

To test the method, a blank matrix sample produced from a heavily coloured napkin was spiked with the six PAAs at various concentrations. All six PAAs could be well separated from the matrix compounds and identified and quantified with the developed method and calibration. 

This application shows that the Agilent 1290 Infinity LC system can detect low amounts of primary aromatic amines by using the 60mm Max-Light high sensitivity cell in the diode array detector, and the large volume injection kit in the autosampler.  

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Environmental

Developing a UHPLC Method for UV-based Detection and Quantification of Primary Aromatic Amines in Low Concentrations

Positron emission tomography (PET) is one of the most powerful non-invasive diagnostic tools for tracing organ functioning. Quality control of the short-livedradiopharmaceuticals is challenging, not least because of the tough time limits, the radiation issue and the near nanomole radiotracer quantities. 

 This article presents a likewise rugged and versatile multichannel radio IC system that controls the production of the radionuclide [

F]fluoride (precursor) and the two radiotracers synthesized from it, [

F]fluorocholine, in accordance with pharmacopoeial regulations. 

Principles of Positron Emission Tomography (PET)

Radiopharmaceuticals are radioactive substances used in nuclear medicine to diagnose, treat or prevent disease. They contain a radioactive isotope, a so-calledradionuclide, attached to a biologically inert or active molecule. 

Radionuclides are unstable isotopes that have an excess of either neutrons or protons and, therefore, radioactively decay, resulting in the emission of gamma rays orsubatomic particles. In proton-rich nuclides, a proton changes to a neutron, whereby a positron (antiparticle of the electron or an electron with a positive charge, also calledβ

  particle) is emitted together with a neutrino (υ) according to  

proton (p) → neutron (n) + positron ( β

While travelling in the surrounding media, the released positron loses its kinetic energy and then combines with an electron. The encounter annihilates both positronand electron and results in two photons (gamma rays) each with an energy of 0.511 MeV that are emitted in opposite directions (Figure 1).  

Sophisticated scanners (detector) can detect such pairs of photons by coincidence detection. From the data collected, threedimensional images of tissue structuresare then calculated. The most commonly used short-lived, cyclotron-produced radionuclides in radiopharmacy are 

F. Their respective half-lives are 20.38, 9.96, 2.03 and 109.7 min. 

To administer the radionuclide to a living human or animal, it is either incorporated in a biologically inert molecule (e.g., the blood flow tracers[

 O]butanol) or in a biologically active molecule that is absorbed by the organ of interest. 

 Figure 1: Principle of positron emission tomography. 

After the radiopharmaceutical is concentrated in the tissue of interest, the patient is placed in the PET scanner. By tracking the photons, computers withsophisticated software generate threedimensional images of the source of the photons. This allows the study of physiological, biochemical and pharmacological functions at amolecular level. Illnesses such as cancer, cardiovascular disease and even neurological disorders can be detected long before symptoms appear. 

Radionuclides used in PET experiments such as 

 F are artificially produced in a cyclotron, where a beam of accelerated charged particles irradiates a prepared target. Subsequently, the resultingradionuclides are isolated and synthetically incorporated into the radiotracer. Since the fluorine atom is similar in size to the hydrogen atom, it acts as a pseudohydrogen andis therefore ideally suited for replacing hydrogen atoms in organic molecules. The positron emitter 

F is thus one of the most important imagingradionuclides in diagnostic nuclear medicine. It is produced by proton bombardment of an 

F reaction, highly accelerated protons (p) react with the 

O atomic nucleus to emita neutron (n) and 

F, which immediately decays by positron emission with a half-life of 109.7 min. The product of the 

F from the target water, the radionuclide is incorporated into thechemical compound required. To this end, the preparation of reactive fluorine radionuclides in organic solvents is an important prerequisite for the synthesis of aliphaticcarbon-fluorine bonds. 

F]Fluorodeoxyglucose, commonly abbreviated [

F]FDG, or simply FDG, is a glucose analogue in which thehydroxyl group  

at the 2' position of the glucose molecule is substituted by [

 F]fluorine (Figure 2a). It throws light on the use and metabolism ofglucose in heart, lungs and brain. Additionally, it is used in oncology to determine abnormal glucose metabolism to characterize different tumour types. After administering[

F]FDG to the patient, it is incorporated into the cells by the same transport mechanism as the normal glucose, but unlike this, once inside thecell, it is not metabolized and thus remains in the cell allowing PET tomographic imaging. Not least because of its many diagnostic uses, the high number of existing labellingprocedures and its advantageous halflife of approximately two hours, which allows the transport to sites that have no cyclotron, [

F]FDG is actuallythe most frequently used organic PET radiopharmaceutical. 

In cells, choline is used as a precursor for the biosynthesis of phospholipids. As the latter are essential cell membrane components and because tumours revealincreased metabolismof cell membrane constituents and increased choline uptake, radiolabelled choline tracers are invaluable diagnostic tools for cancer detection.[

F]Fluorocholine (Figure 2b) is a recently developed PET radiotracer that allows choline metabolism to be imaged 

. It isbased on the tumour-detecting radiotracer [

C]choline. The driving force of the production of the 

F-labelled derivativewas the substantially longer half-life, which allows the distribution of this tracer to PET institutions without on-site cyclotron. 

Figure 2: Chemical structures of two PET radiopharmaceuticals. (a) In [18F]FDG, the hydroxyl group at the 2’ position of normalglucose is substituted by 18F. (b) In [18F]fluorocholine, a [18F]fluoroalkyl group is attachedto the nitrogen atom of N,N-dimethylaminoethanol (DMÃ¦). 

Radio IC is a powerful, fast and sensitive tool for the quality control of a wide range of PET pharmaceuticals. It aims at determining the radiochemical purity ofradiopharmaceuticals after radiosynthesis with cyclotron-produced nuclides. To this end, Radio IC uses a flowthrough mass and radioactivity detector. 

Besides the accuracy and reproducibility of the analytical results, high throughput is a must. One and the same multichannel radio IC takes over the quality controlof three production lines. The analytical unit provides the following benefits: 

The ion chromatography system installed at the ITP (Instituto Tecnológico PET) in Spain combines three quality control systems for PET pharmaceuticals in one.From the very same injection system, the flow can be automatically directed to the three channels. By selecting between an array of different columns, mobile phases anddetectors, [

F]fluorocholine can be separately determined (Table1). All aspects of system operation and data acquisition are controlled by MagIC Net™  software. 

Figure 3: (a) Conductivity and (b) radioactivity chromatogram of cyclotron-produced [18F]fluoride. In the subsequent radiosynthesis(nucleophilic fluorination), tracer (i.e., very low) quantities of [18F]fluoride ions are used to form carbon-fluorine bonds. The IC software convertsthe radiation units, counts per second (cps), to mV. Chromatographic conditions are shown in Table 1. 

The automated sample injection with Metrohm's patented Dosino technology allows the aspiration of very low sample volumes with accuracy and precision. By usingthe MagIC Net™ 

are completely automated without any carryover. The system's modular design supports the installation of the required lead shielding and thus guarantees usersafety. The injection valve is placed inside a 5 cm thick tailor-made lead housing, while radiation from the radiotracers in the separation columns is attenuated to a safe levelby a sufficiently thick lead shielding. In addition, a lead sample holder avoids user exposure to gamma radiation.  

c) Comprehensive Quality Control Using Multichannel Radio IC

Radiopharmaceuticals have unique characteristics and require special tests described in numerous pharmacopoeias. The quality control includes testing for bothchemical and radiochemical purity before the radiotracer is administered to the patient. The radiochemical purity of a radiotracer – the ratio of the radionuclide in boundform (e.g., [

 F]FDG) to the radionuclide in unbound form (e.g., [

 F]fluoride) – guarantees the image quality ofthe PET scan and protects the patient from unnecessary radiation. 

In the first instance, the concentration of the cyclotron-produced radionuclide, the 

 F, which is used as a precursor in the subsequentradiosynthesis, has to be determined (Figure 3). 

Figure 4: (a) IC-PAD chromatogram with the glucose precursor, the carrier-free [18F]FDG and the impurity chlorodeoxyglucose. (b)Radioactivity chromatogram of the [18F]FDG. The IC software converts the radiation units, counts per second (cps), to mV. Chromatographic conditionsare shown in Table 1 

Subsequently, the appropriate chemical purity – as for any other pharmaceutical preparation – and the concentration of the synthesizedradiopharmaceutical, the 

 F, which mostly is in the nanomolar range, have to be determined. In doing so, excessive precursors andradiolabelling-derived impurities have to be quantified also. Figure 4 shows the chromatogram with the glucose precursor, the carrier-free [

 F]FDGand the chlorodeoxyglucose impurity. The analysis is completed in less than 10 min.  

Figure 5 shows the chromatogram of the reaction mixture of [

 F]fluorocholine synthesis. Besides nanomole quantities of the[

 F]fluorocholine radiotracer, considerable amounts of the reactant N,N-dimethylaminoethanol and trace-levels of calcium impurities were detected inthe reaction mixture. 

Figure 5: (a) Conductivity and (b) radioactivity chromatogram of the [18F]fluorocholine reaction mixture.[18F]Fluorocholine is synthesized by 18F-fluoroalkylation of N,N-dimethylaminoethanol (DMÃ¦) using gaseous18F-fluorobromomethane. This labelling reaction results in high levels of residual DMÃ¦. Other potential byproducts such as bromocholine (notdetected) can additionally be determined. The IC software converts the  radiation units, counts per second (cps), to mV. Chromatographic conditions are shown in Table1. 

As most positron-emitting radiopharmaceuticals are characterized by short half-lives, there is a strong drive to reduce the time spent on quality control. Fast andprecise analyses are guaranteed by optimally harmonized and computer-controlled determination and rinsing sequences for detector pathways and the sample injection circuit.

 Table 1: Chromatographic conditions for the quality control of [18F]fluoride, [18F]FDG and [18F]fluorocholine.  

Metrohm's highly customizable chromatography system copes with the tough requirements of the radiopharmaceutical industry and pharmacopoeial regulations. Onesingle multichannel radio IC meets the quality control requirements of various production lines. Besides the high quality, the Metrohm IC presented ensures user safety, lowmaintenance costs and outstanding ruggedness. 

The authors thank the entire group of the Ion Chromatography Department of Gomensoro for their outstanding support and fruitful discussions during this project.

(1) D. Slaets, S. De Bruyne, C. Dumolyn, L. Moerman, K. Mertens and F. De Vos, Reduced dimethylaminoethanol in [

 F]fluoromethylcholine:an important step towards enhanced tumour visualization, 

(2) D. Kryza, V. Tadino, M. Azuzurra Filannino, G. Villeret and L. Lemoucheux, Fully automated [

 F]fluorocholine synthesis in theTracerLab MXFDG Coincidence synthesizer, 

Pharmceutical/Drug Discovery

Radio Ion Chromatography

Insulin was the first recombinant pharmaceutical produced. Currently, one of the critical quality control attributes of therapeutic insulin in the US and European Pharmacopoeia (USP and EP) monograph is the level of covalent high molecular weight (HMW) insulin as determined by HPLC size-exclusion chromatography. Presented in this application are the advantages that may be realized using a125 Å pore size, sub2-µm ethylene-bridged hybrid (BEH) silica packing material and Waters Ultra Performance liquid chromatography (UPLC®) instrumentation for the pharmacopoeial analysis of covalent HMW impurities in therapeutic insulin samples. Among these advantages are faster run times, higher sensitivity and higher resolving separations of insulin and covalent insulin HMW, while at the same time greatly reducing acetonitrile containing waste-stream volumes. 

Instrument: ACQUITY® UPLC H-Class Bio System with TUV detector and titanium flow cell 

Columns: ACQUITY UPLC® BEH125 SEC, 1.7 µm, 4.6 × 300  mm (part number 186006506) and Insulin HMWP HPLC, 10 µm, 7.8 × 300 mm (part number WAT201549)  

Mobile phase: L-arginine (1.0 g/L)/acetic acid (99%)/acetonitrile; 65/15/20 (v/v/v) 

Flow-rate:  0.4 mL/min for the ACQUITY® UPLC BEH125 SEC column and 0.5 mL/min for the HMWP HPLC column 

Injection volume:  10 µL for ACQUITY® UPLC BEH125 SEC column and 100 µL for the Insulin HMWP HPLC column 

For this application a comparison was performed between two SEC columns with particle sizes of 1.7 µm (UPLC) and 10 µm (HPLC) that are of comparable 125 Å pore size and equivalent 30 cm length (Figure 1). While both columns met the EP and USP HMW peak-to-valley ratio (P/V) system suitability criterion of NLT 2, and produced comparable HMW peak quantitation, the average HMW P/V for the UPLC® column (P/V = 37) was more than four times higher than that of the HPLC column (P/V = 8) and an insulin fragment was partially resolved at the tail of the monomer peak. In addition to dramatically increased resolution, the UPLC column also provides a smaller total elution volume (~ 5 mL) than the HPLC column. This decrease in total elution volume provides the analyst with increased sample throughput in addition to a decrease in mobile-phase use. 

 Figure 1: Zoomed view of chromatograms of a biotherapeutic insulin sample (analysed past expiry) generated by the ACQUITY UPLC BEH125 and the Waters HMWP columns. The complete chromatographic profiles are presented in the insets.			

The use of 125 Å pore size, sub-2-µm ethylene-bridged hybrid (BEH) silica packing material and Waters Ultra Performance liquid chromatography (UPLC) instrumentation for this traditional SEC-based analysis provides significant improvements in resolution compared to traditional SE-HPLC methods while reducing analysis time and mobile-phase use. 

Size-Exclusion Ultra Performance Liquid Chromatography Analysis of Insulin

The planar fungicide carbendazim (CASRN 10605-21-7) can be used to control mold on citrus crops but is not approved for use in the US or on imported products. Concentrations in citrus products can be rapidly and accurately determined using QuEChERS extraction with dSPE clean-up. 

a) Add 10 mL of juice to a 50 mL centrifuge tube

e) Centrifuge at 5000 rpm for 5 min at 20 °C

d) Transfer 0.5 mL to 2 mL autosampler vial 

Carbendazim is recovered in high yield and excellent precision from orange juice using this simple, rapid QuEChERS procedure. Limit of detection (LOD) and limit of quantification (LOQ) for this method are determined to be 0.4 and 1.4 ng/mL, respectively. 

 Figure 1: Matrix matched calibration curve.			

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Determination of Carbendazim in Orange Juice Using QuEChERS and LC/MS/MS

The migration to chromatographic methods utilizing smaller particle sizes has been well demonstrated to increase productivity through faster, more efficient separations. While significant improvements have been achieved with the use of sub-2 µm chemistries, the transition to UPLC® may be limited by available resources and regulatory guidelines (i.e., USP regulations). Here we demonstrate the increases in productivity attainable with conventional HPLC configurations, utilizing the new 2.5 µm, e

All data was collected on an Alliance® HPLC System with a PDA detector (254 nm). These evaluations utilized XSelect™  CSH™  C

 columns of various particle sizes and dimensions. The mobile phases were 0.1% formic acid in water and 0.1% formic acid in acetonitrile. A linear gradient from 25% to 35% acetonitrile was used with gradient times and flow-rates scaled according to particle size and column dimension. The sample is a mix of basic tricyclic antidepressants; nordoxepin, doxepin (cis & trans), desipramine, imipramine, nortriptyline, amitriptyline and trimipramine (10 µg/mL each). 

 Figure 1: Gradient separations of tricyclic antidepressants on a  5 Âµm, 4.6 Ã 150 mm, XSelectâ¢ CSHâ¢ C18 (A), a 2.5 Âµm, 4.6 Ã 75 mm, XSelectâ¢ CSHâ¢ C18 XP (B), and a 2.6 Âµm, 4.6 Ã 75 mm, core-shell C18 column. Sample loading on each column was 144, 72 and 72 ng, respectively. Flow rates were 0.99, 1.97 and 1.97 mL/min, respectively. Each chromatogram has been notated with average peak capacity (PC) and resolution for the desipramine/imipramine pair.			

Chromatographic methods can be scaled by maintaining the same ratio of column length to particle size (L/dp). A method developed for a 5 µm, 4.6 × 150 mm column easily transfers to a 4.6 × 75 mm column with a 2.5 µm particle size. This transfer represents an acceptable change in both column length and particle size under current USP guidelines (1). In addition to scaling for the shorter column, scaling the method to a faster flow-rate (based on the smaller particle size) further shortens analysis time as demonstrated in Figure 1 (A and B) for the separation of tricyclic antidepressants on XSelect™  CSH C

 chemistries. This method transfer example yields similar peak capacity and resolution and provides a 70% reduction in analysis time. 

Core-shell particle technology has shown utility in providing faster, more efficient separations. However, because the solid core is not chromatographically active, mass loading is limited for higher concentration samples. This is most dramatic for basic compounds under low ionic strength acidic conditions. This is demonstrated for the sample of basic tricyclic antidepressants on a core-shell C

 column in Figure 1(C). The mass load for each analyte on the 4.6 × 75 mm columns was 72 ng, a concentration typical for routine drug analysis. 

 columns provide the opportunity to scale existing HPLC methods to faster, more efficient methods while utilizing existing HPLC instrumentation and maintaining regulatory compliance. Packed with fully porous particles, better peak shapes are achieved for some applications at higher mass loads relative to coreshell particles. The 

 columns, available in each of the 14 XBridge™  and XSelect™  column chemistries, provide a diverse range of selectivity choices while enabling full scalability between HPLC and UPLC. For full application note, go to 

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tel. +1 (508) 478 2000, fax +1 (508) 872 1990 

(1 )USP Chapter <621> Chromatography, USP34-NF29. The United States Pharmacopeial Convention, official from December 1, 2011. 

Maximizing HPLC Productivity with 2.5 µm Extended Performance (XP) Columns

This application note describes a simple and effective procedure for the extraction of 25-OH vitamin D2 and 25OH vitamin D3 from human serum, using ISOLUTE SLE+  supported liquid extraction plates, with analysis by LC–MS–MS. The procedure incorporates an integral protein binding disruption step which eliminates the need for any offline protein disruption. Supported liquid extraction is a simple, high throughput alternative to traditional liquidliquid extraction for bioanalytical fluid samples, providing high analyte recovery, clean extracts and reduced sample preparation time.

  Dilute human serum (150 µL)  with water:isopropanol (50:50, v/v, 150 uL). Cap and shake for 1 min. 

   Load pre-treated serum (300 µL in total) onto the ISOLUTE SLE+    extraction plate (part number    8200400-P01). Pulse vacuum to initiate flow, then leave sample to absorb for 5 min. 

   Apply heptane (750 µL) and wait 5 min for the solvent to absorb. Apply a second aliquot of heptanes  (750 µL),  allow to soak for a further 5 min, then apply a final pulse of vacuum. 

   Evaporate to dryness at room temperature and re-constitute ininjection solvent (mobile phase A: mobile phase B, 70:30, v/v, 100 µL,). Cap and vortex gently for 60 s. 

Note: analytes are light sensitive and so amber glassware is recommended. 

Waters Acquity UPLC with 20 µL injection loop 

interfaced to Premier XE triple quadrupole  mass spectrometer equipped with an electro  

Isocratic 20%A: 80% B at 0.4 mL/min. Totalrun time 2.0 min.  

2 mM Ammonium Formate (aq) with 0.1% formic acid 

2 mM Ammonium Formate (99% MeOH 1% aq) with 0.1% formic acid 

For full MRM conditions please see AN757 at 

Recoveries of > 90% were achieved for all analytes, with relative standard deviation (RSD) < 10%. Limit of quantifications (LOQs) were 3.75 ng/mL for 25-hydroxyvitamin D2 and 1 ng/mL for 25-hydroxyvitamin D3. Excellent linearity (> 0.99) for both analytes was demonstrated (see Figure 1). DEQAS samples extracted using this method showed excellent correlation with nominal values. 

 Figure 1: Calibration curve for 25-OH Vitamin D3 from serum 

Supported liquid extraction using ISOLUTE SLE+ plates provides a simple, robust sample preparation method for extraction of Vitamin D metabolites from serum. The integral sample pre-treatment step is sufficient to eliminate analyte-protein binding, without any offline steps being required, leading to accurate determination of the analytes in serum samples. 

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Tel. +46 18 56 59 00  fax +46 18 59 19 22

(1) For complete details of this application, see Application Note AN757, available from 

Medical/Biological

Extraction of Vitamin D Metabolites from Human Serum Using Supported Liquid Extraction

Beta blockers can be abused in sports involving little physical activity, such as archery, to reduce cardiac contraction, heart rate and coronary blood flow. They have, therefore, been included in the list of forbidden substances by the International Olympic Committee. Selectivity is the most powerful tool to optimize separations in HPLC. This parameter is changed by using different bonded phases, including C18, C8, polar-embedded and phenyl-bonded phases, or by changing the mobile phase. In this work, Agilent Poroshell 120 columns and the Agilent 1200 Infinity Series LC Multi-Method Solution were used to quickly evaluate method development choices for the analysis of beta blockers. The short column length and high efficiency provided short analysis times and rapid equilibration leading to fast investigations of selectivity. 

An Agilent 1200 Infinity Series LC Multi-Method Solution was used. This system consisted of: 					

1290 Infinity Thermostatted Column Compartment (G1316C)

1260 Infinity High Performance Autosampler (G1367E)  

1290 Infinity Diode-Array Detector (G4212A), equipped with 10 mm MaxLight cartridge flow cell

G6140 Single Quadrupole Mass Spectrometer.

The Agilent 1200 Infinity Series LC Multi-Method Solution is a highly flexible system that can be used for up to 4 (100 mm) columns. In this work 4 Agilent Poroshell 120 columns were used.  

The TCC was fitted with a 6 position/14 port column selection valve. This is a new Quick Change Valve mounted on a slide-out rail to make plumbing and maintenance more convenient. Port 1 was connected to a StableBond C18 column, and port 2 was connected to EC-C18. Port 3 was connected to Bonus-RP, port 4 to PhenylHexyl and port 6 to a bypass connecting capillary. 

The solvent passing into each column was heated using 1 of 4 individual low-dispersion heat exchangers. An additional external 12-solvent selection valve was connected to channel A1 on the 1260 Infinity Binary pump. Together with the internal solvent selection valve of the 1260 Infinity Binary pump, up to 15 solvents could be screened using this system. 

A polar-embedded group inserted into the hydrophobic C

 alkyl chain allows the Bonus-RP phase on totally porous Poroshell 120 to minimize interaction of polar samples with silanols, providing symmetrical peaks for a wide variety of applications. This phase is especially useful at neutral pH where amines can interact strongly with ionized silanols. The polar-embedded group also helps to wet the hydrophobic chains, and prevents phase collapse in highly aqueous mobile phases. 

 Figure 1: Structures of some beta blockers.			

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 Hbond donors, like phenols and acids. Bonus-RP gives retention slightly less than a C18 and 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. 

The Phenyl-Hexyl phase has unique reversed-phase selectivity, especially for polar aromatics and heterocyclic compounds, derived from analyte interaction with the aromatic ring of the bonded phase and its delocalized electrons. Poroshell 120 Phenyl-Hexyl can be orthogonal to both C18 and Bonus-RP phases. More retention and selectivity will often be observed for solutes with aromatic electronwithdrawing groups such as fluorine or nitro groups. 

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 additional selectivity that is desired from a phenyl phase. 

 Figure 2: Separation of beta blockers using Agilent Poroshell 120 columns.			

The separation of all 7 compounds was accomplished on all columns surveyed (Figure 2). The Poroshell 120 EC-C18 column showed very close elution of acebutanol and propranolol and a double peak with the same molecular ion for naldolol. The double peak for naldolol was attributed to a diastereomer. This was not found with the Poroshell 120 Bonus-RP column, as it tended to minimize secondary interactions. The Poroshell 120 Bonus-RP column also reversed peaks 6 and 7 (propranolol and alprenolol), compared to the C18 columns. The Poroshell 120 Phenyl-Hexyl column shared this 6, 7 peak reversal and additionally reversed peaks 4 and 5 (metoprolol and acebutolol). The separation was similar to that shown by the other C18 columns, but was not as retentive. 

This work shows how phases with different selectivity can be used to optimize a separation. While the Poroshell 120 EC-C18 and SBC18 columns provide adequate separation, using an alternative selectivity column such as Poroshell 120 Bonus-RP yields even better results, and can be used for several thousand samples. Automatic setup of methods and sequences for the Poroshell 120 columns was straightforward using the Agilent 1200 Infinity Series LC Multi-Method Solution. 

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Fast Screening Methods for Beta Blockers by HPLC with Agilent Poroshell 120 Columns

Gel permeation chromatography (GPC), also referred to as size exclusion chromatography (SEC), when applied to polyolefins analysis (Polyethylene, PE and Polypropylene, PP) has been considered a demanding task due to the high temperature required for operation and the use of hazardous chlorinated solvents for resin dissolution.  

In the last twenty years the sample preparation process, of major importance in the GPC/SEC analysis of polyolefins, has not advanced significantly; vials are being filled with solvent manually and in most cases external filtration after dissolution is carried out with subsequent manual vial transfer before injection. Sample degradation, especially in the case of polypropylene, has not been fully eliminated and samples still remain at a high temperature longer than required for proper dissolution.  

It has been common practice to place the GPC/SEC columns, injector valves and detectors in the same oven compartment and when loop size or filter needs to be changed or the detector maintained the whole system needs to be cooled down to prevent column damage; thus, requiring slow cooling and heating rates and  a long stabilization time before analysis can be performed.  

Little effort has been applied to investigate chemical composition - molar mass interdependence in the case of polyolefins, and when it was established it demanded off-line FTIR systems or external flow through cells with liquid nitrogen cooled detectors.     

With these considerations in mind, Polymer Char initiated the design of a totally new GPC/SEC system based on a well-known infrared detector (IR4) which provides good stability and sensitivity for the analysis of polyolefins; infrared detection results in equivalent Molar Mass Distributions (MMD) curves to those obtained by DRI and without the presence of negative or spurious peaks after polymer elution.  

Today, the use of the IR4 detector in GPC/SEC analysis is common in many laboratories, meaning a significant breakthrough in detection technology. For the first time simultaneous concentration and composition signals were obtained on a single instrument; the composition signal allows the measurement of branches in PE copolymers, ethylene content in EP copolymers and vinyl acetate in EVA resins, all along the molar mass distribution. This new dimension in GPC analysis (Chemical Composition) comes at a time when new commercial copolymer resins demand such a composition–molar mass interdependence analysis. 

In this application note we describe the full integration of the new infrared detector IR5 MCT, which has outstanding sensitivity and stability, into the GPC-IR instrument redesigned in 2012.			

Besides the IR5 MCT detector and the most advanced sample preparation system, the new GPC-IR incorporates other technological developments, (see Figure 1), to fulfill Polymer Char's design philosophy, based on the principles of the most reliable hardware, full automation, user friendly operation, easy maintenance and proper diagnostic tools: 				

• Three independent thermostated compartments with adjacent walls for reduced length of connecting tubing are included within the main body of the GPC-IR which also holds all the instrument electronics: 

         • independent column oven, 

         • injector and filter compartment, 

         • detectors compartment. 

• Separate high temperature autosampler module, in which sample preparation takes place.

• Solvent management system incorporating Agilent HPLC pump and degasser, syringe dispenser and a software-controlled solvent recycling system.

• Differential viscometer and optional DAWN®  HELEOS™ II Multiple Angle Light Scattering (MALS) detector from Wyatt Technology® , complementing the IR5 MCT detection, and optional IR4.

• Advanced and comprehensive processing software GPC One which integrates all detectors' signals in a single package.

• Virtual Instrumentation Control software with advanced diagnostics tools and prepared for remote control access. 					

High Temperature Gel Permeation Chromatograph (GPC/SEC) with Integrated IR5 MCT Detector for Polyolefin Analysis: A Breakthrough in Sensitivity and Automation

The Application Notebook-07-02-2012