This application note demonstrates an effective and efficient supported liquid extraction (SLE) protocol for the clean-up of urinary cortisol prior to analysis by gas chromatography–mass spectrometry (GC–MS). ISOLUTE SLE+ Supported Liquid Extraction columns offer an efficient alternative to traditional liquid-liquid extraction (LLE) for bioanalytical sample preparation, providing high analyte recoveries, no emulsion formation and significantly reduced sample preparation time.

ISOLUTE SLE+ 200 μL Supported Liquid Extraction Plate, part number 820-0200-P01. 

Load the pre-treated sample (200 μL) onto the plate and apply a pulse of vacuum (VacMaster 96 Sample Processing Manifold, 121-9600) or positive pressure (PRESSURE+ 96 Positive Pressure Manifold PPM-96) to initiate flow. Allow the sample to adsorb for 5 min.  

Apply methyl tert-butyl ether (MTBE) (1 mL) and allow to flow under gravity for 5 min. Apply vacuum or positive pressure to pull through any remaining extraction solvent, collecting into collection plate. 

Evaporate the extract to dryness (40 °C) (SPE Dry 96 Sample Concentrator SD-9600-DHS-EU). Reconstitute in water:methanol (50:50, v/v) (100 μL). 

Waters 2795 Liquid Handling Unit (Waters, Milford, Massachusetts, USA) 

Phenomenex core shell Kinetex C18 2.6 μm, 50 × 2.1 mm (Phenomenex, Macclesfield, UK) 

OAc/0.1% formic acid (aq) (A) and 2 mM NH

OAc/0.1% formic acid in MeOH (B) at a flow rate of 0.2 mL/min  

Waters Ultima Pt triple quadrupole mass spectrometer (Waters, Manchester, UK) equipped with an electrospray interface 

Positive ions were acquired using multiple reaction monitoring (MRM): Cortisol quantifier, 363.1>121.00; Cortisol Qualifier 1, 363.10>97.10; Cortisol Qualifier 2, 363.10>309.10; Cortisol-d4, 367.30>121.00.  

Figure 1: Cortisol calibration curve (lyophilized urine) 25â2000 ng/mL. Chromatogram at 25 ng/mL.			

The method demonstrated high reproducible recoveries 99–101% (n = 7) with all relative standard deviations (RSDs) below 10%. The limit of quantitation (LOQ) was calculated as 25 ng/mL when compared to the calibration curve using lyophilized urine, which showed excellent linearity. Low ion suppression was observed, allowing LOQs below the therapeutic range from 100 μL of urine.  

This application note presents a simple method for the extraction of cortisol from urine demonstrating high recoveries, low ion suppression and acceptable validation parameters.  

For more information on this or to view any of Biotage's extensive database of sample preparation applications please visit 

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Articles

Medical/Biological

Urinary Cortisol Extraction Using Supported Liquid Extraction Prior to LC–MS–MS Analysis

Polysaccharides are very important in nature, occurring in food (starches in rice, wheat, etc.) and plants (cellulose). Some polysaccharides are also produced commercially, for example dextrans, which are manufactured through the fermentation of sugar solutions. These are higher molar mass polysaccharides.  

Dextrans are used in clinical and technical applications, where molecular weight is critical in determining the properties of the final product. Accurate determination of the molecular weight distribution is vital.  

On the other hand, low molar mass saccharides are also very common in food, such as fruits, honey and sweets. Examples of low molar mass sugars are mono- (glucose, fructose), di- (lactose, isomaltose, trehalose) and trisaccharides (maltotriose, isomaltotriose). The separation and identification of low molar mass polysaccharides is a challenge as the compounds have the same chemical formula and only small differences in structure, for example, disaccharides maltose, isomaltose, gentiobiose cellobiose and trehalose C

Columns:  PSS SUPREMA 5 μm 3 × 100 Å (8 × 300 mm)    + precolumn   

Figure 1: Overlay of elugrams of a glucose (red curve) with a low molar mass Dextran T1 (black curve).			

A high resolution and therefore a good separation on the column is necessary for a precise analysis. This is particularly important when new analytical LC coupling methods like GPC/SEC–ESI-MS are used, as the MS detector requires the columns to have a much higher resolution power within an overall smaller column volume.  

Figure 2: Overlay of elugrams of isomaltose (black), maltose (red), gentiobiose (green), cellobiose (dark green) and trehalose (blue).			

The new SUPREMA column, with a reduced particle size of 5 μm, offers a significant improvement in performance compared to 10 μm materials and provides outstanding additional resolution, especially in the low molecular weight area, which is a major consideration when analysing oligomeric polysaccharides.  

The analysis of dextran T1 shows the separation power when a combination of three SUPREMA 5 μm 100 Å columns is used. The oligomers in the low molecular weight are able to be resolved up to P10. A glucose separation is overlaid, as a reference. 

The analysis of different disaccharides shows the ability to separate compounds with the same chemical formula and with only small differences in structure and hence size in solution. 

PSS SUPREMA 5 μm columns can be used for numerous neutral and anionic aqueous applications in the molecular weight area between 100 Da to around 5 million Da. The columns are available in analytical (i.d. 8 mm) and micro (i.d. 4.6 mm) dimensions with different porosities. Linear or mixed columns are also available. 

In der Dalheimer Wiese 5, D-55120 Mainz, Germany  

tel: +49 6131 962390 fax: +49 6131 962390 11  

Food and Beverage

Saccharide and Polysaccharide Analysis

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 methods aspects remained the same. 

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.  

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

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  

Molecular Weight Determination of Low-Molecular-Weight Heparins: SEC/MALS vs. SEC/UV-RI

James Martosella, Phu Duong and Alex Zhu, 

Peptide fragment analysis by liquid chromatography–mass spectrometry (LC–MS) is a crucial requirement for the release of biotherapeutic products and plays an important role in completing the protein characterization strategy. For example, the stability of a recombinant protein therapeutic requires long-term monitoring throughout the shelf life of the product for important modifications, such as glycosylation. Although glycosylation can be analysed on the intact protein, glycopeptide mapping is necessary to provide additional critical information, such as sequence information, mass analyses and identification of the glycosylation sites.  

Here, we used a recombinant human erythropoietin (rhEPO) protein as a model glycoprotein to demonstrate high resolution peptide mapping and glycopeptide analysis by LC–MS using a new 2.7 μm Agilent 

The column was specifically designed to improve separations for peptide mapping applications and provides increased resolution, higher sensitivity and greater selectivity needed for high efficiency peptide profiling, such as that required for rhEPO mapping and enhanced glycopeptide mining. Furthermore, the 2.7-μm superficially porous particles enabled the use of a longer column (2.1 × 250 mm) at a higher flow rate delivering sub-2 μm type resolution within HPLC column pressures.  

Protease digestion was accomplished by adding trypsin protease to a solution including approximately 4.2 mg (2.1 mg/mL, 2 mL) EPO. The ratio of substrate and enzyme was 50:1 (w/w). The mixed solution was incubated at 37 °C for 12 h. The digestion was quenched by storing the sample at –70 °C. After validation, 3.78 mg (2.1 mg/mL, 1.8 mL) of digested EPO was obtained. 

An Agilent 1260 Infinity Bio-inert Quaternary LC System was used, with auto injector (HiP-ALS), quaternary pump, thermostatted column compartment (TCC) and diode array detector (DAD), coupled to an Agilent 6224 TOF LC/MS. Data was processed using Agilent MassHunter Qualitative Analysis software and Agilent MassHunter BioConfirm. 

Spectra were recorded in positive ion and in centroid mode. 

			Acq. mode:  Extended dynamic range mode (2 GHz) 

Column: Agilent AdvanceBio Peptide Mapping, 2.1 × 250 mm, 2.7 μm (p/n 651750-902) 

O + 0.1% formic acid (v/v); B, acetonitrile + 0.1% formic acid (v/v) 

Optimized rhEPO High Pressure Liquid Chromatography (HPLC) Peptide Mapping with an AdvanceBio Peptide Mapping Column 

Systematic method development was performed for mapping the rhEPO digest to determine the best resolved separation under water/acetonitrile (ACN) conditions. During these evaluations, 2.1 × 150 mm and 2.1 × 250 mm AdvanceBio Peptide Mapping columns were evaluated under various flow, temperature and gradient profiles. Additionally, since peptide mapping separations work well under conditions that are favourable for ESI–MS, separations were evaluated with trifluoroacetic acid (TFA) and formic acid (FA) ion-pair additives to determine effects on retention and peak shape. The AdvanceBio Peptide Mapping column delivered excellent separation performance and compatibility with both TFA and FA ion-pair additives. The optimized separation displayed in Figure 1 provided the best separation profile for rhEPO mapping. The 2.1 × 250 mm AdvanceBio Peptide Mapping column and optimized chromatographic conditions with FA as the ion pair additive enabled a high resolution separation of the rhEPO tryptic digest, providing very narrow peak widths, increased sensitivity and unique selectivity across the gradient profile, making this separation highly amendable to ESI–MS analysis.  

Figure 1: Reversed-phase rhEPO peptide map using a 2.1 Ã 250 mm Agilent 2.7 Î¼m AdvanceBio Peptide Mapping column (see chromatographic conditions section).			

Rapid rhEPO Tryptic Digest Mapping by ESI–MS 

Peptide maps often take 2 h or longer using a 2.1 × 250 mm column to produce the needed resolution. Additionally, re-equilibration and run-to-run cycle times can add extended time to the completed analysis, significantly affecting laboratory production. While faster maps are desirable, it is critically important that resolution is not compromised. Figure 2 shows the total ion chromatogram (TIC) during a fast LC–MS analysis on a 2.1 × 250 mm column completed in less than 35 min. Excellent peak retention, peak shapes and resolution across the gradient are all indicators of a robust method for LC–MS validation of the peptide mapping analysis.  

Figure 2: Total ion chromatogram of an LCâMS analysis on a 2.1 Ã 250 mm Agilent AdvancedBio Peptide Mapping column accomplished in under 35 min.			

The faster analysis gained from the AdvancedBio Peptide Mapping column did not compromise the chromatographic map of rhEPO separation. Figure 3 shows the results from the Agilent MassHunter Molecular Feature Extractor (MFE). MFE is an algorithm that finds compounds from complex data and creates averaged MS spectra for each compound. These MFE compound results are then matched back to the rhEPO protein sequence. This 34-min LC–MS method resulted in 100% sequence coverage of the rhEPO protein.  

Figure 3: 100% rhEPO sequence coverage achieved using the 2.1 Ã 250 mm Agilent AdvanceBio Peptide Mapping column. Data generated using Molecular Feature Extractor in Agilent MassHunter qualitative analysis software.			

The rhEPO has a predicted molecular mass of 24,000 Dalton and apparent glycosylated molecular mass of 30,400 Dalton, and thus serves as an effective glycoprotein model to demonstrate glycopeptide profiling using the AdvanceBio Peptide Mapping column. Figure 4 is the extracted peptide glycosylation profile from the total LC–MS analysis shown in Figure 3.  The mass and glycosylated sequence information can be found in the full-length note, available at 

 (pub # 5991-1813EN). The AdvanceBio Peptide Mapping column enabled the identification of 45 unique glycopeptides and demonstrated the performance advantage and utility of this column for highly complex peptide mapping, where post-translational modification information, such as glycosylation, is critical to the complete protein characterization.  

Figure 4: MFE extracted peptide glycosylation profile using the 2.1 Ã 250 mm Agilent AdvanceBio Peptide Mapping column. Data generated using Agilent MassHunter Molecular Feature Extractor (MFE) on an Agilent 6224 TOF LC/MS.			

The new Agilent 2.7 μm AdvanceBio Peptide Mapping column demonstrated excellent utility for fast and efficient LC–MS peptide mapping using a recombinant human erythropoietin protein digest. A 2.1 × 250 mm column dimension and chromatographic conditions optimized for mass spectrometry analysis provided high resolution separation of the tryptic rhEPO digest across the entire gradient profile, demonstrating unique selectivity and retention features critical to generating a well-defined and resolved peptide map. The AdvanceBio Peptide Mapping column enabled 100% rhEPO sequence coverage by ESI–MS and provided 45 glycopeptide matches, making this column an excellent choice for highly complex peptide mapping applications. 

2850 Centerville Road, Wilmington, Delaware 19808, USA  

Pharmaceutical/Drug Discovery

High Resolution Glycopeptide Mapping of EPO Using an Agilent AdvanceBio Peptide Mapping Column

A novel method for Drug Antibody Ratio (DAR) determinations based on size-exclusion chromatography-multi-angle light scattering (SEC-MALS) in conjunction with ultraviolet (UV) absorption and differential refractive index detection.

There has been a significant resurgence in the development of anti-body-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 which 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 Mw 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.  

Figure 1: Molar masses for two distinct ADC formulations are determined using SEC-MALS analysis.			

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 specie or mined from the literature, to calculate the molar mass of the entire complex as well as for each component of the complex. 

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.			

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 which 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.  

tel. +1 (805) 681-9009, fax +1 (805) 681-0123  

Antibody Drug Conjugate (ADC) Analysis

James Hillis, Petra Decker, Ian Sanders and Carsten Baessmann,

The update to the SANCO guidelines in 2009 recognized the power and potential of time-of-flight (TOF) mass spectrometry for pesticide screening applications. When compared with historical screening tools (such as the use of triple-quad mass spectrometry), high performance accurate mass TOF systems offer several advantages for screening applications. These advantages include better resolution, unlimited multiple target capabilities and the possibility to perform comprehensive and/or retrospective analysis on samples 

The maXis impact™  continues the evolution of Bruker's unique TOF accurate mass instruments. The performance of ≥40k resolution, ≤1ppm mass accuracy and high isotopic fidelity is maintained without sensitivity compromise. While acquisition at up to 50 Hz enables maXis impact to handle the fastest of ultra high performance liquid chromatography (UHPLC) separations with ease. Additionally, to aid in quantitative applications, the maXis impact delivers an increase in sensitivity of up to 50-fold over previous generation instruments, especially at the low 

 tandem mass spectrometry (MS–MS) signals important for analyte confirmation.  

The dramatic increase in sensitivity changes the game for pesticide screening applications. With limits of detection (LODs) typically in the sub ng/mL levels, achieving excellent maximum residue levels (MRLs) becomes very easy. The maXis impact enables analysts to obtain both qualitative and quantitative results in a single analysis. Identification data including accurate mass MS and MS–MS, isotopic pattern fitting and Rt fitting can be readily collected and correlated in dedicated software. In the same run, sub ng/mL limits of quantification (LOQs) with dynamic ranges over four orders of magnitude can also be obtained (Figure 1).  

Figure 1: Fluoxastrobin at 50 fg to 1 ng on column.			

Taken together, the key qualitative and quantitative performance attributes of the maXis impact make it an excellent choice for routine pesticide screening applications with confirmation and quantitation in a single run. The maXis impact can provide comprehensive data quality that is unmatched by any other technology currently available. 

A mixture of 200 pesticides was run on the PesticideScreener™  application. This mixture has previously been well characterized and publicized. This same mixture was used to assess the performance of the maXis impact and specifically to assess the increase in sensitivity observed.  

The maXis impact changes the game in small molecule pesticide screening. Sensitivity increases of 20- to 50-fold versus previous systems are routine. This enhanced sensitivity, coupled with the additional capabilities of the maXis impact, allows both high quality qualitative and quantitative data to be acquired in a single run (Figure 1). The capabilities of the maXis impact are ideally suited to pesticide screening and quantification, and provides an unrivalled level of confidence at sub ppb levels. 

To further optimize the use of the maXis impact for pesticide screening, the PesticideScreener application platform is a fully developed protocol, allowing the use of accurate mass and isotopic pattern fitting and retention time fitting, for straight-out-of-the-box implementation of this outstanding analytical system (Figure 2).  

Figure 2: Overlapped hrEIC traces showing the evolution of TOF sensitivity.			

The maXis impact sets the new gold standard for pesticide screening through full utilization of the capabilities of ultrahigh-sensitivity TOF accurate mass spectrometry. 

40 Manning Road, Billerica, Massachusetts, USA  

tel. (978) 663-3660, fax (978) 667-5993  

Environmental

Accurate Mass Screening for Pesticide Residue Testing

2,4,6-trichloroanisole (TCA) emitted from wine corks can taint wine and cause an objectionable odour. Because of the low threshold value for sensing the odour, highly sensitive measurements are required for monitoring. Conventionally, TCA has be measured using methods such as purge-and-trap, which is very effective in concentrating samples, or solid-phase microextraction (SPME) or thermal desorption analysis. The HS-20 headspace sampler with an incorporated trap function enables concentration of the headspace sample and ultimately highly sensitive analysis. The trap is filled with an adsorption material and is electronically cooled, thereby avoiding the need for coolant consumption. This application note describes an example of high-sensitivity measurement of TCA in wine using a HS-trap GC–MS system. The structure of TCA is illustrated in Figure 1 and the mass spectrum is shown in Figure 2.  

Figure 1: Structure of 2,4,6-trichloroanisole (TCA).			

In this application the GCMS-QP2010 Ultra used was equipped with the novel technology of the HS-20 trap headspace sampler. Without any need for further preparation, the wine sample was placed in the headspace vials and analysed using the method parameters shown in Table 1. As a high concentration of ethanol is present in the wine sample, a trap temperature of 80 °C is used as a compromise to reduce the influence of ethanol in the results obtained.  

A wine sample spiked with the equivalent of 1 ng/L TCA was measured by selected ion monitoring (SIM) using the HS-trap method (Figure 3). The results clearly demonstrate the system power for analysis of very low concentrations of TCA with high sensitivity.  

Figure 3: Selected ion monitoring (SIM) chromatogram of TCA in wine measured using HS-20Trap (wine spiked with 1 ng/L TCA).			

A wine sample spiked with the equivalent of 100 ng/L TCA was measured by SIM using the headspace-GC–MS method and HS-trap method, as illustrated in Figures 4 and 5. A comparison of both shows that the HS-trap method provided about 10 times greater sensitivity.  

Figure 4: SIM chromatogram of TCA in wine measured using conventional HS-20 GCâMS (wine spiked with 100 ng/L TCA).			

Figure 5: SIM chromatogram of TCA in wine measured using HS-20Trap (wine spiked with 100 ng/L TCA).			

Linearity was confirmed by adding specific concentrations of TCA to wine (from 1–100 ng/L, see Figure 6). The calibration curve obtained with R

  = 0.9991 demonstrates the excellent linearity.  

Figure 6: Linearity of TCA (wine spiked with 1â100 ng/L TCA).			

3 ng/L of TCA was added to wine to test the repeatability (n = 5) of the peak area (Table 2). The results show good repeatability, with a coefficient of variation (CV) value not exceeding 5%.  

Table 2: Area repeatability of TCA (n = 5, wine spiked with 3 ng/L TCA).			

The application describes an example of high-sensitivity measurement of TCA in wine using a HS-trap GC–MS system. The results showed that the system was easily able to measure even a few nanograms per litre. This also confirmed that a HS-trap-GC–MS system using the HS-20 trap sampler is an effective and easy tool for monitoring of TCA in wine. The extremely low carry-over should be mentioned as an outstanding feature, based on the patent pending design of the sampler.  

Albert-Hahn-Strasse 6-10 47269 Duisburg, Germany  

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