	Propylene glycol (PG) can be used as a food additive and is widely accepted as safe for human consumption at low levels. The WHO (World Health Organization) have set the maximum permissible daily intake of propylene glycol as a food additive at 25 mg/kg bodyweight. PG comes in several grades with food grade PG, often used in the external chilling systems of breweries, as well as ice cream production, amongst others. The brewing industry standard requires that PG used within the chilling system must be food grade at a concentration no less than 25% by volume. This mix of PG and water enables the brewer to achieving cooling temperatures between -2.7 °C and -3.3 °C (25–27 °F). Contamination of beer can occur as a result of a damaged vessel wall or pipework, resulting in PG from the coolant jacket entering the beer stream. In Europe, authorities are very cautious about acceptable uses of PG within the cosmetics and food industry and is limited to mostly nonâfood applications. Those that are permitted are very limited and it is not currently approved to add to beer. There is, however, an EU 1 g/kg limit in food (additive number E1520).

	An Ellutia 200 Series gas chromatograph with a flame ionization detector (FID) was used for the analysis. The only sample preparation required was for the sample of beer degassed. This can be achieved by sonicating the sample or by repeatedly pouring the sample between two beakers. Once degassed the sample can be directly injected into the GC.

	Column flow (mL/min): 3.0 simulated constant flow

	Column type: 15 m × 0.25 mm, 0.25-µm df EL-WAX

	Initial temperature (°C)/hold time (min): 50/0

	Final column temperature (°C)/hold time (min): 200/0

	Analysis of propylene glycol content in lager can be achieved using an Ellutia 200 Series gas chromatograph with an FID. Initial testing has indicated that there are low PG concentrations in commercially available lagers. Sources of PG found in these samples cannot be established off-site and any unintentional contamination of these samples would need to be investigated. Intentional PG content should be monitored and levels should be kept within permissible levels. As daily exposure levels are subjective to an individual’s bodyweight and average consumption of lager, and other potential food sources of PG, it would seem prudent to reduce the levels of PG that an individual could be exposed to.

	12–16 Sedgeway Business Park, Witchford, Cambs, CB62HY, UK

	Tel: +44 (0)1353 669916  Fax: +44 (0)1353 669917

Articles

Food and Beverage

Propylene Glycol (PG) in Alcoholic Beverages

Institute of Inorganic and Analytical Chemistry, University of Münster, Münster, Germany, 

	In recent decades, the use of artificial sweeteners greatly increased. As sugar substitutes they have become popular in today’s calorieâconscious society. The most commonly used artificial sweeteners in beverages and food are acesulfame, aspartame, cyclamate, neohesperidin dihydrochalcone (NHDC), neotame, saccharin, and sucralose. Some of them show resistance to wastewater treatment and distribute in the aquatic environment as emerging contaminants. In addition, concerns about health risks from the artificial sweeteners have been raised as well (1–3).

	The analytical challenge is to simultaneously determine the artificial sweeteners with diverse physicochemical properties. Considering the polar and hydrophilic nature of these compounds, hydrophilic interaction liquid chromatography (HILIC) should be considered as an alternative to commonly used reversed-phase liquid chromatography. In this application, we used one iHILIC-Fusion(+) column packed with charge modulated hydroxyethyl amide silica, which combines hydrophilic partitioning, weak electrostatic interactions, and hydrogen bonding in HILIC separation.

Advance UHPLC with a CTC PAL autosampler and Bruker EVOQ™ Elite triple quadrupole system with electrospray ionization; multiple reaction monitoring mode (MRM) in ESI(+) and ESI(-) was used for selective and sensitive detection, see details in Table 1.

150 × 2.1 mm, 3.5-µm 100Å iHILIC-Fusion(+) (P/N 100.152.0310, HILICON AB, Sweden)

 A) acetonitrile; B) 10 mM ammonium formate, pH = 3.5; gradient elution from (95/5) A/B to (84/16) A/B in 8.5 min.

	Seven artificial sweetener standards can be simultaneously determined by the combination of iHILIC-Fusion(+) HILIC column and EVOQ™ Elite triple quadrupole MS–MS in a 12 min run, as shown in Figure 1. The developed method was verified to detect artificial sweeteners in the diet cola of a well-known brand. After a simple and fast sample preparation, we can identify acesulfame, cyclamate, and aspartame in the tested diet cola (Figure 2).

	This work describes an efficient HILIC–MS method for separation and detection of artificial sweeteners. It can be further developed to a routine analysis method with a combination of HILIC and aerosol-based detectors (ELSD, NQAD, and CAD) and used in different application areas.

		M. Scheurer, H.J. Brauch, and F.T. Lange, 

		F.T. Lange, M. Scheurer, and H.J. Brauch, 

		R.A. Lorenzo, M.T. Pena, P. Fernandez, P. Gonzalez, and A.M. Carro, 

	E-mail: info@hilicon.com  Website: www.hilicon.com

Analysis of Artificial Sweeteners by HILIC–MS Method

	This application note outlines a QuEChERS procedure for the detection of patulin in processed food products. It typically grows on fruit, including apples, pears, peaches, and grapes, but has also been reported in vegetables and cereal grains. Patulin has been implicated as a possible carcinogen and teratogen, although an official designation has not yet been made. An apple-based baby food product was used as a representative sample matrix. The inclusion of graphitized carbon black (GCB) in the dispersive solid-phase extraction (dSPE) cleanup step produces a clean sample extract. Analysis is carried out by ultrahigh-pressure liquid chromatography coupled to tandem mass spectrometry (UHPLC–MS–MS) using the polyaromatic Selectra® DA column.

	            (a) Weigh 10 g of sample into a 50 mL polypropylene centrifuge tube.

	            (b) Add 10 mL of acetonitrile.

	            (c) Add the contents of the ECMSSC-MP Mylar pouch and shake for a minimum of 1 min (by hand or mechanically).

	            (d) Centrifuge the samples at ≥ 3000 × g for 5 min.

	            (a) Transfer 1 mL of supernatant to a 2 mL dSPE tube 

	            (b) Vortex the sample for 30 s.

	            (c) Centrifuge the samples at ≥ 3000 × g for 5 min.

	            (d) Transfer 500–600 µL of purified supernatant into an autosampler vial (filter if desired).

	LC–MS–MS:     Thermo Scientific™ Dionex™ Ultimate™3000 UHPLC and TSQ Vantage™ (MS–MS)

	Column: UCT Selectra® DA UHPLC column 50 × 2.1 mm, 1.8-μm

	Guard column: UCT Selectra® DA guard column 10 × 2.1 mm, 1.8-μm

	Excellent recoveries ranging from 83% to 90.1% were obtained using UCT’s QuEChERS salts 

 clean-up procedure in conjunction with the Selectra® DA HPLC column 

for the analysis of patulin in processed foods. For increased sensitivity, clean-up of a 5 mL extract using a 15 mL dSPE tube 

	2731 Bartram Road, Bristol, Pennsylvania 19007, USA

Determination of Patulin in Processed Foods Using QuEChERS Extraction and UHPLC–MS–MS Analysis

Laura McGregor, Matt Edwards, Stefan Koschinski, and David Barden

In this study, we demonstrate the enhanced separation and unparalleled sensitivity that can be achieved in the characterization of cigarette smoke using thermal desorption (TD) and GC×GC with time-of-flight mass spectrometry, aided by Tandem Ionisation for simultaneous acquisition of hard and soft electron ionisation (EI) data.

	The hazardous constituents of cigarette smoke have attracted considerable attention lately, especially with increasing regulation around the world limiting or banning smoking in public places - and even in private cars if children are present.

	From an analytical perspective, however, there is much that remains to be learnt about the composition of cigarette smoke, because of its high degree of complexity - tobacco smoke is thought to contain thousands of components across multiple chemical classes and wide concentration ranges.

	Comprehensive two-dimensional gas chromatography (GC×GC), when coupled with time-of-flight mass spectrometry (TOF-MS), is a powerful approach for the analysis of complex samples, improving chemical fingerprinting in areas of study as diverse as petrochemical analysis and fragrance profiling.

	In this study, we use flow-modulated GC×GC–TOF-MS to enable smoke constituents to be routinely and confidently sampled, separated, and identified. The use of Tandem Ionisation® is also harnessed to increase the analytical resolution of the system, by providing both reference-quality 70 eV spectra and soft electron ionisation (EI) spectra simultaneously in a single analysis.

	Cigarette smoke (~50 mL) was drawn (on the fourth “puff”) directly onto an inert-coated Tenax® TA sorbent tube using an ACTI-VOC™ low-flow pump (Markes International).

	Analysis of the tubes was performed by TD–GC×GC–TOF-MS, using a TD100-xr™ thermal desorber (Markes International), a BPX5 1st-dimension column, a DB17MS 2nd-dimension column, an Insight™ flow modulator (SepSolve Analytical), and a BenchTOF-Select™ mass spectrometer (Markes International).

	TOF-MS instrument control and data acquisition used TOF-DS™ (Markes International), and GC×GC data visualization and processing used the ChromSpace® plug-in (Markes International).

	The GC×GC–TOF-MS plots of the three collected smoke samples showed the same major components, with nicotine and triacetin unsurprisingly the dominant peaks. Sample A is shown as an example (Figure 1).

	The expanded region shown in Figure 2 shows the efficient modulation and excellent separation provided by this GC×GC method, with average peak widths of ~200–300 ms in the second dimension. At one point, at least five components would be expected to co-elute in a one-dimensional separation (dotted red line), which would likely have resulted in identification difficulties. Here, however, confident matches against the NIST 14 database were obtained in every case.

	Although the “reference-quality” spectra generated by BenchTOF enable the confident characterization of cigarette smoke described above, challenges may still remain in cases where compounds exhibit similar spectral characteristics at conventional 70 eV ionisation energy.

	Markes’ Tandem Ionisation technology addresses this problem by multiplexing two ionisation energies within a single analysis, and was applied here at 70 eV and 14 eV EI. This created two independent data files with the obvious advantage of eliminating the need to collect a duplicate sample, and results in two perfectly aligned chromatograms from a single analytical run, simplifying both the acquisition and processing of soft-ionisation data. 

	In tobacco smoke analysis, the availability of complementary soft EI spectra can be of considerable value, as illustrated by the spectra in Figure 3. Neophytadiene is a particularly interesting example because this diterpene is present at low levels in green tobacco leaf, but it is known to increase in concentration during yellowing and curing. Neophytadiene undergoes a high degree of fragmentation at 70 eV, making confident identification problematic, but acquiring data at 14 eV using Tandem Ionisation enhances the structurally-significant peaks, enabling its identity to be verified. Triacetin is the most extensively used plasticizer in cigarette filters, but the significant ions in the 70 eV spectrum have very low intensity. Tandem Ionisation provides enhancement of the higher-m/z ions (such as m/z 145 and 158) for simpler identification of this compound in complex matrices, and making it possible to achieve reliable quantitation using these diagnostic ions, rather than the more common m/z 43.

	In this study, we have shown that flow modulation for GC×GC can provide excellent separation of trace-level analytes while minimizing breakthrough and tailing of highly concentrated compounds. Furthermore, Tandem Ionisation using BenchTOFâSelect provides another level of confidence in compound identification, through the simultaneous acquisition of NIST-quality 70 eV spectra and complementary soft EI spectra with enhanced molecular (and other structurally-significant) ions.

	When these GC×GC–TOF-MS technologies are combined with the sensitivity enhancement possible with thermal desorption, the result is a highly-automated platform with a high degree of analytical resolution. As demonstrated here, such capabilities make it very well-suited for tackling the analytical challenges encountered in the tobacco industry, whether in matters of regulatory compliance or research and development.

	Gwaun Elai Medi-Science Campus, Llantrisant, Wales, UK

General

Comprehensive Analysis of Tobacco Smoke Using GC×GC–TOF-MS with Tandem Ionisation

Shun-Hsin Liang, Paul Connolly, and Ty Kahler

	Thyroid hormones are essential for the regulation of development and growth in humans and animals. The thyroid gland produces thyroxin (T4) and tri-iodothyronine (T3) and quickly releases these compounds into the circulatory system. The concentration of circulating T4 is 50–60 times higher than T3 and the majority of these molecules are bound to blood proteins. The unbound or “free” T3 and T4 are the active forms of the hormone which only represent a small portion (less than 1%) of total thyroid hormones. Accurate and sensitive measurement of low pg/mL levels of free hormones is necessary to assess thyroid function for both veterinary and human diagnostics. Reverse tri-iodothyronine (rT3) is an inactive form that results from T4 biotransformation. Since rT3 functions as the feedback inhibitor of thyroid hormone production, the measurement of rT3 can be an important diagnostic marker with clinical implications.

	The intent of this application was to develop an LC–MS–MS method for the analysis of thyroid hormones at the free form levels using the highly efficient and selective Raptor™ Biphenyl LC column. The clinical applicability of this method was demonstrated by analyzing fortified thyroid hormone in phosphate buffered saline (PBS) containing 4% human albumin.

	The instrument and analytical conditions are listed in Table 1. The analyte MRMs are shown in Table 2.

	Human albumin was dissolved in PBS solution to a final concentration of 4%. This solution was used to prepare calibration standards ranging from 2 to 400 pg/mL. Standard solutions (0.5 mL) were fortified with 5 µL of internal standard (T4-13C6 [1 ng/mL]) and mixed with 1 mL of acetonitrile in a 4 mL glass vial. A 2 mL aliquot of ethyl acetate was added, stirred for 2 min, and then centrifuged for 10 min at 4300 rpm. The organic phase was removed and placed into a 4 mL glass vial, then evaporated to dryness at 55 °C under a gentle stream of nitrogen. The dried extract was reconstituted with 80 µL of a 30:70 water:methanol solution and injected (10 µL) into the LC–MS–MS for analysis.

Chromatographic Separation of Thyroid Hormones

	Since the most sensitive mass transitions for T3 and rT3 are identical, it is necessary to chromatographically separate these two compounds for accurate quantitation. With independent injections of T3 and rT3, it was shown that T3 and rT3 were completely resolved with the Raptor™ Biphenyl column (Figure 1). An example chromatogram (Figure 2) shows the baseline resolution of three forms of thyroid hormone from extracted sample (20 pg/mL) within 2 min.

	Good linearities (1/x weighted) were obtained for all three forms of thyroid hormones with coefficient of determination (R2) values
	> 0.990 from 2 to 400 pg/mL (for T3) or 5 to 400 pg/mL (for T4 and rT3). The %deviation was < 15%.

	The Raptor™ Biphenyl column is excellent for rapid and sensitive analysis of thyroid hormones. With the method described here, concentrations of thyroid hormones as low as 2 pg/mL (T3) or 5 pg/mL (T4 and rT3) can be determined with less than 3.5 min of total analysis time. The analytical method is thus applicable to the clinical analysis of free thyroid hormone at low pg/mL levels.

	110 Benner Circle, Bellefonte, Pennsylvania 16823, USA

	Tel: (800) 356 1688  fax: (814) 353 1309

Medical/Biological

A Rapid and Sensitive LC–MS–MS Method for the Analysis of Three Forms of Thyroid Hormones Using Raptor™ Biphenyl LC Columns

This application note demonstrates the analysis of charge variants of Rituximab (both innovator and biosimilar molecules) using multiple heart-cutting two-dimensional liquid chromatography (2D-LC) with mass spectrometry (MS) detection.

	Monoclonal antibodies (mAbs) represent a class of highly advanced, but expensive, pharmaceutical products. The increasing demand for these biopharmaceuticals has led to the development of less costly biosimilars (1). To qualify the charge variant pattern of the mAbs, identification with mass spectrometry (MS) is necessary. However, identification using online MS analysis after ion exchange chromatography is not a straightforward procedure. This is mostly because of the incompatibility of the most common ion exchange buffers with the electrospray ionization process.

	The Agilent 1290 Infinity 2D-LC solution enables automated desalting, and offers denaturation and, if necessary, extra separation by the addition of reversed-phase chromatography in the second dimension.

	1D - Mobile phase:   A: 10 mM ammonium formate pH 6.2

	1D - Mobile phase:   B: 500 mM ammonium formate, pH 6.2

	2D - Mobile phase:   B: acetonitrile with 5% FA    

	                    30 min - 40% B

	                    35 min - 100% B

	                    38 min - 0% B

	                     2.5 min - 60% B

	                     2.75 min - 90% B

	Peak width 1D: 0.025 min (0.5 s response time) (10 Hz)

	Peak width 2D: > 0.0063 min (0.13 s response time) (40 Hz)

	Figure 1 shows the results of the multiple heart-cutting 2D-LC analysis presented in the 2D-LC heart-cut viewer.

	Figure 2 shows the deconvoluted spectra of charge variant main peaks 1–3 (Figures 2a–c). The mass of the first biosimilar main peak for G0F/G0F is 147,082 Da, identical to G0F/G0F of the innovator. The G0F/G0F of the second (Figure 2b) and third (Figure 2c) charge variant peak are shifted with an interval of about 128 Da, representing the mass of lysine. This leads to the assumption that the three main peaks are C-terminal lysine variants.

	Innovator and biosimilar Rituximab charge variants were analyzed using weak cation exchange chromatography. Direct qualification of the charge pattern using online MS detection was enabled using a two-dimensional workflow with reversed phase in the second dimension for straightforward MS detection.

	For research use only, not for use in diagnostic procedures.

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

Pharmaceutical/Drug Discovery

2D-LC–MS Characterization of Charge Variants Using Ion Exchange and Reversed-Phase Chromatography

	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 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
	size-exclusion chromatography combined with multi-angle light scattering (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.

	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.

	6300 Hollister Avenue, Santa Barbara, California 93117, USA

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

Antibody Drug Conjugate (ADC) Analysis with SEC–MALS

A.J. Freitag, K. Wittmann, G. Winter, and J. Myschik

	Proteins - especially monoclonal antibodies (MABs) - have become increasingly important in pharmaceutical work. However, there are some important differences between conventional, chemically-synthesized drugs and proteins. Because of the complex and weak structure of proteins, even a slight change in conditions, such as pH value, temperature, or mechanical stress, may lead to aggregation and a loss of activity or stability.

	A separation of MABs before analysis is preferred because a detailed investigation of a fraction would be much easier and provide a better insight into the physical and chemical properties, compared to an analysis in the presence of a coexisting monomeric protein.

	Asymmetrical flow field-flow fractionation (AF4) is a well-established method for sizing and quantifying different aggregate species in protein formulations. A major advantage of AF4 is the use of the formulation buffer of the protein as the mobile phase. The separation is independent of the ionic strength of the buffer.

	The capability of AF4 as a preparative tool for the separation of aggregates, fragments, and monomeric species was investigated in this study. The system consisted of an Eclipse (Wyatt Technology Europe GmbH, Dernbach, Germany), equipped with DAWN multiâangle light scattering (MALS), refractive index (RI) and ultraviolet (UV) detectors, and a fraction collector. The separation was achieved by using a semiâpreparative SP channel with 350 μm height.

	A monoclonal antibody sample was exposed to light, resulting in the formation of fragments and aggregates (Figure 1). A sample of 1 mg of protein per run was injected into the channel and collected in tubes made of glass, using a Gilson FC-203B fraction collector. The collected fractions were concentrated and the final concentration was determined by UV absorbance at 280 nm using a Micro-BCA Assay.

	AF4 is a useful method for analytical and preparative separation of protein aggregates since the separation can be performed in each buffer or even pure water, thus facilitating subsequent activities. Furthermore, an investigation of possible immune responses triggered by protein aggregates in comparison to monomeric proteins can be performed, which is currently of major interest in administrative organizations and the pharmaceutical industry at large.

	The results presented in this application note have been published before in A.J. Freitag, K. Wittmann, G. Winter, and J. Myschik, 

	6300 Hollister Ave., Santa Barbara, California 93117, USA

The Application Notebook-07-01-2016