February 2019 Volume 32 Number 2 www.chromatographyonline.com

Fake News Modern methods to identify counterfeit medicines and health products

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© 2019 Wyatt Technology. All rights reserved. All trademarks and registered trademarks are properties of their respective holders. COVER STORY February | 2019 88 PHARMACEUTICAL PERSPECTIVES The Role of Liquid Chromatography and Gas Chromatography in the Analysis of Illegal Medicines Volume 32 Number 2 and Health Products Yaxin Tie, Celine Vanhee, Erwin Adams, andd EricEric Deconinck This review intends to give a general overview of the liquid chromatographic (LC) and gas chromatographic (GC) methods, utilized by regulatory authorities, for the detection and characterization of suspectedd illegal medicines and health products, including lifesaving drugs, lifestyle drugs, and biotechnology drugs.

Features 62 Ultrahigh Resolution Semipreparative Liquid Chromatography: Application to Structure Elucidation of Drug Impurities Fabrice Gritti, Sylvain Cormier, Ronald Morris, Frank Riley, and Qi Yan The performance of a research prototype twin column recycling liquid chromatography (TCRLC) process was investigated regarding the preparation of drug impurities.

94 Analysis Focus: Pharmaceutical Analysis Going Green in Pharmaceutical Analysis Alasdair Matheson LCGC Europe spoke to Yong Liu and Adam Socia from MSD about the cost-saving benefi ts of implementing green chromatography in the pharmaceutical sector, the importance of AMVI, and effective practices to reduce solvent consumption and replace harmful solvents, including SFC, fast chromatography, and “cocktail chromatography”. Columns 72 LC TROUBLESHOOTING Reversed-Phase Liquid Chromatography and Water, Part 1—How Much is Too Much? Dwight Stoll When can we use completely aqueous eluents with reversed-phase stationary phases, and what happens if we make a mistake?

80 GC CONNECTIONS GC×GC: From Research to Routine Nicholas H. Snow This instalment of “GC Connections” begins with a brief introduction to GC×GC, follows with examples of how GC×GC opens additional avenues of analysis, and it concludes with information about how to learn more.

102 THE ESSENTIALS The Essential Guide to Optimizing Sensitivity in GC–FID Editorial Policy: Analysis All articles submitted to LC•GC Europe Tips to improve signal-to-noise ratio when using standard GC are subject to a peer-review process in association equipment with the magazine’s Editorial Advisory Board.

Cover: Departments Original materials courtesy: photokozyr/ 97 Products stock.adobe.com 101 Events

56 LC•GC Europe February 2019 A Higher Level of Sensitivity. Every Time. High-purity UHPLC-MS LiChrosolv® solvents for rapid and reliable results.

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Selected highlights of digital content from LCGC Europe and LCGC North America:

INTERVIEW PEER-REVIEWED ARTICLE Entering the Second Dimension Molecular Imprinting for LCGC Europe spoke to Alina Sample Preparation Muscalu about the evolving role of Sample preparation is the most comprehensive two-dimensional crucial step for the development gas chromatography (GC×GC) of an analytical method. It is also in environmental analysis. the most time-consuming step and Read Here: https://goo.gl/63SLcc should be deliberately optimized to enhance extraction selectivity

and detection capability. Photo Credit: thaporn942/ Read Here: https://goo.gl/Vi7cNQ stock.adobe.com

LCGC BLOG NEWS Gas Chromatography–Vacuum Novel Flow-Confi nement Ultraviolet Spectroscopy Concept for LC×LC for Fatty Acid Analysis A novel flow-confinement Although GC–MS may have concept designed to remove the greater sensitivity, GC–VUV offers performance sacrifice associated Photo Credit:deomis/stock.adobe.com some distinct advantages for with current comprehensive speciation of fatty acid mixtures. two-dimensional liquid chromatography (LC×LC) Read Here: https://goo.gl/jteUhU techniques has been tested by researchers. Read Here: https://goo.gl/wC2ehz

E-BOOK WEBCASTS Techniques for Improving Keep Up-to-Date Biopharmaceutical LC–MS Analysis with Upcoming and This e-book explores how analysts can On-Demand Webcasts address some common issues related Working in partnership to the analysis of biomacromolecules with industry leaders, like monoclonal antibodies and LCGC broadcasts antibody–drug conjugates. live technical tutorial-style Read Here: https://goo.gl/WiJK3K webcasts, as well as application-based tutorials. A wide range of topics are covered and the full list of upcoming and on-demand webcasts can be found on our website at www.chromatographyonline.com/LCGCwebseminars

CHROMACADEMY CONNECT WITH LCGC CHROMacademy is now free for all university Stay in Touch with LCGC and Keep students and staff. Sign up today for our Updated with the Latest News animated and interactive e-learning courses. Follow us on social media to keep up-to-date Find Out More Here: www.chromacademy. with the latest troubleshooting tips and com/Agilent-uni.html technical peer-reviewed articles featured on our website. Follow @LC_GC on Twitter, join our LCGC Magazine LinkedIn group, or Like our page on Facebook. You are also free to post your questions or discussions for other members to view and comment on!

60 LC•GC Europe February 2019

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Fabrice Gritti1, Sylvain Cormier1, Ronald Morris2, Frank Riley2, and Qi Yan2, 1Waters Corporation, Milford, Massachusetts, USA, 2Pfizer, Groton, Connecticut, USA

A research prototype high performance liquid chromatography (HPLC) system was built to prepare approximately 1 mg of drug impurities (purity > 90%) for structure elucidation by liquid state nuclear magnetic resonance (NMR). The system is based on alternate pumping (or twin column) recycling liquid chromatography. The system is coupled to a fraction collector and is designed to cope with severe experimental constraints, including situations where the impurity is barely separated from the active pharmaceutical ingredient (API); the drug diluent is strong relative to the eluent; viscous fi ngering occurs; the impurity-to-drug concentration ratio is extremely small (< 1/100); and where the yield and purity levels required should be larger than 99% and 90%, respectively. The combination of LC–mass spectrometry (MS) data and ultraviolet (UV) absorption spectra enabled the impurity to be identifi ed.

The isolation and preparation of approximately 1 mg concentrations (1), while high resolution chromatography of unknown impurities present in concentrated drug handles small and diluted samples (5). Interestingly, solutions is required by the pharmaceutical industry complex separation problems can be solved by alternate for identification purposes. Unambiguous structure pumping or twin column recycling liquid chromatography elucidation is commonly achieved by liquid state nuclear (TCRLC) (6). The general principle of TCRLC is to virtually magnetic resonance (NMR) experiments with 1 mg of increase the column length while still operating at material. However, serious separation and production optimum velocity and standard pressure. This technique problems can arise when: was applied in the past to fractionate polymer mixtures • The targeted impurity nearly coelutes with the drug; (7), prepare isotopes and isomers by gas chromatography • The relative abundance of the targeted impurity is (GC) (8,9), and to collect optically-active compounds (10). very small (<1/100) with respect to that of the active This technique was recently used for various purification pharmaceutical ingredient (API); processes (11–16) and implemented with modern • The sample diluent is much stronger than the eluent, adsorption and size-exclusion columns for the automated and viscous fingering occurs; separation of shape isomers, enantiomers, isotopes, • The yield needs to be be larger than 99%; • When the purity level required for successful NMR experiments is larger than 90%.

For cases where sufficient resolution is lacking, standard preparative processes, such as batch KEY POINTS • A high-resolution recycling chromatography system chromatography (1), simulated moving bed (SMB) with semipreparative capability is described. (2), steady state recycling (SSR) (3), or multicolumn • The technique targets very challenging drug and countercurrent solvent gradient (4), cannot fully solve this trace impurity separation problems. problem. • The technique also solves problems resulting from The solution to the problem is to combine diluent-to-eluent strength and viscosity mismatches. high-resolution performance with semipreparative • Proof-of-concept for a 1/5000 impurity to drug capabilities into a single purification process. In essence, concentration ratio is reported. these two characteristics are incompatible: preparative

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 Gritti et al.

Figure 1: Principle of a twin column recycling process. polymers, and of monoclonal antibodies (mAbs) from their The targeted separation zone is transferred multiple times undesirable aggregated forms (17–20). Even though the between two columns of finite length, L, by switching the production rate of such discontinuous TCRLC is clearly valve. This recreates the conditions of an infinitely long lower than that of continuous SMB or semicontinuous SSR column that can still be run at optimum speed and at processes, it has several advantages: the experimental standard allowable pressures. setup can be easily assembled by a non-expert (17), it can solve extremely challenging separation problems (selectivity factor α < 1.2 with analyte-to-analyte

Column 1 abundance ratio < 1/100) (18,19), it can cope with strong (a) sample diluent and viscous fingering effects by applying a large enough number of cycles, and its long-time automation is straightforward when maintaining the eluent and column temperature steady (20). For these reasons, Pump/ Injector a TCRLC process coupled with a fraction collector is a POSITION 1 potential solution to critical separation problems, such as Detector those faced by the pharmaceutical industry. In this article, the performance of a research prototype TCRLC process was investigated regarding the preparation of drug impurities. It was tested to isolate Column 2 an unknown and poorly resolved impurity present in a concentrated (10 g/L) solution (60 mL) of the drug estradiol. In this particular case, the analyst faced (b) a low drug–impurity selectivity factor (1.166), a very Column 1 low impurity-to-drug abundance ratio (< 1/000), band deformation as a result of sample diluent and viscous fingering effects, and also had to comply with yield and purity levels larger than 99% and 90%, respectively. The identification of the impurity was revealed by either NMR Pump/ Injector POSITION 2 (if 1 mg can be collected with > 90% purity) or by liquid Detector chromatography–mass spectrometry (LC–MS) with single ion monitoring (SIM) and UV absorption spectra of the concentrated collected fractions.

Column 2 Experimental Recycling Experiments: The general principle of TCRLC is presented in Figure 1. Two identical columns of length

Figure 2: (a) Schematics and (b) photograph of the different components assembled to build a research prototype semipreparative (4.6 mm × 150 mm length columns) and high resolution twin column recycling liquid chromatography system. Adapted with permission from reference 20. (a) (b) RECYCLE

Column 1 Collection Recycling Detector Detector Recycling Detector

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64 LC•GC Europe February 2019 Gritti et al.

L are connected to a two-position recycling valve (six-port if no detection is performed between columns, eight-port if a detection cell is placed between them), enabling the transfer of the separation zone from one column to the other before the sample is either collected or sent the micro-Chip Chromatography Company to waste. As a result, TCRLC mimics an infinitely long column that can still be operated at optimum velocity and low pressures. Figure 2 shows (a) the schematics and (b) the photograph of the actual research prototype TCRLC system assembled and used to solve critical chromatographic separation and preparation problems. The whole system is made of three sub-units performing sample injection, impurity–API separation by recycling chromatography, and impurity collection. It includes: a binary solvent pump mixer (red); a flow-through needle sample manager (blue) equipped with 10-mL vials, a 100-μL extension loop; a two-column oven compartment (purple) with two positions (column 1 and column 2), six-port selection valves (the left standard six-port Changing the ART of analytical valve was replaced with a low-dispersion eight-port recycling valve, the right six-port valve is unused); a chromatography with fraction manager analytical (green) equipped with 10-mL μPAC™ Pillar Array Columns: collection vials and a two position (waste and collect) four-port valve; two low-dispersion (50-nL volume) detection cells and their module box (light source, These micro-Chip columns feature photodiode, and fibre optics to carry light, orange). All the components are connected via Zenfit perfect connection a perfectly-ordered separation bed (face seal) tubings (Waters). The whole system is automated by the software Empower version 3.0 (Waters). of free-standing pillars ensuring: The principle of the semipreparative TCRLC system is straightforward: the entire chromatographic band of the • excellent separation power targeted impurity is recycled as many times as necessary • unprecedented reproducibility between the twin columns until it is fully separated from that of the main drug and of other nontargeted impurities. • unrivalled robustness Finally, the isolated targeted impurity is collected. The schematics in Figure 2(a) provide a clear visualization of the fluidic paths related to the real research recycling system shown in Figure 2(b), from the pump to the sample Enhance the data productivity manager, the recycling unit, the detection units, and to the fraction collector. of your nano-LC/MS system The sample solution was a stock solution of estradiol (10 g/L) dissolved in a mixture of 50:50 (v/v) acetonitrile for complex biological samples. and methanol (strong elution strength). The mobile phase was a 65:35 (v/v) mixture of acetonitrile and water (weaker elution strength). The flow rate was set at 0.7 mL/min. Discover our products on The oven and eluent temperatures were maintained at www.pharmafluidics.com 30 +/- 0.1 oC. The pressure drop along the two columns was measured around 4000 psi. The 4.6 mm × 150 mm or meet us at twin columns were packed with 3.5-μm Sunfire-C18 Pittcon (March 17-21, Philadelphia, USA) particles (Waters), which maximized the speed-resolution or ABRF (March 23-26, Texas, USA) performance of the TCRLC process at 4000 psi pressure drop (see Figure 3). All the details regarding the extension of the construction of the well-known speed-resolution plots (21) to recycling chromatography are given in reference 17. The production rate of the TCRLC process was maximized by fixing the cycle number at n = 6 and the largest injection volume at 100 μL. Details and rationale for these optimized parameters are given in reference 20. The sample content was sent to waste = = between t 1.0 min and t 2.9 min, and between FOLLOW US t = 4.0 min and t = 8.8 min (elimination of early and late nontargeted impurities). The targeted impurity and www.chromatographyonline.com 65 Gritti et al.

Figure 3: Calculated speed-resolution (hold-up time per Figure 4: Overloaded band profiles of the API when resolution factor unit as a function of the resolution factor) increasing the injection volume of the API stock solution or kinetic plots of the recycling process at a constant from 3 μL to 6 μL, 12 μL, 24 μL, 48 μL, 74 μL, and 100 μL. pressure drop of 4000 psi and for two twin columns of Note the increasing fronting of both the API and impurity length L = 15 cm packed with particles of size increasing bands, which is no longer separated from the API for from 1.5 μm (circles) to 2.0 μm (squares), 2.5 μm injection volumes larger than about 10 μL. This illustrates (crosses), 3.5 μm (triangles), 5.0 μm (full triangles), 7.5 μm the complexity of the purification problem associated with (pluses), and to 10 μm (diamonds). All the details for the small selectivity factors, viscous fingering effect, sample construction of the speed-resolution plot for recycling diluent effect, and very low impurity-to-API concentration chromatography are given in reference 10. Each empty ratio. Adapted with permission from reference 20. circle accounts for a particular number of cycles (or for a number of passages through one column length) from 1 1.8 to the maximum allowable number of cycles. The x-axis 3 μL represents the resolution factor for any number, n, of 1.6 AU (λ = 254 nm) 6 μL cycles. The y-axis represents the ratio of the hold-up time 12 μL to the resolution factor. The thick solid black line locates 1.4 the highest resolution factors expected for the maximum 24 μL allowable number of cycles. Note a first optimum particle 1.2 48 μL size that maximizes resolution and is located at the unique 74 μL intersection between the solid black line and a vertical 1.0 Targeted impurity 100 μL tangent (not drawn): this would correspond to a 2.5-μm 0.8 particle size. Note also a second optimum particle size, API which maximizes speed-resolution performance and is 0.6 located at the unique intersection between the solid black Early impurity line and a tangent of slope +1 (not drawn) in a log-log 0.4 scale: this would correspond to a 5.0-μm particle size. In this work, a 3.5-μm particle size was selected. 0.2

0.0 10000 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 ∆P=4000 psi 1.5 μm Time (min)

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/ solvent B–solvent A for 1.0 min followed by a linear 0 t 5.0 μm gradient to 95:5 solvent B–solvent A in 6.0 min returning

100 n =14 to initial conditions in 0.1 min with column re-equilibration max 7.5 μm n =13 max for 1.9 min. The flow rate was set at 0.4 mL/min. The

n =12 max 10 μm sample was analyzed with an Orbitrap Fusion Lumos n =10 max Mass Spectrometer (Thermo). This mass spectrometer Max was equipped with a heated electrospray ionization 10 1 10 probe (HESI) to enhance assay sensitivity. Analytes were Resolution Factor, Rs detected in the positive ion mode under a full mass scan range from 150 to 1000 daltons with a resolving power of 60,000. The source temperature was set to 350 oC, and the ion spray voltage was 3500 V. The vaporizer the nearly coeluting impurities and API were transferred temperature was set at 100 oC with a sheath gas flow at for the first time between columns from t = 2.9 min to 20 L/min, auxiliary gas at 15 L/min, and sweep gas at 2 L/ t = 4.0 min. This entire zone was transferred another four min. times between the twin columns. The isolated impurity was finally collected after six cycles between t = 20.3 min Results and Discussion and t = 21.3 min. This very same method was repeated Volume Overload Without Recycling (n = 1): Figure 4 600 times over a non-stop period of one and a half weeks shows the analytical chromatogram (6 μL injection) of to collect the entire mass of the targeted impurity present the stock solution containing the API estradiol (10 g/L) in the 60 mL stock solution of estradiol. and many unknown impurities. The UV wavelength was Gradient LC–UV–MS Experiments: The set at λ = 254 nm because it maximized the targeted chromatographic separation was achieved on impurity-to-estradiol signal ratio (1 to 67). The targeted a 2.1 mm × 100 mm, 1.7-μm Acquity CSH C18 impurity eluted at t = 3.55 min while the API estradiol was = column (Waters). The very-high performance liquid detected at 3.79 min (hold-up time t0 2.04 min). The chromatography (UHPLC) system was the Agilent 1290. selectivity factor was then α = 1.16. Several nontargeted Column temperature was controlled at 40 oC. The mobile impurities were detected before and after (not shown) the phase was composed of solvent A (0.1% formic acid in elution of the targeted impurity and all were eliminated water) and solvent B (acetonitrile). The vHPLC pump to waste during the TCRLC process. Figure 4 shows the

66 LC•GC Europe February 2019 Gritti et al. overloaded band profiles of the API when increasing the for n = 6 cycles and a 100 μL injection volume (23 min injection volume of the API stock solution from 3 μL to run). The top and bottom chromatograms were recorded 6 μL, 12 μL, 24 μL, 48 μL, 74 μL, and to 100 μL. Figure 4 by the low-dispersion cell connected to the recycling illustrates the serious separation problem faced by the valve in between the two twin columns (see the recycling analyst: it is impossible to get a decent production rate of UV cell in Figure 2 monitoring in real time the progress the targeted impurity with a single column batch process of the separation between the targeted impurity and the because its band becomes more and more buried under API estradiol) and by the second low-dispersion cell the API band, which dramatically fronts and gets distorted placed in between the recycling valve and the collection as a result of sample diluent and viscous fingering effects. valve (see the collection UV cell in Figure 2 monitoring in The retention factor of the impurity and estradiol were real time the separation status immediately before waste both reduced by a factor ~ 4 when the mobile phase was or fraction collection), respectively. As indicated by the replaced with the sample diluent (20). dotted red vertical lines at t = 2.9 min and t = 4.0 min, the Theoretical Recycling and Optimization of the targeted impurity was transferred between columns from n = n = = Production Rate ( opt): The fundamental question t 2.9 min to t 4.0 min, while the nontargeted impurities is then: Should small (and many runs) or large (and less runs) sample volumes be injected to maximize the production rate of the TCRLC process? To answer that question, the calculations of the concentration profiles of the targeted impurity and estradiol were performed for a series of cycle numbers from 1 to 14 (the maximum allowable number BioLC Innovations... of cycles shown in Figure 3 for 3.5-μm particles). For each cycle number, the injected volume was ...with Incredible Reproducibility! maximized so that yield and purity levels were at least 99.7% and 99.0%, respectively. It is important to note that the calculations take into consideration the impact of the strong sample diluent on the retention of the targeted impurity and API (19). Accordingly, the calculated bands were close to those observed. The calculations reveal that the band shape was fronting when the injected sample volumes became larger than about 50 μL (number of cycles larger than 2). Figure 5 shows the corresponding variation of the production rate as a function, n. It Proteins can be seen that the production Antibodies rate first rapidly increased from Oligonucleotides ~ 3 μg/h (n = 1) to ~ 5.5 μg/h (n = 6), then plateaued, and Peptides finally slightly decreased for cycle • SEC for high resolved MAbs numbers larger than 7. The largest production rate was expected • HIC with exceptional effi ciency for n = 7 at 5.6 μg/h. The corresponding injection volume was 159 μL. Accordingly, in the • RP-C4-Widepore with superior stability next experiments (Figure 6), n = 6 cycles for an injection volume • IEX for high recovery of 100 μL will be selected (the sample manager used is equipped with a 100 μL extension loop). Experimental Recycling: Figure 6 shows the recycling experiments www.chromatographyonline.com Discover more at www.ymc.de Gritti et al.

Figure 5: Prediction of the largest production rate of Figure 7: Comparison between the original the targeted impurity (99.7% yield and 99.0% purity) chromatogram recorded for the API stock solution as a function of the number of cycles when the elution (solid black chromatogram) and that recorded for the strength of the sample diluent is much stronger than that concentrated fractions of the unknown impurity (solid of the eluent. Note that the optimum conditions consist red chromatogram). Twelve consecutive fractions in operating at a number of cycles of 7 and by injecting were collected and concentrated by a factor 20 as much as 160 μL of stock solution. The maximum in pure water. Note the enrichment of the impurity production rate is then about 5.5 μg/h. (t = 3.55 min) relative to the API (t = 3.79 min) by a factor close to 2000. The collected fraction is not 90% pure (see Figure 8). Production Rate 0.0075 n =7 opt Concentrated (x20) Collected Fractions (6 μL) API stock solution (6 μL) 0.012 Targeted 0.0050 Impurity Estradiol =254 nm)

Vp,opt=159 μL λ 0.008 AU ( Pr (mg/h) 0.0025

0.004

0.0000 0 24 6 8 10 12 14 n cycle 0.000

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Time (min)

Figure 6: Experimental chromatograms (number of cycle = 6, injection volume of the stock solution = 100 μL) of were eliminated to waste before and after as can be the targeted impurity (low unknown concentration) and seen from the bottom blue chromatogram. The targeted API (10 g/L) recorded by the recycling low-dispersion impurity was transferred four more times by actuating the detection cell after the first 5 cycles (top chromatogram, recycling valve at t = 8.9 min, 12.4 min, and 15.9 min. solid black line) and by the collection detection cell after From the top black chromatogram, it is striking to observe the last and sixth cycle (bottom chromatogram, solid that the impurity is fully buried under the API band until blue line). The unknown impurity is collected after the sixth cycle during the time interval from t = 20.3 min to the fourth cycle is completed after which the apex of the t = 21.3 min (see solid vertical black lines). The impurity impurity band becomes visible. After the fifth cycle, the band is baseline separated from the API band based on impurity is almost baseline separated from the API and its the UV absorption signal (254 nm). The red dotted vertical band is clearly fronting as predicted by the calculations. lines indicate the six consecutive times at which the After the sixth cycle (bottom blue chromatogram), the recycling valve was switched in order to clean the sample impurity band is baseline separated from the estradiol from early and late impurities and to transfer the impurity band (based on absorption signals recorded at the UV band from one to the second twin column. Adapted with wavelength λ = 254 nm). It is ready to be collected from permission from reference 20. t = 20.3 min and t = 21.3 min as shown by the solid black vertical lines.

0.048 Switch Repeatability of the Twin Column Recycling Switch API estradiol recycling recycling Switch #2 #3 recycling Separation Process (TCRSP), Production Rate, and Switch Switch Switch #4 recycling recycling 0.036 recycling #1 #5 #6 Purity Levels Achieved: In order to collect all the

Impurity targeted impurity present in the 60-mL stock solution AU 0.024 of estradiol, 600 recycling runs were repeated sequentially. The repeatability of the recycling run is 0.012 Transfer #2 Transfer Transfer #4 Transfer Transfer #1 Transfer Transfer #3 Transfer

Transfer #5 Transfer critical to achieving excellent purity levels. The relative

0.000 0.0125 standard deviation of the retention times observed at the Switch Switch collect collect #1 apex of the collected band of the impurity was as low as 0.0100 #2 0.1% because the temperature of the twin columns and Clean Clean Clean Clean Clean 0.0075 that of the mobile phase was actively controlled at AU + o 0.0050 30 /-1 C. Clean All the collected fractions were concentrated by a factor 0.0025 Collect ~ 20 after total and partial evaporation of acetonitrile 0.0000 and water, respectively. Acetonitrile is totally removed 0.00 4.00 8.00 12.00 16.00 20.00

Time (min) under low-vacuum at room temperature. Water is partially removed at low-vacuum and moderate heating (60 ºC).

68 LC•GC Europe February 2019 Gritti et al.

Figure 8: (a) Photodiode array UV max plot and total ion chromatogram. (b) Gradient LC–extracted ion monitoring MS experiments unambiguously revealing the masses, m/z = 271.1691 and 253.1588, for the molecular ion of the unknown targeted impurity and of its dehydrated form, respectively. It is fully consistent with the exact molecular weight (MW = 271.16926) of either the ketone derivative of estradiol, estrone (bottom right structure), or with the enol tautomeric form of estrone (bottom left structure). XIC chromatograms (bottom graphs) for 252.50 < m/z < 253.50 (dehydrated molecular ion) and 270.50 < m/z < 271.50 (molecular ion) revealing the mass of the targeted impurity.

(a) (b) 4.23 100 4.23 000 Extracted Ion Monitoring 000 80 Dehydrated Molecular Ion 000 m/z=253.1588 000 60 000 PDA UV Max Plot 4.20 4.33 4.34 40 3.46 3.64 000 3.29 3.34 3.43 3.57 3.77 3.77 3.78 3.91 4.03 4.12 4.36 4.61 4.42 4.49 4.61 4.70 4.88 000 Impurity API 4.79 4.92 4.96 20 3.27 4.22 100 100 O OH API Extracted Ion Monitoring 80 80 Impurity H Total Ion Chromatogram Molecular Ion H 60 m/z 60 =271.1691 H H H H

+ + H O 40 H O 2 4.85 2 40 3.33 3.37 4.33 4.33 4.85 3.42 3.48 3.63 3.74 4.34 + Chemical Formula: C H O + 3.75 4.11 4.22 Chemical Formula: C H O 18 23 2 3.80 3.95 3.97 4.64 18 23 2 4.63 4.65 4.88 Exact Mass: 271.16926 4.36 4.39 20 Exact Mass: 271.16926 4.70 4.77 4.99 5.07 3.27 4.33 4.34 20 3.39 3.48 3.51 3.583.66 3.71 3.80 3.89 3.92 3.94 4.07 4.18 4.48 4.51 4.58 4.70 4.79 4.884.93 4.98 3.3 3.4 3.5 3.6 3.7 3.9 4.0 4.1 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5.0 3.8 4.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4.0 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5.0 Time (min) Time (min)

Figure 7 shows the chromatogram (red colour) recorded the API. However, the purity of the collected fractions after a 6-μL injection of a concentrated fraction (gathering is still unknown because the UV signal is recorded at a total of 12 collected fractions or 12 × 1 min × 0.7 mL/min λ = 254 nm, which maximizes the impurity-to-estradiol = 8.4 mL reduced into a 0.42 mL concentrated fraction in absorbance. Complementary LC–UV–MS experiments pure water). The reference black chromatogram is for the shown in Figure 8 estimate that the purity level of the injection of 6 μL API stock solution. The impurity-to-API collected fractions is rather close to 50% (see the top peak area increases from 1/67 (in the API solution) to total PDA scan and total ion chromatogram, Figure 8[a]). 26/1 (in the collected fractions); for example, the targeted After complete drying of all the collected fractions, a impurity was enriched by a factor ~ 1750 relative to total mass residue of 220 μg was measured indicating

DryLab® 4. Right First Time. Every Time. Pittcon Software for U(H)PLC Modeling Booth #3201 Saves DryLab reduces HPLC runtime up up to 40-fold while delivering to 97% improved selectivity.[1] Method Runtime [1] Schmidt, Molnár / J. Pharm. Biomed. Anal. 78–79 (2013) 65–74

and With DryLab, the time needed for HPLC method development is up [2] to92% reduced up to 12-fold. Development Time [2] Kochling et al. / J. Pharm. Biomed. Anal. 125 (2016) 130–139

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www.chromatographyonline.com 69 Gritti et al.

that the concentration of the impurity in the API solution (8) P. Kucera and G. Manhus, J. Chromatogr. A 219, 1–12 (1981). × = (9) A. Martin, Gas Chromatography (Academic Press, Ed., New is ~ 0.5 220 μg/0.060 L 1.85 mg/L. The actual York, New York, USA, 1958). production rate is then ~ 120 μg/252 h ~ 0.5 μg/h. The (10) J. Dingenen and J. Kinkel, J. Chromatogr. A 666, 627–650 relative abundance of the impurity relative to estradiol is (1994). (11) J. Zhao, T. Hooker, and J. Jorgenson, J. Microcolumn then ~ 1/5500 in the stock solution. Separations 11, 431–437 (1999). Identification of the Targeted Impurity: The total (12) L. Lim, H. Uzu, and T. Takeuchi, J. Sep. Sci. 27, 1339–1344 (2004). amount and the purity of the targeted impurity extracted (13) L. Lim, H. Uzu, and T. Takeuchi, Chromatography 28, 131–135 from the 10 g/L estradiol stock solution were 110 μg and (2007). (14) G. Nagy, T. Peng, D. Kabotso, M. Novotny, and N. Pohl, Chem. 50%, respectively. This is clearly insufficient to perform Commun. 52, 13253–13256 (2016). reliable NMR experiments for unambiguous structure (15) M. Trone, M. Vaughn, and S. Cole, J. Chromatogr. A 1133, elucidation since a minimum mass of 1 mg and a purity 104–111 (2006). (16) Q. Liu, J. Xiao, J. Yu, Y. Xie, X. Chen, and H. Yang, J. of at least 90% is needed. Alternatively, gradient LC–MS Chromatogr. A 1363, 236–241 (2014). using extracted ion chromatograms (XIC) for the masses (17) F. Gritti, M. Leal, T. McDonald, and M. Gilar, J. Chromatogr. A under consideration were performed to measure the mass 1508, 81–94 (2017). (18) F. Gritti, S. Besner, S. Cormier, and M. Gilar, J. Chromatogr. A of the impurity. The bottom mass spectrograms reveal 1524, 108–120 (2017). that the experimental masses of m/z = 253.1588 and (19) F. Gritti and S. Cormier, J. Chromatogr. A 1532, 74–88 (2018). 271.1691 match with the elution time of the impurity. The (20) F. Gritti, M. Basile, S. Cormier, M. Fogwill, M. Gilar, T. latter mass is consistent with the exact mass of estrone McDonald, F. Riley, and Q. Yan, J. Chromatogr. A 1566, 64–78 = (2018). (M 271.16926), a derivative of estradiol in which a (21) A. Fanigliulo, D. Cabooter, G. Bellazi, B. Allieri, A. Rottigni, and double bond replaces a single bond and two hydrogen G. Desmet, J. Chromatogr. A 1218, 3351–3359 (2011). atoms are eliminated. Complementary UV absorption (22) R. Chowdury, Solar degradation of estrone and 17β-estradiol (The University of Western Ontarion, London, Ontario, Canada, spectra demonstrated that the targeted impurity is not 2010). estrone because its secondary maximum absorption wavelength is 260.7 nm instead of 279.6 nm (22). Most Fabrice Gritti is a Principal Research Scientist at likely, the targeted impurity is the enol tautomeric form of Waters Corporation in Milford, Massachusetts, USA. estrone, which is either an intermediate or a by-product of He received his Ph.D. in chemistry and physics of the synthesis reaction of estradiol. condensed matter from the University of Bordeaux (France) in 2001. He worked with Prof. Georges Conclusions Guiochon as a Research Scientist until 2014 at the This research work has demonstrated that a high University of Tennessee Knoxville. Dr. Gritti’s research resolution, twin column recycling chromatography interests involve liquid/solid adsorption thermodynamics system coupled in series to an analytical fraction and mass transfer in heterogeneous media for collector successively isolated a significant amount of characterization and design optimization of new liquid trace impurities present in concentrated API solutions. chromatography instrument and columns. He has made The system is particularly well suited to solve very fundamental contributions to separation science with challenging separation problems involving poor resolution over 80 invited keynote lectures and 270 peer-reviewed levels (low selectivity, low efficiency), low impurity-to-API publications leading to the Chromatographic Society relative abundance, sample volume overload and strong Jubilee Medal in 2013. sample diluents, viscous fingering, high yield, and high Sylvain Cormier has been a Senior Development purity levels. The optimum production rate delivered Engineer at Waters Corporation in Milford, Massachusetts, by 4.6 mm × 150 mm twin columns is ~ 0.5 μg/h for a USA, for over 30 years. ~ 2 mg/L impurity concentration and for near coelution Ronald Morris is a Principal Scientist at Pfizer Inc. in (selectivity factor 1.16) with the API (10 g/L). Groton, Connecticut, USA, with over 20 years experience The delivery of higher production rates will only be in small molecule structure elucidation using mass possible provided that the separation and collection spectrometry. He has worked in the pharmaceutical recycling system is scaled up to operate larger internal industry since obtaining a B.S. in chemistry from Purdue diameter columns (1 cm × 250 mm, 5-μm particles). This University in 1983. task is currently under investigation in order to achieve a Frank Riley is an Associate Research Fellow at Pfizer production rate of ~ 2.5 μg/h in the same time period. in Groton, Connecticut, USA, in the Pharmaceutical Sciences Small Molecule Structure Elucidation Group. References He has worked in the pharmaceutical industry for over (1) G. Guiochon, A. Felinger, A. Katti, and D. Shirazi, Fundamentals 20 years in the area of separation sciences supporting of Preparative and Nonlinear Chromatography (Academic Press, Ed., Boston, Massachusetts, USA, 2006). early drug discovery to late-stage development (2) J. Araujo, R. Rodrigues, M. Eusebio, and J. Mota, J. Chromatogr. activities. A 1217, 5407–5419 (2010). Tony Q. Yan currently works for Pfizer in Groton, (3) C. Grill, L. Miller, and T. Yan, J. Chromatogr. A 1026, 101–108 (2004). Connecticut, USA, in the field of impurity isolation for (4) M. Krattli, F. Steinebach, and M. Morbidelli, J. Chromatogr. A 1293, 51–59 (2013). structure elucidation in the Department of Pharmaceutical (5) J.C. Giddings, Dynamics of Chromatography (Marcel Dekker, Science. He has worked in drug research and Ed., New York, New York, USA, 1965). development in the area of chiral and achiral purification (6) J. Porath and H. Bennish, Arch. Biochem. Biophys. 1, 152–156 (1962). and impurity isolation since he graduated from the (7) K. Bombaugh and R. Levangie, Separation Sci. 5, 751–763 Department of Chemistry at the University of Missouri in (1970). Rolla with a Ph.D. degree in 1995.

70 LC•GC Europe February 2019 Unleash the impossible

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Reversed-Phase Liquid Chromatography and Water, Part 1—How Much is Too Much?

Dwight R. Stoll, LC Troubleshooting Editor

When can we use completely aqueous eluents with reversed-phase stationary phases, and what happens if we make a mistake?

Reversed-phase liquid taking the time here to refresh couple of decades, the prevailing chromatography (LC) is an incredibly our perspective on the topic as idea seems to have been that the powerful mode of separation that column manufacturers introduce observed decrease in retention is applicable to a wide variety new stationary phase chemistries time was due to a change in the of applications ranging from the and particle morphologies, and conformation of C18 chains bonded separation of small organic acids to our knowledge of what goes on to the particle substrate in aqueous 150 kDa proteins. Reversed-phase inside the column improves through eluents from highly extended separations have limitations, however, fundamental research. In this first chains (that is, perpendicular to the with one of the most practically segment on this topic, I will review the substrate surface), to ones that laid significant ones being low retention basic concepts that are important for down on themselves (that is, parallel for compounds that are highly reversed-phase separations in highly to the surface). The latter state was water soluble (that is, hydrophilic). aqueous eluents, summarize recent commonly referred to as “phase Understanding the general trend advances in our understanding of collapse” (1–3). These observations for reversed-phase separations that what goes on inside the column, and also led to the notion that operating retention increases as the fraction provide examples of bad column reversed-phase columns in of water in the eluent increases, behaviour and potential remedies. completely aqueous eluents was and encountering situations where generally a really bad idea, so much retention is too low for an analyte of It has been observed so that this idea made it into the Top interest pushes us to use eluents with since the early days of LC 10 Myths of LC addressed by Ron higher and higher levels of water. Majors just five years ago (4). Around This, then, leads to the question,“How that operating a typical the late 1990s, however, experimental much water is too much?” Jumping C18-type reversed-phase evidence led a number of groups to the end of this article, the short column in a completely to adopt the idea that the decrease answer will be “It depends”. in retention time was due instead to In some cases, using completely aqueous eluent can lead “dewetting” of the stationary phase aqueous eluents (that is, containing to gradual or sudden (5). Specifically, the idea is that the no organic solvent such as methanol decreases in retention high surface tension of an aqueous or acetonitrile) may be completely time. eluent in contact with a hydrophobic acceptable and in fact can provide surface causes the eluent to extrude very useful separations of highly from the pores of the stationary hydrophilic molecules. In other Basic Concepts for phase particle. If there is no liquid cases, completely aqueous eluents Reversed-Phase Separations in the pores of the particle, this can cause some reversed-phase with Aqueous Eluents effectively reduces both the column stationary phases to behave in It has been observed since the early dead volume (that is, the volume of undesirable ways and should be days of LC that operating a typical mobile phase inside the column, Vm) avoided. This is not a new topic by C18-type reversed-phase column and the volume of stationary phase any means (1,2), but the options we in a completely aqueous eluent can that is accessible to the analyte. have for handling such situations lead to gradual or sudden decreases Indeed, significant decreases in Vm are constantly changing. It is worth in retention time (3). For at least a have been measured for C18-type

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Pure Chromatography LC TROUBLESHOOTING

Figure 1: Retention factor for uracil measured before and after stopping the flow for 10 minutes. Chromatographic conditions: Columns, HALO AQ-C18 (black points) or HALO C18 (red points) (both Advanced Materials Technology, Inc.), 50 mm × 2.1 mm, 2.7-μm superficially porous particles; Eluent, 10 mM phosphoric acid in water; Flow rate, 0.40 mL/min; Temperature, 40 °C. Retention factors were calculated using thiourea as a dead time marker, and injections of the thiourea and uracil analyte mixture were made once per minute.

0.7 AQ-C18 0.6 0.5

0.4 Flow 0.3 Stopped 0.2 C18

Retention Factor (k) 0.1 0.0 0 5 10 15 20 25 30 Experiment Time (min)

-2γcosθ phases when switching from phases in an effort to better P = organic-rich to completely aqueous understand how reversed-phase r [1] eluents, and these decreases are separations work (7). The results correlated with decreases in retention of these studies complement Since the contact angle of water observed for analytes of interest (3,6). experimental work, and provide on a hydrophobic surface, such as The extent to which this extrusion insights that cannot be obtained C18-modified silica, is greater than of the eluent occurs under actual easily by experiment. Among 90°, a positive pressure is required separation conditions depends on a number of topics, they have to force water into the pore of a a number of factors, including the addressed the question of what particle. Under conditions typical of particle pore size, stationary phase happens to reversed-phase modern LC separations, the pressure chemistry, column temperature, and stationary phases in aqueous eluents. required to push the eluent through operating pressure (through this They have found that the results of the particle bed will exceed the last factor, we could say dewetting simulation support the idea that the pressure required to push the eluent depends on particle size and flow observed decreases in retention must into the pores of the particles at rate, as well). be due to loss of eluent from the most points along the column length. particle pores, rather than physical However, when the flow is stopped The equation of Young collapse of the stationary phase (for example, if the LC is not operated chains onto themselves (8). overnight), an aqueous eluent can and Laplace is most The equation of Young and be spontaneously extruded from the commonly used to Laplace is most commonly used to pores of a hydrophobic stationary rationalize the effects of rationalize the effects of different phase particle, and the retention different chromatographic chromatographic variables on the behaviour will look very different the dewetting phenomenon (1,5,6,9). This next time the column is used. With variables on the dewetting equation, shown in equation 1 (10), this framework in mind, we can think phenomenon. provides a relationship between the about how different chromatographic pressure (P) required to force a liquid variables will affect this behaviour. For over a decade, Siepmann, into a capillary, the contact angle of As the stationary phase becomes Schure, and coworkers have been the liquid on the interior surface of less hydrophobic, the contact angle using Monte-Carlo molecular the capillary (θ), the surface tension for water will decrease. When the simulations to study the microscopic of the liquid (γ), and the radius of the angle is less than 90°, the pressure details of mobile and stationary capillary (r): indicated by equation 1 becomes

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Figure 2: Representative chromatograms for the thiourea and uracil analyte mixture obtained before and after stopping the flow. Conditions are as described in Figure 1. (a) AQ-C18 before flow stop, (b) AQ-C18 after flow stop, (c) C18 before flow stop, (d) C18 after flow stop.

35 AQ-C18 (a) 35 C18 (c) 30 Before Flow Stop30 Before Flow Stop 25 25

20 20

15 15 thiourea uracil 10 10 mAU (210 nm) mAU (210 nm) 5 5

0 0 0.0 0.2 0.4 0.6 0.0 0.2 0.4 0.6 Time (min) Time (min) 35 AQ-C18 (b)35 C18 (d) After Flow Stop 30 After Flow Stop 30 25 25

20 20

15 15

10 10 mAU (210 nm) mAU (210 nm) 5 5

0 0 0.0 0.2 0.4 0.6 0.0 0.2 0.4 0.6 Time (min) Time (min)

negative, meaning that the eluent chromatographic particles are was no measurable retention loss. On will spontaneously be drawn into not ideal by any means. The pore the other hand, they found that very the pores of the particles. Among structure itself is heterogenous, with a little retention loss was measured for chromatographers, we refer to this distribution of diameters and shapes, a phenyl-type stationary phase, even as “wetting” of the pores. Equation 1 and the chemistry of the pore surface for particles with 80 Å pores. Walter also suggests that the particle is locally heterogeneous with some and coworkers observed similar pore size ought to play a role (that unbonded silanol sites, stationary trends, and also described results is, through r), with the pressure phase ligands (for example, C18), for the dependence of retention loss required to force eluent into the and endcapping functional groups on stationary phase bonding density, pore increasing as the pore size (for example, trimethylsilyl groups). the concentration of methanol in the decreases. And so, we look to experimental eluent, and use of a post-column results for the definitive answer to restrictor to increase pressure inside One good way to assess the question, “How much water is the column (5). too much?” The chromatographic whether or not a reversed- literature provides useful data that The good news is that phase stationary phase is at least establish trends, even if the dewetting does not have susceptible to dewetting results are not exactly transferrable to be a death sentence for under a particular set to a particular set of conditions of interest. For example, Bidlingmeyer reversed-phase columns. of conditions is by fi rst and Broske showed results that equilibrating the column speak to the effect of particle pore under conditions where the size, stationary phase chemistry, and Testing for Dewetting, column temperature on the extent to and Some Remedies stationary phase is highly which dewetting occurs in aqueous One good way to assess whether solvated, or fully wetted. eluents (11). They found that, for or not a reversed-phase stationary one type of C18 stationary phase, phase is susceptible to dewetting The Washburn equation provides a there was a retention loss of 80% under a particular set of conditions is helpful framework for thinking about for particles with a pore diameter of by first equilibrating the column under the effects of these parameters, 80 Å, but, with a diameter of 150 Å conditions where the stationary phase but, of course, the pores of and the same stationary phase, there is highly solvated, or fully wetted.

76 LC•GC Europe February 2019 LC TROUBLESHOOTING

For most reversed-phase stationary Figure 3: Comparison of chromatograms obtained for the analyte butyrophenone phases, this could be an eluent high in 50:50 acetonitrile–10 mM phosphoric acid in water using the C18 (a) before, and in methanol or acetonitrile content. (b) after the dewetting experiment. All other conditions are the same as in Figure 1. Flushing the column at a modest flow rate for a time equivalent to about 400 (a) 20 column volumes should be more Before Dewetting than enough. Then, we switch to the 350 aqueous eluent (or whatever eluent 300 is useful for the application at hand), 250 begin injecting a mixture of two or 200 three probe compounds that have 150 reasonable retention under these

mAU (254 nm) 100 conditions, and monitor the change 50 in retention factor (k) as the column equilibrates. One could simply wait 0 0.6 0.8 1.0 1.2 1.4 for 20 column volumes to pass first Time (min) before injecting the test sample, 400 but, if we start injecting right away, After Dewetting (b) we also learn something about how 350 quickly the column equilibrates when 300 switching from the organic-rich to 250 the aqueous eluent. Then, once the 200 retention has stabilized after this 150 initial equilibration step, turn the flow mAU (254 nm) 100 off, wait 10 minutes, then turn the flow 50 back on, and start injecting the test 0 mixture again. If there is a significant 0.6 0.8 1.01.2 1.4 difference between the retention Time (min) before and after stopping the flow,

www.chromatographyonline.com 77 LC TROUBLESHOOTING

dewetting is likely to be a serious when dewetting happens, effectively reversed-phase stationary phases). problem under these conditions. decreasing the dead volume of the In a future instalment, I will discuss Retention will vary from day to day, column. the implications of using solvent depending on the post-column flow The good news is that dewetting gradient elution involving these highly restriction in the system, and peak does not have to be a death sentence aqueous eluents. shapes may deteriorate and become for reversed-phase columns. As is variable. the case when recovering a column Acknowledgements that has dried out during storage (12), I’d like to thank Dr. Stephanie Columns that have columns that have dewetted can be Schuster of Advanced Materials dewetted can be recovered recovered by flushing with several Technology for providing the columns column volumes of organic-rich used in this work, and Dr. Mark by fl ushing with several eluent. Figure 3 shows a comparison Schure and Dr. Richard Henry for column volumes of of chromatograms obtained for helpful discussions on the topic of organic-rich eluent. the analyte butyrophenone before dewetting. and after the dewetting experiment summarized in Figures 1 and 2. In References Figures 1 and 2 show this case, simply pumping 50:50 (1) M. Przybyciel and R.E. Majors, LCGC North Amer. 20, 516–523 representative results from such a acetonitrile–water through the column (2002). test, conducted with two C18-type at 0.4 mL/min for 5 minutes (pressure (2) J.W. Dolan, LCGC Europe 21(12), stationary phases that are otherwise drop across the column was about 624–627 (2008). very similar (that is, they use the 100 bar) was enough to restore both (3) P. McDonald, Advances in Chromatography (Marcel Dekker, New same base silica), but one (AQ-C18) the retention and peak shape for this York, USA, 2003) pp. 323–375. is designed for use in highly aqueous column to a “like new” state. (4) R.E. Majors, LCGC Europe 26(10), eluents. In other words, the AQ-C18 584–592 (2013); R.E. Majors, LCGC Europe 26(11), 630–636 (2013). is engineered to avoid the dewetting It is a good idea to test (5) T.H. Walter, P. Iraneta, and M. phenomenon in completely aqueous Capparella, J. Chromatogr. A 1075, eluents. Figure 1 shows the retention for dewetting using 177–183 (2005). DOI:10.1016/j. factor for uracil measured on these the stop-fl ow during chroma.2005.04.039. (6) K. Nakamura, H. Nakamura, S. Saito, two columns in a completely aqueous method development of and M. Shibukawa, Anal. Chem. eluent, injecting sample once per applications involving an 87, 1180–1187 (2015). DOI:10.1021/ minute, before and after stopping the ac503802q. alkyl bonded phase if they (7) M.R. Schure, J.L. Rafferty, J.I. flow for 10 minutes. For the very first Siepmann, and L. Zhang, LCGC Europe injection, the retention is slightly lower will be used with eluents 27(1), 18–27 (2014). than the rest of the points, because (8) L. Zhang, L. Sun, J.I. Siepmann, containing less than 5% and M.R. Schure, J. Chromatogr. A the column has not equilibrated from organic solvent. 1079, 127–135 (2005). DOI:10.1016/j. the 50:50 organic–water flushing chroma.2005.03.124. solvent to the completely aqueous (9) M. Schure, N. Devitt, R. Moran, J.M. eluent. After equilibration, the Summary Godinho, and B. Wagner, J. Chromatogr A Submitted (2019). retention of uracil on the two phases In this instalment of “LC (10) A.W. Adamson and A.P. Gast, Physical is remarkably similar. After turning the Troubleshooting”, I have described Chemistry of Surfaces (Wiley, New York, flow off for 10 minutes, we observe the phenomenon that has USA, 6th Ed., 1997). (11) B.A. Bidlingmeyer and A.D. Broske, that the two phases behave very become known as “dewetting” in Journal of Chromatographic Science differently. For the AQ-C18, there is reversed-phase chromatography. 42, 100–106 (2004). DOI:10.1093/ no statistically significant change in I have discussed the basic chromsci/42.2.100. the retention of uracil. On the other principles that explain why and (12) D.R. Stoll, LCGC Europe 30(7), 352–357 (2017). hand, the retention of uracil on the when this occurs, so they can be C18 column decreases by about used as a guide during method Dwight R. Stoll is the editor of 75%. development. Since the extent of “LC Troubleshooting”. Stoll is a Representative chromatograms dewetting depends on a number professor and co-chair of chemistry for the two columns before and of factors, including particle pore at Gustavus Adolphus College in St. after stopping the flow are shown in size, stationary phase chemistry, Peter, Minnesota, USA. His primary Figure 2. The chromatograms for the and operating conditions, it is a research focus is on the development AQ-C18 column are indistinguishable, good idea to test for dewetting of 2D-LC for both targeted and as expected. In the chromatograms using the stop-flow during method untargeted analyses. He has for the C18 column, though, we see development of applications authored or coauthored more than 60 that there is not only a change in the involving an alkyl bonded phase peer-reviewed publications and three retention factor of uracil, but also (for example, C8 or C18), if they will book chapters in separation science a 23% decrease in the measured be used with eluents containing and more than 100 conference dead time. This is consistent with less than 5% organic solvent. presentations. He is also a member the idea discussed above, that This piece has been restricted to of LCGC ’s editorial advisory eluent is extruded from the pores isocratic conditions (that is, the use board. Direct correspondence to: of the stationary phase particles of completely aqueous eluents with [email protected]

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GC×GC: From Research to Routine

Nicholas H. Snow, Department of Chemistry and Biochemistry, Seton Hall Univeristy, New Jersey, USA

Comprehensive two-dimensional gas chromatography (GC×GC) is becoming increasingly popular. Since its inception in the 1990s and its original commercial availability about 15 years ago, GC×GC has moved from a strict research realm into more routine applications and use. While not yet a standard capability on all gas chromatographs, GC×GC is rapidly becoming the technique of choice for analysis of highly complex samples as diverse as petroleum, pharmaceuticals, biological materials, food, fl avours, and fragrances. This instalment of “GC Connections” begins with a brief introduction to GC×GC, follows with examples of how GC×GC opens additional avenues of analysis, and it concludes with information about how to learn more.

Comprehensive two-dimensional very short (often 1–2 m), to perform into these slices, which are then gas chromatography (GC×GC) a fast separation. As analytes elute organized side-by-side, to generate is continuing to see increased from the first column, they are the three-dimensional plot shown attention throughout the scientific refocused at the head of the second below. community. Until fairly recently, column and injected very rapidly, with Figure 2 shows a photograph of the most GC×GC work was performed second dimension separations lasting column oven in a GC×GC instrument. and published by chromatography for only a few seconds. This transfer The most important difference versus experts. Much like traditional gas is performed by the modulator, which GC is the addition of the second chromatography, which is now is programmed to perform this step at column oven and the modulator routinely used by analysts with a wide regular intervals, essentially cutting hardware. In this configuration, the variety of scientific backgrounds, the chromatogram into short slices. second column oven is installed GC×GC is finding its way out of As seen in Figure 1, GC×GC has one within the main column oven; usually, the chromatographer’s laboratory. major instrumental advantage over the second column is maintained “Should I try GC×GC?” is one of the other multi-column techniques in that at the same or higher temperature most common questions I receive it employs only one detector. LCGC ’s as the main oven. The two columns when discussing GC or GC×GC. ChromAcademy provides a summary are connected with a simple Following a short introduction, of the various configurations of capillary column butt-connector. this article discusses scenarios in multidimensional GC that are The modulator sits at the head of which GC×GC has proven useful available (1). the second column. In this system, a and applicable to routine analyses, two-stage thermal modulator is used and some avenues for obtaining Comprehensive two- with alternating cold liquid nitrogen additional help and information. and heated gaseous nitrogen gas A simple schematic of the basic dimensional gas jets that focus and inject the analytes instrumentation and data processing chromatography (GC×GC) into the second column, without in GC×GC is shown in Figure 1. is continuing to see allowing unwanted carryover from The instrumentation is based on increased attention the first. There are numerous reviews a traditional GC system, with the and vendor websites that provide same inlet, main column oven, and throughout the scientifi c more detail about instrumentation, detector. A second column oven, community. modulators and data analysis in usually smaller for ease of heating GC×GC (2). A simple online search and cooling, is added to house the using “GC×GC” as the keyword will second-dimension column within The right side of Figure 1 shows point you to major vendors. a separate heated zone. Also, a the data analysis process performed One major advantage of GC×GC modulator is added to provide the by the data system. As there is one is that the additional components sample transfer between the two detector, a single chromatogram is (modulator and second column columns. These are all controlled generated, shown at the top. The oven) are installed between the by a specialized data system. A solid vertical lines represent the traditional inlet and detector in a traditional column is used in the first beginning and end of each slice. gas chromatograph, allowing them dimension, and the second column is The chromatogram is separated to be used without modification. As

80 LC•GC Europe February 2019 GC CONNECTIONS

GC×GC is most commonly used alternatives, such as heart-cutting or • First, there are a large number of for analysis of extremely complex additional sample preparation, should peaks in the chromatogram; some mixtures, most applications have also be considered. GC×GC has been or all may be of interest. been developed using split injection, described as a “super-resolution” • Second, there are a large number which ensures a rapid transfer of the technique, so the need for high of peaks, they are of interest, and analyte mixture to the first-dimension resolution or peak capacity (the full separation must be generated. column. Other injection techniques, number of peaks that can theoretically • Third, there are a few very closely and nearly all on- and off-line sample fit into the chromatogram) is the main spaced or overlapping peaks, and preparation techniques, have been driver for choosing it (3). they are not separated following attempted with GC×GC. In this article, This main consideration leads optimization of the method. examples using split, splitless, and to three situations where a solid-phase microextraction (SPME) multidimensional approach to the The first major application for injections are shown. In detection, column in GC, leading to GC×GC, GC×GC was in petroleum analysis, there is one important caveat in mainly apply. perhaps the most extreme example of GC×GC: the detector must be fast. The short second-dimension column generates peaks that elute with widths of 100–200 ms or less. If 10 data points are required to generate a properly symmetrical peak for good quantitative analysis, the detector must be able to respond by providing a signal within 10–20 ms. The data collection rate must therefore be set at a minimum of 50–100 Hz for proper peaks to be generated. A flame ionization detector (FID) is easily capable of this, but most benchtop mass selective detectors in full-scan mode are not without careful optimization. Most GC×GC– mass spectrometry (MS) applications are therefore performed using

One major advantage of GC×GC is that the additional components (modulator and second column oven) are installed between the traditional inlet and detector in a gas chromatograph, allowing them to be used without modifi cation. time-of-flight (TOF-) MS, which allows extremely fast full-scan rates. The decision to consider GC×GC should be based on the overall needs and goals of the project, and on the other capabilities that can be built into the method. There is also a cost consideration, as the hardware for GC×GC and for GC×GC–TOF-MS requires larger capital and ongoing expenses than for traditional GC and GC–MS, and additional training and skill development for system operators are needed. Less expensive www.chromatographyonline.com 81 GC CONNECTIONS

Figure 1: Simplified schematic of a GC×GC instrument and the data analysis process.

Inlet FID

Oven 1 Oven 2

Modulator Dimension nd 2 1st Dimension

The sample has the physical Figure 2: Photograph of the oven in a GC×GC system showing the primary column, consistency of chocolate mousse, column connection, modulator, and secondary oven. and the odour of another well-known brown substance. Extraction and chromatographic conditions are included with the figure. This chromatogram is typical of petroleum analysis in one dimension. The spikes represent signals from straight chain alkanes, and the “hump” results from a huge number, perhaps thousands, of overlapping hydrocarbon isomers. It is very difficult to detect individual compounds or compound classes of interest from such a complex one-dimensional chromatogram. Figure 4 shows a GC×GC–TOF-MS separation of the same extract. The chromatogram is viewed as a contour plot, with the bright spots representing peaks against the blue baseline. The horizontal axis represents the traditional retention time on the first-dimension column; the total run time is 40 min, similar to the one-dimensional example. The vertical axis represents the short second-dimension retention time. As described above, the full the first situation. Petroleum-related chromatogram of a sample of chromatogram was separated into samples often contain thousands “brown mousse” obtained from 8 second slices, and reassembled of compounds, yet often only a the surface of the Gulf of Mexico into the three-dimensional plot. The few are of interest. They must still following the explosion of the brighter and more red the spot, the be separated from the rest of the Deepwater Horizon oil platform in taller the peak, while the blue colour extremely complex sample matrix. 2010. A photograph of a brown represents the baseline. The black Figure 3 shows a traditional GC–MS mousse sample in a jar is included. dots represent the apexes of all the

82 LC•GC Europe February 2019 GC CONNECTIONS

Figure 3: One-dimensional GC–MS total ion chromatogram of brown mousse sample from the surface of the Gulf of Mexico following the Deepwater Horizon oil spill.

individual peaks identified by the Figure 4: GC×GC–TOF-MS chromatogram of brown mousse sample from the surface data system. In this case, it is of the Gulf of Mexico following the Deepwater Horizon oil spill. possible to generate a mass spectrum of each of these peaks, if 8 desired. Columns: 1D: DB-5MS 30mx 0.25mm x 0.25 μm The decision to consider 2D: Rtx-200 1.5m x 0.25 mm x 0.25 μm GC×GC should be based Temperature Programs: on the overall needs and 1D: 40ºC to 300ºC at 10ºC /min goals of the project, and 2D: 45ºC to 305ºC at 10ºC /min on the other capabilities that can be built into the Modulator: 0.90 sec hot pulse method. 1.60 sec cold pulse

We were interested in polycyclic MS: El, full scan 40-600 amu aromatic hydrocarbons (PAHs). The (sec) Second Dimension Retention Time 0 540 PAHs were easily identified using First Dimension Retention Time (min) extracted ion chromatograms; some are circled on the plot. This is the first and most important benefit of GC×GC: individual compounds or also shows a few peaks near the high selectivity that can be obtained compound classes of interest are bottom of the chromatogram. This when combining the separation much more easily separated from the is an example of “wraparound”, an power of a traditional first-dimension remaining matrix components. With effect caused by second dimension column with the added selectivity judicious choices of column sets, peaks being retained longer on the of the second-dimension column the chromatography can be made second-dimension column than the followed by a selective detector. very highly selective for specific modulation period (in this case 8 s). The bright spots represent the compounds of interest. In this case, They then appear in the next slice. peaks that included signals for the PAHs are found together near Since the analytes of interest were m/z 128. Note that the large signals the “top” of the chromatogram in the PAHs and related compounds, we for the alkanes no longer appear. circled region. In our application, chose to extract m/z 128, which is The black dots, which represent the GC×GC was used to separate representative of naphthalene and maxima of all the peaks, are left and identify a small number of related compounds. Figure 5 shows in the figure to demonstrate the components of interest that share the extracted ion chromatogram unique selectivity obtainable in a common chemistry, from a large (EIC) of an oil spill sample. This GC×GC, especially when using MS and complex sample matrix. Figure 4 chromatogram demonstrates the very detection, that enables the isolation www.chromatographyonline.com 83 GC CONNECTIONS

Figure 5: Extracted ion GC×GC–TOF-MS chromatogram (m/z 128) of brown mousse the class II, moderately toxic sample from the surface of the Gulf of Mexico following the Deepwater Horizon oil spill. solvents. These include common Conditions identical to Figure 4. moderately toxic solvents, such as methanol, ethanol, acetonitrile, 8 dichloromethane, and others. A full list of the analytes can be found sec)

( in the article. In this example, the analytes were dissolved in methanol, Time

which is not a traditional diluent for residual solvents analysis. As seen in the figure, the components are all well-separated from each

Retention other, from matrix components

and from interferences inherent in gas chromatographic analysis. The inset shows the separation of

Dimension × GC GC has been described as a “super- resolution” technique, Second 0 5 40 so the need for high First Dimension Retention Time (min) resolution or peak capacity (the number of peaks that can theoretically fi t into the Figure 6: GC×GC–FID chromatogram of ICH Class II pharmaceutical residual chromatogram) is the main solvents. Inset shows three-dimensional plot of the separation of acetonitrile and dichloromethane as system suitability test. Adapted with permission from driver for choosing it (3). reference 5. acetonitrile and dichloromethane, as an important system suitability test. Note the large solvent peak, methanol. It is also fully separated from these two analytes, including

3 the long tail. In GC, most solvent peaks can exhibit a detectable tail long after the solvent has eluted. In

2 traditional one-dimensional GC, the 2.38 solvent peak tail would lie underneath 154 1.38 0.38 Second dimension time all analyte peaks, possibly affecting × Second dimension time 1 quantitation. In GC GC, the analytes are fully separated from the solvent peak tail. Also note the row

0 of small, sharp peaks in the lower 0 500 1000 1500 right quadrant of Figure 6. These First dimension time are indications of a small amount of septum bleed. In a traditional separation they would appear as of a few peaks from hundreds or Pharmacopeia, General Chapter <467>, “extra” peaks, possibly interfering thousands. USP <467> (4). In 2008, Crimi and with the analysis. In GC×GC, like the In the second situation, there are Snow published an article in LCGC solvent peak, they are fully separated a large number of analytes, and they North America describing the use from the analytes. A complete must be fully separated, either for good of GC×GC–FID for this analysis (5). separation of all the International quantitative analysis (perhaps detection This article is available freely on-line. Council for Harmonization (ICH) is performed with a FID or other A two-dimensional separation of the Class I, II, and III residual solvents traditional detector, not a mass selective complete set of class I (high toxicity), (57 compounds) and full description detector [MS]) or the resolution must class II (moderate toxicity), and class of the separation conditions is meet regulatory requirements. One III (low toxicity) residual solvents is provided in the article. In this case, example of this type of problem is described, along with discussion of the GC×GC allowed separation of all the analysis of residual solvents in method development process. 57 analytes, with separation from pharmaceuticals, which is governed in Figure 6 shows the the solvent peak tail and other the United States by the United States two-dimensional separation of interferences.

84 LC•GC Europe February 2019 GC CONNECTIONS

In the third situation, GC×GC can be used to separate several Figure 7: Selected-ion monitoring (SIM) chromatogram for SPME–GC–MS analysis of nine drugs. The drugs are identified in the article. Adapted with permission from closely eluting or overlapping peaks reference 6. that are not separated in more traditional method development. This situation may arise, especially 1200000 7 in cases where the first-dimension separation is optimized for speed. 6 A faster separation generally 1000000 means less separating power and lower resolution. Figure 7 shows 800000 5 a one-dimensional separation, really a non-separation, of several 1 steroids on a traditional 5% phenyl 600000 3 polydimethylsiloxane column, Signal × 4 15 m 0.25 mm, 0.25-μm (6). I use 400000 this stationary phase and column 8 dimensions for many separations in 9 my own laboratory, as it represents 200000 an excellent compromise between 2 separating power and analysis time. 0 It is a good starting point for method development. There are about 10 0246810121416 1820 overlapping peaks eluting in a very Time (min) short time window at about 12 min. This situation is common with close structural isomers and closely related power of GC×GC in a similar run time to GC, with the formerly structures. There are several possible approaches overlapping analytes now fully separated. Also note that SPME for optimizing this separation, including further was used for the sample preparation and injection. optimizing the temperature program (see Hinshaw’s recent “GC Connections” column using a computer simulation [7]), changing the column to one with greater selectivity for steroids, or heart-cutting, a classical form of multidimensional GC in which the small portion of the chromatogram with the overlapping peaks is transferred to a second, more selective column (8). Heart-cutting is often still the method of choice in this situation, as the instrumentation required is simpler than SERVING ROYALTY. EXCEEDING EXPECTATIONS. EVERY MOMENT. for GC×GC.

GC×GC can be used to separate several closely eluting or overlapping peaks that are not separated in more traditional method development. This situation may arise, especially in cases where the fi rst-dimension separation is optimized for speed.

In this case, we used GC×GC to separate the steroids. The resulting chromatogram is shown in Figure 8. The • Provider of top brand HPLC instrumentation products first-dimension column was the same as in the single • Equivalent to corresponding OEM products dimension separation, so the retention times of the overlapping • Serving customers for over 30 years peaks are about the same and the total run time is about the • Reduce product repair expenses by 30% same. The second-dimension column was 1.5 m × 0.25 mm, • Lifetime Warranty on manufacturing defects 0.25-μm trifluoro propyl methyl polysiloxane, which is more polar, and has better selectivity for steroids and other drugs www.sciencix.com 800.682.6480 [email protected] due to greater opportunity for dispersive and dipole-dipole interactions between the trifluoropropyl methyl moieties on the stationary phase and the various functional groups on the drugs. This separation demonstrates increased resolving www.chromatographyonline.com 85 GC CONNECTIONS

References Figure 8: SPME–GC×GC–TOF-MS contour plot chromatogram of 14 drugs extracted (1) https://www.chromacademy.com/ from tap water. Drugs are identified in the article. Adapted with permission from gc-training.html (Accessed January reference 6. 2019). (2) M. Adahchour, J. Beens, R.J.J. Vreuls, and U.A.T. Brinkman, Trends Anal. Chem. 25(5), 438–454 (2006). (3) P. Marriott and Y. Nolvachai, The Analytical Scientist 67 (2018). (4) General Chapter <467> “Residual 4 Solvents” in United States

s) Pharmacopeia 40 (USP 40) (United (

r t

States Pharmacopeial Convention, Rockville, Maryland, USA) https://hmc. 3 usp.org/sites/default/files/documents/ HMC/GCs-Pdfs/c467.pdf (Accessed January 2019). Dimension (5) C.M. Crimi and N.H. Snow, LCGC North

nd 2

2 Amer. 26(1), 62–70 (2008). http://www. chromatographyonline.com/analysis- pharmaceutical-residual-solvents- 1 using-comprehensive-two-dimensional- gas-chromatograhy (Accessed January 2019). (6) P.C.F.L. Gomes, B.B. Barnes, A.J. 0 Santos-Neto, F.M. Lancas, and N.H. 6.710 16.7 Snow, J. Chromatogr. A 1299, 126–130 (2013). 1st Dimension t (min) r (7) J.V. Hinshaw, LCGC Europe 30(11), 632–637 (2017). http://www. chromatographyonline.com/fast-gas- There are now myriad applications of elbows and discuss chromatography chromatography-2 (Accessed January GC×GC in the chemical literature, and in an informal setting with the leaders 2019). × (8) P.Q. Tranchida, D. Sciarrone, P. Dugo, several instrumental configurations. in the field. The GC GC meeting and L. Mondello, Analytica Chimica Most major instrument manufacturers begins with a GC×GC short course Acta 716, 66–75 (2012). offer multidimensional GC in some on Sunday 12 May, followed by the (9) http://scholar.google.com (Accessed 10 form, ranging from simplified opening of the GC×GC symposium on January 2019) (10) K.V. Schug, LCGC North Amer. heart-cutting to fully implemented Monday and the rest of the capillary 36(12), 891–893 (2018). http://www. GC×GC. A simple search in Google chromatography programme on chromatographyonline.com/deep- Scholar using “GC×GC” as the Tuesday. This is an unparalleled heart-texas-chromatography-advances (Accessed January 2019). keyword yielded about 5870 results opportunity to learn about GC and (11) https://www.isccgcxgc.com/ (Accessed (9). To quickly learn more about GC×GC directly from leaders and January 2019). GC×GC, consider attending the pioneers. Nicholas H. Snow is the Founding International Symposium on Capillary Endowed Professor in the Department Chromatography and GC×GC If “super-resolution” of Chemistry and Biochemistry at Symposium in Fort Worth, Texas, is a priority, GC×GC is Seton Hall University. He is also the USA, from 12–17 May 2019 (10,11). university’s Director of Research This is the continuation of an annual clearly ready to take its and Adjunct Professor of Medical series of small meetings of the best place alongside GC as a Science. During his 30 years as a minds in capillary chromatography major technique for both chromatographer, he has published that began in Hindenlang, Germany, more than 60 refereed articles and in the 1970s. The meeting is now research and routine book chapters and has given more than held in alternating years in Ft. Worth analysis in its own right. 200 presentations and short courses. (USA) and Riva del Garda (Italy). One He is interested in the fundamentals of this year’s plenary lectures, titled GC×GC is fully quantitative and applications of separation science, “Don’t Bring a Football to a Baseball and automated as easily as GC. If especially GC, sampling, and sample Game: Get GC×GC to Have its Own “super-resolution” is a priority, GC×GC preparation for chemical analysis. His Rules, Field, and Fan Base”, is being is clearly ready to take its place research group is very active, with given by Chris Reddy of Woods Hole alongside GC as a major technique for ongoing projects using GC, GC–MS, Oceanographic Institute. Chris is one both research and routine analysis in two-dimensional GC, and extraction of the leaders in bringing GC×GC its own right. methods including headspace, liquid– from the realm of chromatography liquid extraction, and SPME. research laboratories into the “real Acknowledgement John V. Hinshaw is a Senior Scientist world”. Unlike most conferences, The author is grateful to Prof. John at Serveron Corporation in Beaverton, this symposium (of about 500 R. Sowa, Jr. (now at Governor’s State Oregon, USA, and a member of attendees) is built around discussion, University) for providing the brown LCGC Europe’s editorial advisory well-attended poster sessions, and mousse sample and to Dr. Brian board. Direct correspondence about a strong social programme where all B. Barnes (now at ExxonMobil) for this column to the author via e-mail: attendees have opportunities to rub obtaining the chromatograms. [email protected]

86 LC•GC Europe February 2019 CONNECT WITH LCGC ON SOCIAL MEDIA

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The Role of Liquid Chromatography and Gas Chromatography in the Analysis of Illegal Medicines and Health Products

Yaxin Tie1,2, Celine Vanhee1, Erwin Adams2, and Eric Deconinck1, 1Scientific Direction Chemical and Physical Health Risks, Section Medicines and Health Products, Sciensano, Brussels, Belgium, 2KU Leuven, University of Leuven, Department of Pharmaceutical and Pharmacological Sciences, Pharmaceutical Analysis, Leuven, Belgium

Falsifi cation or adulteration of medicines and health products is a long-standing issue posing a serious menace to public health. These substandard and falsifi ed (SF) medical products could result in treatment failures, drug resistance, and serious adverse drug reactions. In response to the increasing concern about the occurrence of falsifi ed medical products in the European Union, the Falsifi ed Medicines Directive 2011/62/EU will be implemented in the beginning of 2019. This directive creates a preventive system for falsifi ed medicines. Alternatively to the global regulatory efforts that are being made, regulatory authorities, including official medicine control laboratories (OMCL), also need efficient analytical methods to monitor drug quality and survey the (illegal) market. This review article will give a general overview of the liquid chromatographic (LC) and gas chromatographic (GC) methods used by these analytical laboratories for the detection and characterization of suspected illegal medicines and health products, including lifesaving drugs (antimicrobials and antimalarials), lifestyle drugs (erectile dysfunction drugs), and biotechnology drugs (doping peptides and skin-tanning peptides). Literature published from 2015 until early 2019 will be surveyed.

Over the past five years, incidents medical products from entering the of at least 48 nonrelated incidents involving pharmaceutical crime legal supply chain of the European that affected the health of thousands increased by 60%, posing a Union (EU) because obligatory safety of adults and children worldwide, growing threat to public health (1). features (a unique identifier and with a similar number of incidents in Pharmaceutical falsification is a tamper-evident packaging) will be developing and developed countries lucrative business. The profits of the used to guarantee the authenticity (7). In the battle against SF medical illegal pharmaceutical market ranged of medical products. Moreover, products, it stands to reason that from 75 billion to 200 billion US dollars additional strict rules are prescribed besides preventive strategies and in 2012, which resulted in the growing for the import of active substances stricter regulations and controls, trend of pharmaceutical crime (2). and strengthened requirements efficient analytical methodologies In order to come to a global of record-keeping for wholesale are also paramount because they uniform semantics, the World Health distributors will be put in place. enable the surveillance of (illegal) Organization (WHO) has adopted Furthermore, internet pharmacies medical products on the market. the term “substandard and falsified should display a common EU-wide Chromatographic and hyphenated (SF) medical products’’, to represent logo on the website (4). techniques allow a comprehensive three mutually exclusive classes, According to the WHO, SF medical analysis of SF medical products namely substandard medical products influence every region in terms of active pharmaceutical products, unregistered or unlicensed of the world (5). The International ingredients (APIs), impurities, and medical products, and falsified Criminal Policing Association (Interpol) residual solvents, giving them a medical products (3). Furthermore, estimated that an annual death toll of prominent role in the supervision of SF the Falsified Medicines Directive more than one million people was due medical products. 2011/62/EU will be executed in to SF medical products (6). Recently, February 2019 to combat the threat of a survey on the available scientific Analytical Methods falsified medicines (4). The directive peer-reviewed publications since The increased awareness of the

focuses on the prevention of falsified 2006 demonstrated the occurrence potential dangers of SF medical Photo Credit: Oleksii Fedorenko/Shutterstock.com

88 LC•GC Europe February 2019 PHARMACEUTICAL PERSPECTIVES products has resulted in numerous Figure 1: Overlay of UHPLC–MS2 total ion chromatograms of 35 antimicrobials articles, in which chromatographic and 1 beta-lactamase inhibitor. 1 = cefepime, 2 = clavulanic acid, and spectroscopic techniques 3 = amoxicillin, 4 = cefadroxil, 5 = lincomycin, 6 = ceftazidime, 7 = cefaclor have been employed for the and trimethoprim, 8 = cefalexin and ampicillin, 9 = cefradine and polymyxin = = = detection and characterization of B1, 10 ofloxacin, 11 norfloxacin, 12 tetracycline and ciprofloxacin, 13 = azithromycin, 14 = ceftriaxone and cefotaxime, 15 = clindamycin and SF medicines and illegal health nitrofurantoin, 16 = bacitracin, 17 = doxycycline, 18 = erythromycin and cefazolin, products. In general, spectroscopic 19 = clarithromycin, 20 = roxithromycin, 21 = sulfamethoxazole, 22 = penicillin G, techniques, such as Fourier-transform rifampicin and cefuroxime axetil, 22(2) = benzathine (of benzathine penicillin G), infrared spectrometry (FT-IR), 23 = penicillin V, 24 = griseofulvin, 25 = cloxacillin, 26 = gentamicin and neomycin. Adapted with permission from reference 15. near infrared spectroscopy (NIR),

and Raman spectroscopy, are x109 13 1.50 often used for a first evaluation or 19 15 18 screening of suspected products, 1.25 since they are fast and require less 5 or no sample preparation. However, 1.00 20

10 14 spectroscopic techniques are not 0.75 16 17 12 22 Intensity always able to detect the presence 8 7 of illicit APIs because of matrix 0.50 interference, and they are of limited 21 0.25 9 11 25 use for the quantification of APIs and 3 6 24 26 1 4 23 22 (2) 2 the detection of impurities. Nuclear 0.00 0 2 4 6 81012 14 16 magnetic resonance (NMR) allows Time (min) proper identification and quantification without utilization of standards, but this technique is rather complicated analytical laboratories responsible for be tackled by hyphenation with mass and requires milligrams of material. the analysis of SF medical products, spectrometry (GC–MSn or LC–MSn). Nowadays, gas chromatography despite the fact that reference This article will discuss (GC) and liquid chromatography (LC) substances are necessary for chromatographic techniques and methods are the gold standards in quantification. Identification issues can their applications in lifesaving drugs

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adipate, chlorzoxazone, ciprofloxacin, Figure 2: Elution of nine peptides on a ZIC-HILIC column as illustrated by Janvier et al. Adapted with permission from reference 44. and acetaminophen. Lifestyle Medicines: Erectile 0.5 Dysfunction Drugs: Falsification of erectile dysfunction drugs is widespread in developed countries. 0.4 LC, GC, and hyphenated techniques have been proposed for the

0.3 surveillance of this illegal market. Lee et al. (20) developed a simple, fast, and selective LC–quadrupole Absorption 0.2 time-of-flight (QTOF)-MS method for the screening of 40 analogues of sildenafil and the creation of a 0.1 spectral library, including accurate mass, ion fragmentation patterns, and retention times. This powerful 0 246810 12 14 LC–QTOF-MS-based spectral Time (min) CJC-1295 Epitalon GHRP2 GHRP6 Ipamorelin Leuprorelin Melanotan ||Oxytocin Sermorelin library provided comprehensive and accurate mass data, which was necessary and useful in the analysis of complex SF erectile dysfunction (antimicrobials and antimalarial MS2 method was able to detect drugs. A case report described drugs), lifestyle drugs (erectile 35 antimicrobials and one that Kamagra tablets (containing dysfunction drugs), chemical beta-lactamase inhibitor (Figure 1). sildenafil, but not registered in adulteration of dietary supplements, A longer UHPLC–diode array the EU) seized by the German and biotechnological drugs. detection (DAD) method was applied police contained large amounts of Lifesaving Medicines: to quantify the 35 antimicrobials 2-mercaptobenzothiazole (MBT), Antimicrobials and Antimalarials: and one beta-lactamase inhibitor. which is known as a human contact According to the WHO, since 2013, The results showed that half of the allergen and a rubber vulcanizing antibiotics and antimalarials have collected samples were noncompliant accelerator. High-resolution UHPLC– been the most commonly reported with the regulation. In this case, DAD orbital trap MS2 was used for the SF medical products (8), and most was preferred over 3D ion trap MS identification of sildenafil and the are associated with African and Asian for quantification because the former detection of MBT, although the initial regions (9–13). Fadeyi et al. (10) and provides accurate results without screening using handheld FT-IR gave Frimpong et al. (12) investigated the the need for expensive isotopically a negative result (21). Subsequent quality of antimicrobials in Ghana by labelled internal standards. quantifications were achieved by using LC–UV for the determination of Antimalarials are of the utmost using the LC–DAD method described the API content. More than half of the importance as lifesaving drugs. As in the European Pharmacopoeia (22). samples purchased in Ghana were stated by the WHO, half of the global With regard to quantification, Fidan found to be of poor quality. Recently, population was at risk of malaria et al. (23) developed and validated Islam et al. (14) evaluated the quality in 2016 (16). It has been reported a LC–UV method to determine of antimicrobial drugs in Myanmar that SF-antimalarial medicines are simultaneously sildenafil and tadalafil using pharmacopoeia methods prevalent in Africa and Southeast in SF erectile dysfunction drugs (LC–UV). Out of 177 samples, 36 Asia (9,13,17,18). In the past, often within 6 min. The method was fast, (20.3%) failed the assay tests. The thin-layer chromatography (TLC) but was unable to detect impurities. use of LC–UV for the analysis of SF was used in these regions for the For the detection and identification antimicrobials is more common in quality evaluation of antimalarials of analogues and impurities, a Africa because of limited access to (19) because it is a cheap and easy technique for compound structure sophisticated equipment. technique, but, more recently, LC elucidation is required, for example, SF antimicrobials are also methods have also been applied high-resolution (HR)MS and NMR. gaining in popularity in developed (9,13,17). Generally, LC–MS is used to Although most of the studies on countries. In a very recent work confirm claimed APIs and determine the evaluation of erectile dysfunction of the authors, chromatographic potential impurities, while LC–UV/DAD drugs use LC systems because of the methods were developed for is used for subsequent quantification. high boiling point and high molecular the screening and subsequent For example, Kaur et al. (17) used weight of these drugs, Jeong et al. quantification of suspected illegal LC–DAD and MS to find out that (24) applied high-temperature GC– antimicrobial drugs encountered falsified artemisinin-containing MS (HTGC–MS) for the simultaneous on the Belgian market (15). Taking antimalarial samples contained no determination of sildenafil, tadalafil, advantage of the additional selectivity labelled API, but other potentially and vardenafil in erectile dysfunction of MS, a short ultrahigh-pressure deleterious compounds, such as drugs. Full scan mode and an internal liquid chromatographic (UHPLC)– bis (2-ethylhexyl) adipate, dioctyl standard were used to obtain an

90 LC•GC Europe February 2019 PHARMACEUTICAL PERSPECTIVES

efficient qualitative and quantitative The use of UHPLC–MS/MS for with chemometrics. This method method for routine analysis. HTGC–MS target screening and quantification of was able to discriminate slimming generated improved peak shapes and 26 different AAS in food supplements capsules to which sibutramine and provided faster analysis compared to was reported by Paíga et al. (27) and phenolphthalein had been illegally conventional GC. Moreover, HTGC–MS Cho et al. (28). Internal standards added. For the adulterants found could avoid matrix effects. Concerning were employed by Paíga et al. to in dietary supplements for sexual the in-field evaluation of SF erectile quantify the adulterants, whilst Cho performance enhancement, GC–MS dysfunction drugs, a portable GC–MS et al. used the most sensitive product (31) and UHPLC–QTOF-MS (32,33) system was introduced to analyze ion for quantification. In addition, were described for the screening. residual solvents (25). Neves et al. (29) presented a Moreover, Kim et al. (32) extended Chemical Adulteration of Dietary GC–MS method for the identification the UHPLC–QTOF-MS full-scan Supplements: Since dietary and quantification (using internal screening method for erectile supplements are sold directly to standards) of 13 AAS in medicines dysfunction drugs to the detection consumers and regulations are not and dietary supplements. Seventeen of multiclass illegal adulterants, as strict as for pharmaceuticals, dietary supplements from the including synthetic steroids, anabolic they are vulnerable to adulteration. black market were analyzed, of steroids, and antihistamine drugs. Many types of adulterants have which five contained unlabelled Six out of 70 samples analyzed been reported in food supplements. AAS at a pharmacological relevant were found to be positive for illegal Commonly, the majority of adulterants quantity. The use of an internal adulterants. Taken together, these found in dietary supplements standard for quantification can reports clearly demonstrate the utility can be classified as sports solve problems caused by matrix and importance of GC or LC coupled performance enhancers (for example, effects. All of the above-mentioned to MS for the detection of adulterants anabolic-androgenic steroids methods require sample cleanup in dietary supplements. [AAS]), weight loss enhancers procedures; however, Xia et al. It has been reported that various (for example, sibutramine with or (30) proposed a rapid screening other classes of adulterants were without antidepressant), and sexual method of SF slimming capsules detected in dietary supplements, performance enhancers (for example, without sample pretreatment using that is, diuretics, antidiabetics, phosphodiesterase type 5 inhibitor an approach based on the electronic cognition enhancers (amphetamine [PDE-5i]) (26). nose and flash GC combined or analogues and vinpocetine), and

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synthetic hair-growth drugs (34–37). biopharmaceuticals, extended with a with trypsin), for the characterization Ki et al. (34) utilized LC–QTOF-MS to subsequent quantification method via of large biotherapeutics (46). screen 35 diuretics and antidiabetics UHPLC–DAD. Although quantification using an in-house library. Avula via LC–MS2 is more sensitive, in this Conclusion et al. (35) used UHPLC–DAD to case LC–UV was sufficient to quantify This review gives a general overview identify and quantify vinpocetine and the amount of peptides in illegal of the literature published from 2015 picamilon in dietary supplements products. The method was able to on the application of LC and GC sold in the United States and never selectively detect 25 peptides and methods for the analysis of popular approved by the US Food and Drug was applied to analyze 65 suspected SF medicines and health products. Administration. illegal samples confiscated by the In general it can be said that LC and However, over the past five years, Belgian Federal Agency for Medicines GC are able to tackle the majority of it has been reported that some new and Health Products (FAMHP). The the challenges encountered during PDE-5i analogues were detected analytical results disclosed that almost the analysis of a broad range of in dietary supplements (38). The all collected illegal peptides were SF medicines and illegal dietary identification of these new analogues underdosed and the most frequently supplements. These chromatographic is more challenging, since their detected illicit peptides were doping methods display several advantages structures are unknown. This often agents. However, by applying this of high sensitivity and accuracy in requires an approach using multiple reversed-phase LC method, some terms of API and impurity (either API- techniques. Generally, most studies more polar peptides (epitalon and or process-related) or contamination employed HRMS or NMR techniques protirelin), which eluted at the very analysis. APIs, potential impurities, for structure elucidation. Kern et al. beginning of the gradient, could not and excipients are first separated (39) and Yun et al. (40) identified a be quantified. In addition, GHRP-2 through LC or GC and then detected new tadalafil analogue and a new coeluted with leuprorelin. To solve and quantified using mainly UV sildenafil analogue, respectively, in these problems, Janvier et al. (44) and different modes of MSn for dietary supplements. They both used envisaged an alternative separation further identification and structure LC–DAD to isolate the unknown target strategy, based on hydrophilic elucidation. It should be noted that and evaluated the compound via the interaction chromatography (HILIC) techniques like LC and GC require UV spectrum. However, while Yun et (Figure 2), without any need for sample reliable reference substances to al. confirmed the structure with the preparation. The aforementioned perform proper quantification. synthesized reference using LC– reversed-phase LC method combined The Falsified Medicines Directive QTOF-MS, NMR, and FT-IR, Kern et with the HILIC method allowed the 2011/62/EU will be implemented in al. used LC–HRMS and GC–FT-IR–MS routine analysis of illegal peptide February 2019, and pharmaceutical for structure elucidation. For another drugs frequently encountered by manufacturers and associated supply case, in which a certified reference controlling agencies. In another case chain distributors will have to comply. was unavailable, Vanhee et al. (26) study, the polypeptide epidermal Tamper-evident packaging techniques disclosed the presence of adrafinil (a growth factor (EGF) was found in will be developed to curtail the synthetic cognition enhancer) in food an illegal injection sample that was manufacturing, distribution, and supply supplements utilizing GC–MS, labelled to contain insulin-like growth of SF medicines and illegal health LC–MS2, and NMR analysis. factor-1 (IGF-1) (45). EGF can bind products. In this context, the drug Peptide and Biotechnology Drugs: to the EGF-receptor and possibly tablet film can be coded. LC and GC In recent years, a new type of promote tumour cell motility, posing could be used in the coding analysis unregistered or unlicensed drug has potential danger to consumers. The of tablet coating to protect genuine emerged in the form of peptides, presence of EGF was identified by drug products from falsification or which are gaining popularity as screening with low-resolution adulteration. For example, Ilko et al. demonstrated by Venhuis et al. (41). LC–MS2 techniques and subsequent (47) proposed a novel strategy of In order to identify the substances confirmation using LC–HRMS (45). adding monodisperse polyethylene present in that type of sample, either It is evident that HRMS devices play glycol (PEG) into tablet coating NMR or LC–MS are utilized. a pivotal role in the identifications of solutions to create a code on individual Gaudiano et al. (42) applied unknown peptide drugs. tablet film. These writing codes could both high-resolution LC–MS and In addition to peptide drugs, be easily recognized and confirmed NMR to identify GHRP-2, an biotech drugs, in the form of through LC–MSn analysis. With the unauthorized synthetic peptide used proteins, including monoclonal advances in LC and GC, analytical predominantly for illegal doping antibodies (mAbs), are also methods are continuously being purposes. In addition to doping gaining in popularity. Although until developed and optimized for in-depth peptides, skin-tanning peptides, recently only doping proteins (for scrutiny of SF medical products and potential anti-ageing peptides (for example, human growth hormone, illegal dietary supplements. example, epitalon), and potential erythropoietin, human chorionic cognition-enhancing peptides gonadotropin) were encountered, SF References have been encountered (43,44,26). mAbs have been found across the (1) PSI, Pharmaceutical Security Institute, n (2018). http://www.psi-inc.org/ Vanhee et al. (43) developed and globe. LC–MS is once again the geographicDistributions.cfm (accessed 2 validated a reversed-phase LC–MS preferred methodology, upon sample 30 October 2018). method to identify illegal peptide pretreatment (for example, digestion (2) World finance, Trade in illegal medicine

92 LC•GC Europe February 2019 PHARMACEUTICAL PERSPECTIVES

hits pharmaceutical sector, (2012). Feineis, L. Hoellein, U. Holzgrabe, and Alimonti, L. Rufini, and L. Valvo, Ann Ist https://www.worldfinance.com/special- G. Bringmann, Drug Test. Anal. 10, Super Sanità 52, 128–132 (2016). reports/trade-in-illegal-medicine-hits- 1599–1606 (2018). (43) C. Vanhee, S. Janvier, B. Desmedt, G. pharmaceutical-sector (accessed 30 (19) M. Lalani, F.E. Kitutu, S.E. Clarke, Moens, E. Deconinck, J.O. De Beer, October 2018). and H. Kaur, Malar. J. 16, 1–14 and P. Courselle, Talanta 142, 1–10 (3) World Health Organization, WHO (2017). (2015). global surveillance and monitoring (20) S. Lee, D. Ji, M. Park, and K.H. (44) S. Janvier, E. De Sutter, E. Wynendaele, system for substandard and falsified Chung, Forensic Sci. Int. 257, 182–188 B. De Spiegeleer, C. Vanhee, and medical products, Geneva (2017). (2015). E. Deconinck, Talanta 174, 562–571 http://www.who.int/medicines/ (21) P.H.J. Keizers, A. Wiegard, and B.J. 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Akimoto, Delerue-Matos, Eur. J. Pharm. Sci. 99, Celine Vanhee has a Ph.D. in and K. Kimura, Trop. Med. Int. Heal. 23, 219–227 (2017). biochemistry and works for the 1294–1303 (2018). (28) S.H. Cho, H.J. Park, J.H. Lee, J.A. Do, (8) World Health Organization, Substand S. Heo, J.H. Jo, and S. Cho, J. Pharm. Belgian official medicines control and falsified medical products, (2018). Biomed. Anal. 111, 138–146 (2015). laboratory, part of Sciensano, http://www.who.int/mediacentre/ (29) D.B.D Neves and E.D. Caldas, Forensic Brussels. She is, together with Eric factsheets/fs275/en/(accessed 31 Sci. Int. 275, 272–281 (2017). October 2018) (30) Z. Xia, W. Cai, and X. Shao, J. Sep. Sci. Deconinck, responsible for the (9) H. Kaur, E.L. Allan, I. Mamadu, Z. Hall, 38, 621–625 (2015). (bio)chemical analysis of falsified M.D. Green, I. Swamidos, P. Dwivedi, (31) S.U. Mokhtar, S.T. Chin, C.L. Kee, M.Y. medicines, including peptide drugs M.J. Culzoni, F.M. Fernandez, G. Low, O.H. Drummer, and P.J. Marriott, Garcia, D. Hergott, and F. Monti, BMJ J. Pharm. Biomed. Anal. 121, 188–196 and biotherapeutics. Glob Health 2, e000409 (2017). (2016). Erwin Adams is a professor at the (10) I. Fadeyi, M. Lalani, N. Mailk, A. Van (32) E.H. Kim, H.S. Seo, N.Y. Ki, N.H. Park, KU Leuven, Belgium. He performs Wyk, and H. Kaur, Am. J. Trop. Med. W. Lee, J.A. Do, S. Park, S.Y. Baek, research on pharmaceutical analysis Hyg. 92, 87–94 (2015). B. Moon, H. Bin Oh, and J. Hong, J. (11) N. Alotaibi, S. Overton, S. Curtis, J.W. Chromatogr. A 1491, 43–56 (2017). with a focus on analytical method Nickerson, A. Attaran, S. Gilmer, and (33) X.B. Wang, J. Zheng, J.J. Li, H.Y. Yu, development and validation for P.M. M ayer, Am. J. Trop. Med. Hyg. 99, Q.Y. Li, L.H. Xu, M.J. Liu, R.Q. Xian, Y.E. quality control of medicines. 477–481 (2018). Sun, and B.J. Liu, J. Food Drug Anal. (12) G. Frimpong, K. Ofori-Kwakye, N. 26, 1138–1153 (2018). Eric Deconinck has a Ph.D. in Kuntworbe, K.O. Buabeng, Y.A. Osei, (34) N.Y. Ki, J. Hur, B.H. Kim, K.H. Kim, B.J. pharmaceutical sciences and is M. El Boakye-Gyasi, and O. Adi-Dako, Moon, H. Bin Oh, and J. Hong, J. Food currently head of the scientific J. Trop. Med. 2018, 1–14 (2018). Drug Anal., in press (2018). service Medicines and Health (13) S. Yeuchaixiong, H. Kaur, P. Tabernero, (35) B. Avula, A.G. Chittiboyina, S. Sagi, S. Sengaloundeth, I. Swamidoss, Y.H. Wang, M. Wang, I.A. Khan, and Products of Sciensano. This M.D. Green, F.M. Fernández, M. P. A . C ohen, Drug Test. Anal. 8, service acts as the Belgian Official Khanthavong, P. Dwivedi, M. Mayxay, 334–343 (2016). Medicine Control Laboratory and C. Vilayhong, E.L. Allan, P.N. Newton, (36) J.H. Lee, G. Kang, H.N. Park, J. Kim, M.J. Culzoni, C. Phonlavong, and C. N.S. Kim, S. Park, S.K. Park, S.Y. Baek, National Reference Laboratory for Sichanh, Am. J. Trop. Med. Hyg. 92, and H. Kang, Food Addit. Contam.-Part the quality control of medicines. His 95–104 (2015). A 35, 191–199 (2018). research interests are mainly in the (14) M. Islam, N. Yoshida, K. Kimura, C. (37) P.A. Cohen, C. Bloszies, C. Yee, and R. Uwatoko, M. Rahman, S. Kumada, J. Gerona, Drug Test. Anal. 8, 328–333 analysis and risk evaluation of illegal Endo, K. Ito, T. Tanimoto, T. Zin, and H. (2016). medicines and health products. Tsuboi, Pharmacy 6, 96 (2018). (38) C.L. Kee, X. Ge, V. Gilard, M. Deirdre Cabooter is the editor of (15) Y. Tie, C. Vanhee, E. Deconinck, and Malet-Martino, and M.Y. Low, J. Pharm. “Pharmaceutical Perspectives”. E. Adams, Talanta 194, 876–887 Biomed. Anal. 147, 250–277 (2018). (2019). (39) S.E. Kern, L.M. Lorenz, A. Lanzarotta, She is an associate professor at (16) World Health Organization, 10 facts E.A. Nickum, and J.J. Litzau, J. Pharm. the Department of Pharmaceutical on malaria, (2016). http://www.who.int/ Biomed. Anal. 128, 360–366 (2016). and Pharmacological Sciences of features/factfiles/malaria/en/ (accessed (40) J. Yun, K.J. Shin, J. Choi, K. Kwon, 22 November 2018). and C.H. Jo, J. Chromatogr. B 1072, KU Leuven, in Leuven, Belgium. (17) H. Kaur, E.L. Allan, I. Mamadu, Z. Hall, 273–281 (2018). She is also a member of LCGC O. Ibe, M. El Sherbiny, A. Van Wy, S. (41) B.J. Venhuis, P.H.J. Keizers, R. Europe’s editorial advisory board. Yeung, I. Swamidoss, M.D. Green, P. Klausmann, and I. Hegger, Drug Test. Direct correspondence about this Dwivedi, M.J. Culzoni, S. Clarke, D. Anal. 8, 398–401 (2016). Schellenberg, F.M. Fernández, and O. (42) M.C. Gaudiano, L. Manna, M. column to the editor-in-chief, Alasdair Onwujekwe, PLoS One 10, 1–13 (2015). Bartolomei, A.L. Rodomonte, P. Matheson, at alasdair.matheson@ (18) J.P. Mufusama, K. Ndjoko Ioset, D. Bertocchi, E. Antoniella, L. Romanini, S. ubm.com www.chromatographyonline.com 93 PHARMACEUTICAL ANALYSIS FOCUS Going Green in Pharmaceutical Analysis LCGC Europe spoke to Yong Liu and Adam Socia from MSD about the cost-saving benefi ts of implementing green chromatography in the pharmaceutical sector, the importance of analytical method volume intensity (AMVI), and effective practices to reduce solvent consumption and replace harmful solvents, including supercritical fl uid chromatography (SFC), fast chromatography, and “cocktail chromatography”.

Interview by Alasdair Matheson, Editor-in-Chief, LCGC Europe

Q. What is the definition of green liquid chromatography (UHPLC) does not depend on the use of chromatography? or conventional HPLC systems hazardous organic solvents (such Yong Liu: Green chromatography with minor modifications, such as acetonitrile). Supercritical is an important part of green as smaller diameter columns for fluid chromatography (SFC) uses analytical chemistry and originates greener separation or new column pressurized carbon dioxide in the from the 12 green chemistry technologies, such as fused-core subcritical or supercritical state as principles developed to reduce the particle columns. The second a chromatographic mobile phase environmental impact of chemical approach is to use microflow and and has been adopted for both synthesis and analysis (1). Green capillary HPLC. The third strategy preparative separation and analytical chromatography incorporates is solvent replacement, which purposes. practices to reduce the amount of is achieved by using ethanol to solvent consumption and waste replace acetonitrile, super-heated Q. You recently published generation mainly through separation an article on a greener and sample preparation. chromatography method for Green chromatography is dissolution testing on solid Q. What green approaches are an important part of green pharmaceutical formulations. being used to reduce the amount What is novel about your approach of solvents being used in liquid analytical chemistry and and what benefits does it offer the chromatography (LC)? originates from the 12 analyst? YL: From our perspective, the first green chemistry principles Adam Socia: Green chromatography step is to provide a metric tool for developed to reduce the has been practiced to support small measurement of the “greenness” of molecule drug substance synthesis a liquid chromatographic method. environmental impact of and release in the pharmaceutical Hartman, Helmy, and coauthors chemical synthesis and industry for more than 10 years. developed analytical method volume analysis. However, the application of green intensity (AMVI), a simple metric chromatography in formulation that offers quick measurement development for drug products is of total solvent consumption water chromatography, and carbon not so common. Our work represents for a high performance liquid dioxide-based chromatography. one of the first applications of green chromatography (HPLC) method (2). Recently, a work by Welch, Regalado, chromatography in drug product AMVI enhances the awareness of and coauthors demonstrated that development (5). We provided green chromatography and, more HPLC and LC–mass spectrometry an improved chromatographic importantly, makes this concept an (MS) experiments can be performed protocol combining the utilization integral part of method development using distilled alcohol spirits, of smaller internal diameter (i.d.) for bench analytical scientists. such as cachaça, rum, vodka, columns, superficially porous From a technical point of view, and aguardiente, as well as column technology, injection cycle the review article by Welch, Wu, other common household items, time for gradient re-equilibration, and coauthors offered an excellent including vinegar and ammonia, as system dwell volume understanding, summary of major green approaches mobile phases and additives (4). and basic separation concepts in chromatography (3). The first The former has been nicknamed for optimization for a greener, is solvent reduction through “cocktail chromatography”. faster, and robust way to conduct fast chromatography by using This methodology provides a dissolution testing. This methodology conventional HPLC with elevated low cost and environmentally provides 70−80% reduction in

pressures, ultrahigh-performance sustainable alternative in LC that solvent consumption and waste Tumik/Shutterstock.com Abel Credit: Photo

94 LC•GC Europe February 2019 PHARMACEUTICAL ANALYSIS FOCUS generation, as well as run times with equivalent accuracy, precision, and robustness based on current LC instruments commonly used in our industry.

“Cocktail chromatography” provides a low cost and environmentally sustainable alternative in LC that does not depend on the use of hazardous F-DGSi- SMART SOLUTIONS WITH ‘‘GEN SECURE’’ organic solvents.

Q. Have you applied this approach to “real life” SECURE MODULAR ADVANCED samples? AS: Yes, we have implemented this approach to several phase I programmes. We strongly believe this approach should be adapted at the earliest point possible in drug product development to achieve the greatest REMOTE CONTROL TRUSTED savings.

Q. Are there any other practical examples of green COSMOS GAS chromatography you have developed in your GENERATORS FOR GC laboratory that illustrate the benefits in terms of sustainability and cost‑effectiveness? SECURE YOUR GC WORKFLOW WITH YL: Yes. From a sample preparation perspective, we— TECHNOLOGY LIGHT YEARS AHEAD ! with work led by Nowak and Regalado— developed a method that significantly reduced organic solvent use in standard preparation for residual solvent analysis, a test required by regulatory agencies for a drug substance developed in the pharmaceutical industry (6). Typically, H2 the analyst makes fresh residual solvent standard in volumetric flasks and only uses a small portion of solution (an HPLC vial, for example) for every new sample submission. We showed that a multisolvent standard mixture can be stored in the crimped HPLC vials at -10 °C for at least 31 months with excellent recovery for all 25 solvents (over 97% with overall relative standard deviation [RSD] below 5%). The multisolvent standard in crimped N2 HPLC vials are made in a large quantity and are provided to analysts to support almost all the projects in our department. This practice greatly reduces the repetitive standard preparation and organic solvent consumption and waste generation to support residual solvent analysis for in-process samples. AIR Q. What green chromatography techniques are being adopted in gas chromatography (GC)? YL: At MSD, small bore and short GC columns were adopted to reduce the analysis time for fast COMP chromatography and bring the benefit of lower instrument energy and gas consumption (6). The improved instrument output also offers the possibility to use fewer instruments. The other practice adopted for GC is in standard sample preparation, which we mentioned earlier. Another green chromatography effort in GC is to replace the carrier gas helium with renewable clean gas, such as hydrogen. WWW.F-DGS.COM Bernardoni et al. recently published a GC–flame ionization detection (FID) method using hydrogen as carrier gas for the analysis of 30 common solvents in pharmaceutical VISIT OUR BOOTH : · PITTCON PHILADELPHIA N° 2811 · ARABLAB DUBAI N° 658 synthesis (7). www.chromatographyonline.com 95 PHARMACEUTICAL ANALYSIS FOCUS

Q. SFC was widely touted as always be a social concern for an (10) W. Schafer, T. Chandrasekaran, Z. Pirzada, C. Zhang, X. Gong, M. a greener technique. Is this analytical scientist. Science and Biba, E.L. Regalado, and C.J. Welch, approach being used more in the technology advances always offer Chirality 25, 799–804 (2013). pharmaceutical sector? new opportunities for greener (11) A.G. Perrenoud, C. Hamman, M. Goel, J.L. Veuthey, D. Guillarme, and S. YL: SFC uses pressurized carbon chromatography, such as the invention Fekete, J. Chromatogr. A 1314, dioxide to replace hexane or of UHPLC, analytical‑scale SFC, and 288–297 (2013). heptane, which are commonly microflow and capillary HPLC to name (12) M. Hicks, E.L. Regalado, F. Tang, used, nonpolar eluents in normal just a few examples. Chromatography X. Gong, and C.J. Welch, J. Pharm. Biomed. Anal. 117, 316−324 (2016). phase chromatography. SFC was is widely used in different areas of first widely adopted for preparative drug discovery and development in Adam Socia is an purification of chiral molecules the pharmaceutical industry. A lot of Associate Principle (8). Recently, analytical SFC has great work has already been done Scientist in the become more popular for chiral in supporting chemical synthesis of Analytical Sciences separation to support synthetic drug substances, particularly small group at Merck process development, monitor molecules. In the areas of formulation & Co., Inc., West chiral purification, and even as an development, PK determination, Point, Pennsylvania, analytical release assay to support vaccines, and biologics, LC is heavily USA. He completed regulatory filing (9–11). At MSD, SFC employed. Green chromatography, his Ph.D. at Drexel University and became the “go-to” analytical tool for however, is not so common. The his MS at Sacred Heart University, chiral separation for bench analysts authors believe that there are good focusing on the development of novel several years ago in the area of opportunities in these areas to separations for amino acids and other process chemistry development (12). implement green chromatography. charged analytes. Adam’s current role is in providing analytical support for both solid‑dosage and sterile‑liquid Another green Being green and early‑stage pharmaceuticals. Adam has written about his research in several chromatography effort in GC protecting our peer‑reviewed journals and presented is to replace the carrier gas environment by doing both oral and poster presentations at helium with renewable clean good science should numerous conferences. gas, such as hydrogen. always be a social concern Yong Liu is a Principal Scientist in for an analytical scientist. the Department of Q. Are there any other Analytical Sciences examples in your organization at Merck & Co., that illustrate the benefits of References Inc., West Point, green chromatography in the (1) J. Namiesnik and M. Tobiszewski, Pennsylvania, LCGC Europe 27(8), 405–408 (2014). pharmaceutical sector? (2) R. Hartman, R. Helmy, M. Al-Sayah, USA. He has AS: Purity and content analysis, and C.J. Welch, Green Chem. 13, been in Merck for 17 years in the primarily performed by HPLC, is 934–939 (2011). field of analytical chemistry and a required test for drug products (3) C.J. Welch, N. Wu, M. Biba, R. quality control for small molecule Hartman, T. Brkovic, X. Gong, R. in development and release, Helmy, W. Schafer, J. Cuff, and Z. pharmaceuticals, siRNAs, lipids, regardless of formulation type. Green Pirzada, TrAC, Trends Anal. Chem. peptides, and proteins both in chromatography is still relatively new 29(7), 667−680 (2010). drug substance and drug product. (4) C.J. Welch, T. Nowak, L.A. Joyce, and and we are working on implementing E.L. Regalado, ACS Sustainable Chem. Currently, he is leading a group in existing best practices, such as AMVI Eng. 3(5), 1000−1009 (2015). providing analytical support for drug and fast chromatography, into this area. (5) A. Socia, Y. Liu, X. Gong, O. White, product development covering phase A. Abend, and P. Wuelfing, ACS I through phase III and drug product Sustainable Chem. Eng. 3(5), 1000−1009 (2015). degradation impurity structure (6) T. Nowak, G.C. Graffius, Y. Liu, N. elucidation by mass spectrometry. Our work represents one Wu, X. Bu, X. Gong, C.J. Welch, and He has 40 publications with research E.L. Regalado, Green Chem. 18(13), of the first applications of 3732−3739 (2016). interests in separation science, green chromatography in (7) F. Bernardoni, H.M. Halsey, R. structural elucidation of degradation Hartman, T. Nowak, and E.L. Regalado, impurities of drug molecules, and drug product development. J. Pharm. Biomed. Anal. 165, 366–373 (2019). studying of drug–drug and (8) C.J. Welch, W.R. Leonard Jr., J.O. Da drug–excipient interactions via Q. Green chromatography seems Silva, M. Biba, J. Albaneze-Walker, D.W. MS. He obtained bachelor’s degree to go in and out of fashion. Why is Henderson, B. Laing, and D.J. Mathre, from Wuhan University, a master’s LCGC Europe 18(5), 264–272 (2005). this and is it back in fashion? (9) R. Helmy, M. Biba, J. Zang, B. Mao, K. degree from University of Central YL and AS: Being green and Fogelman, V. Vlachos, P. Hosek, and Florida, and a Ph.D. degree from protecting our environment C.J. Welch, Chirality 19(10), 787–792 Rutgers University, both in organic (2007). by doing good science should chemistry.

96 LC•GC Europe February 2019 PRODUCTS Field-fl ow fractionation LC accessories The Eclipse DualTec system Restek has expanded the company’s offers both hollow-fi bre flow-FFF line of liquid chromatography (HF5) and asymmetric fl ow-FFF accessories for chromatographers. (AF4) techniques. Both High-quality couplers, fi ttings, systems may be integrated into unions, tees, and crosses; PEEK and one instrument and coupled stainless steel tubing; mobile phase seamlessly to an advanced maintenance and safety products, light scattering detector for including bottle tops, valves, fi lters, and spargers are now determination of molar mass available. and nanoparticle size, Wyatt’s Dawn Heleos II. www.restek.com/LCacc https://www.wyatt.com/separation Restek Corporation, Bellefonte, Pennsylvania, USA. Wyatt Technology, Santa Barbara, California, USA.

GC×GC system UHPLC system The Pegasus BT 4D reportedly offers enhanced sensitivity by Shimadzu’s new “Nexera Bio” coupling the benchtop Pegasus solution, a biocompatible ultrahigh BT with a high performance performance liquid chromatography GC×GC. The system is (UHPLC) system, offers the same available with fl ow modulation, superior reliability, robustness, an option that replaces thermal and expandability as other Nexera modulation. According to the series UHPLC systems. The system company, both confi gurations is particularly well suited for analyzing protein-based give the Pegasus system the ability to interrogate challenging pharmaceuticals, antibody drugs, and other substances samples where the best sensitivity is needed. Software and developed or manufactured using biotechnologies. hardware features simplify quantitation, while also making The system is compatible with mobile phase solvents GC×GC easy to use and understand. containing high concentrations of salts or acids. www.leco.com www.shimadzu.eu Leco Corporation, Saint Joseph, Michigan, USA. Shimadzu Europa GmbH, Duisburg, Germany.

Mass spectrometer Antibody titer analysis Knauer has launched a single In the many stages quadrupole mass spectrometer, of mAb development, the Knauer 4000 MiD, which harvest cell culture covers a mass range of samples must be 50–800 m/z. As a result of the screened for their IgG integrated vacuum system, titers. TSKgel Protein the 4000 MiD has a very small A-5PW is specifi cally designed for the fast analysis of footprint and can be operated mAb concentration in cell culture supernatant. The wide in laboratories with limited dynamic range allows equally accurate determination of space. Together with the MiDas comparatively low mAb titers in early development and automated sampling unit, the high mAb titers of optimized cell lines in production. 4000 MiD enables high-throughput preparative HPLC. The www.separations.eu.tosohbioscience.com/solutions/ Azura prep LC for mass-directed fractionation is controlled with hplc-products/protein-a ClarityChrom, a chromatography data system for workstations. Tosoh Bioscience, Griesheim, Germany. www.knauer.net Knauer Wissenschaftliche Geräte GmbH, Berlin, Germany.

www.chromatographyonline.com 97 PRODUCTS

Method modelling software FID gas station

Molnár-Institute’s DryLab Flow rate Start [%B] End [%B]

The VICI FID gas station software has a 35-year history T [°C] Start [%B]

T [°C] combines the reliability of in scientifi c method modelling. T [°C] Flow raterat [mL //min] m in] e [min] tG [min] End [%B] the VICI DBS hydrogen Using a DoE of twelve input tG tG [min] and zero-air generators runs, the software integrates the into one compact and theory of solvophobic interactions and linear solvent strength convenient package. Available in high and ultrahigh (LSS) to predict the movements of peaks, selectivity changes, purity for all GC detector and carrier gas applications. and retention times of any multidimensional design space. The generator is available in two styles: fl at for placement The software’s automation module creates method sets in under a GC, or the Tower. Available in H2 fl ow ranges up the most economic and ecologic order, executes runs, and to 1 L/min and 10.5 bar. acquires results from the CDS. Mass and other integrated data www.vicidbs.com are retrieved and ambiguity in peak tracking is reduced to a VICI AG International, Schenkon, Switzerland. minimum. www.molnar-institute.com Molnár-Institute, Berlin, Germany.

HILIC columns (U)HPLC columns Hilicon offers a broad range of hydrophilic interaction liquid chromatography (HILIC) YMC-Triart Bio C4 is a new products to separate polar compounds. wide-pore phase for (U)HPLC. As Three column chemistries in UHPLC and a result of its 300-Å pore size, this HPLC, iHILIC-Fusion, iHILIC-Fusion(+), stationary phase is designed for and iHILIC-Fusion(P), provide customized peptide and protein separations. and complementary selectivity, excellent Excellent reproducibility, high pH durability, and ultralow column bleeding, according to the (1–10), and high temperature (up to company. The columns are suitable for the LC–MS analysis 90 °C) stability are provided. of polar compounds in “omics” research, food and beverage www.ymc.de analysis, pharmaceutical discovery, and clinical diagnostics. YMC Europe GmbH, Dinslaken, Germany. www.hilicon.com Hilicon AB, Umeå, Sweden.

Thermal desorption unit Microchip column The Gerstel Thermal Desorption Unit μPAC is PharmaFluidics’ (TDU 2) performs multiple sample chip-based chromatography introduction techniques including column for nano-liquid headspace, SPME, SBSE, thermal chromatography. Perfect desorption, dynamic headspace, order in the separation bed is pyrolysis, and liquid introduction. For achieved by etching a regular extreme sensitivity, the multi-desorption pattern of pillars into a silicon mode can be used. The MPS robotic wafer using micromachining adds automation for up to 240 samples, technology, according to the company. The column allows including barcode reading and high-resolution separation of tiny, complex biological tube-spiking options. Multiple techniques samples, with an unprecedented robustness. μPAC is can be performed in one automated sequence. suitable for lipidomic, metabolomic, and peptide profi ling. www.gerstel.com www.pharmafl uidics.com Gerstel GmbH & Co. KG, Mülheim an der Ruhr, Germany. PharmaFluidics, Ghent, Belgium.

98 LC•GC Europe February 2019 PRODUCTS

Sample prep HPLC system LCTech has introduced a new The Agilent 1260 Infi nity II Prime automated system designed to clean LC system provides the highest up samples that need to remain convenience standard in the 1260 melted in PCB and dioxin analysis. Infi nity II LC portfolio, as well as Three specifi cally designed heating an extended pressure range (up to zones keep the sample liquid from 800 bar), excellent quaternary mixing, sample vial to the fi rst column. The and specifi cally designed columns, DEXTech Heat, which is based according to the company. The automated instrument on the established DEXTech Pure features reportedly increase analytical laboratory efficiency system, processes difficult samples and the Intelligent System Emulation Technology (ISET) such as stearin or PFADs. Excellent ensures seamless method transfers from many Agilent and automated, reliable results, without third-party legacy instruments. The improved convenience clogging are produced, according to level of the system is highlighted with a local user the company. interface—the Agilent Infi nityLab LC Companion. www.LCTech.de www.agilent.com LCTech GmbH, Obertaufkirchen, Germany. Agilent Technologies, Inc., California, USA.

Isocratic pump Triple detection The Verity 3011 isocratic pump is a liquid delivery solution for Postnova has introduced the T r i p l e D e t e c t i o n Online Coupling to FFF and SEC chemical reaction monitoring in Triple Detection for thermal

petroleum applications and for fi eld-fl ow fractionation PN3621 FFF PN3310 + MALS Viscometer + Detector SEC Detector gel permeation chromatography (FFF) and GPC/SEC. Triple Particle Sizee Intrinsic Viscositycosity Rg / Molar Mass Branching (GPC). According to the company, Detection is the combination PN3150 RI Detector the pump is highly accurate and of multi-angle light scattering Concentrationon delivers virtually pulse-free, stable (MALS), viscosity detection, solvent fl ow for a variety of liquids, refractive index detection, and UV detection. In a single including high-viscosity solvents, and allows for fl ow rates from separation experiment Triple Detection provides molar 0.01 0 mL/min to 10 mL/min and pressures of up to 600 bar. mass distribution, molecular size distribution, and www.gilson.com/verity3011 molecular structure (branching, composition) of polymers, Gilson, Middleton, Wisconsin, USA. biopolymers, polysaccharides, proteins, and antibodies. www.postnova.com Postnova Analytics GmbH, Landberg, Germany.

GPC system Sample automation GPC-QC delivers the Markes’ new Centri multitechnique complete molar mass platform is an advance in sample distribution, short chain automation and concentration for branching, and intrinsic GC–MS, according to the company, viscosity information for one and offers four sampling modes: HiSorb sample in 30 min, including high-capacity sorptive extraction, dissolution, according to the headspace, SPME, and thermal company. GPC-QC reportedly desorption. The company reports allows more accurate control analyte focusing allows increased of production thanks to a precise and complete characterization sensitivity in all modes, state-of the-art robotics increase of the resins, saving costs because off-grade material production sample throughput, and sample re-collection allows is reduced. repeat analysis without having to repeat lengthy sample http://www.polymerchar.com/gpc-qc extraction procedures. Polymer Char, Valencia, Spain. http://chem.markes.com/Centri Markes International Ltd., Llantrisant, UK.

www.chromatographyonline.com 99 PRODUCTS

Nitrogen generator Seals Genius XE Nitrogen is a cutting-edge The Bal Seal provides excellent nitrogen generator combining advanced sealing performance in a broad range technology with refi ned and robust of pressures, temperatures, fl ow rates, engineering, according to the company. and media, helping pump designers Two model options are available: XE 35 meet PM requirements of more than with up to 35 L/min and XE 70 with up 1 M cycles/60 L. The seal allows for to 70 L/min. The generator reportedly precise control of frictional forces, provides a premium standalone resulting in reduced contamination. Designs for UHPLC, nitrogen solution for high performance LC–MS and other with pressures greater than 20,000 psi, are also available. mission-critical laboratory applications where performance and www.balseal.com reliability are paramount. Bal Seal Engineering, Foothill Ranch, California, USA. www.peakscientifi c.com/genius Peak Scientifi c, Scotland, UK.

Electrochemical detector Storage rack The Decade Elite from Antec Scientifi c is designed as an easy-to-use For the safe storage electrochemical detector that can of their ergonomic and integrate with any LC system on the electronic crimping tools, market, according to the company. Macherey-Nagel offers a The system can reportedly handle fast foam rack that is chemically eluting peaks in (U)HPLC and deliver resistant. According to the fast stabilization from dedicated fl ow company, the crimping tools cells. When used with the SenCell, are properly organized and well protected in the rack. With the system is a highly sensitive their standing position the tools are kept handy for next electrochemical detector. use. The storage rack completes the range of vials, caps, www.AntecScientifi c.com and chromatography accessories. Antec Scientifi c, Zoeterwoude, www.mn-net.com Netherlands. Macherey-Nagel GmbH & Co. KG, Düren, Germany.

Mass spectrometer Pesticide reference materials The Thermo Scientifi c ISQ EM single Spex CertiPrep has introduced quadrupole mass spectrometer for a new pesticide mix to address high performance and productivity the European Commission’s standards has a mass range of Regulation 2017/170. The 10–2000 m/z. The system offers the Commission is amending power to detect and quantify small Annexes II, III, and V to and large molecules, and supports Regulation (EC) No 396/2005 analytical needs across an extensive of the European Parliament range of applications, from drug and of the Council as regards to maximum residue levels development to manufacturing support and quality control. for bifenthrin, carbetamide, cinidon-ethyl, fenpropimorph, The system’s heated electrospray ionization (HESI) and dual and trifl usulfuron in or on certain products. HESI/atmospheric pressure chemical ionization (APCI) probes www.spexeurope.com facilitate the measurement of polar and nonpolar analytes, Spex Europe, Dalston Gardens, Stanmore, UK. enabling application fl exibility. It is integrated with HPLC systems and fully controlled by Thermo Scientifi c Chromeleon Chromatography Data System. www.thermofi sher.com/ISQEM Thermo Fisher Scientifi c, California, USA.

100 LC•GC Europe February 2019 EVENT NEWS

The Chromatographic Society and Separation Science 25–28 March 2019 Group of the Royal Society of Chemistry Analytical 35th International Symposium on Microscale Separations and Division: Emerging Separations Technologies (2019) Bioanalysis (MSB2019) Oregon State University, Oregon, USA The third meeting in the “Emerging Separations E-mail: [email protected] Technologies” series will take place on Thursday Website: https://msb2019.org 28 March 2019 at RSC Burlington House, Piccadilly, London. The meeting will provide 6–7 May 2019 an insight into the latest innovations in the field Method Development for the of separation science, including instrument and column technologies, novel Separation of Therapeutic Proteins Photo Credit: Royal Society of Chemistry applications, and utilization in life sciences. The programme is designed to cover (Biopolymers) a range of topics, including multidimensional chromatography, hyphenated Molnár-Institute, Berlin, Germany techniques including mass spectrometry (MS) and ion-mobility spectrometry E-mail: [email protected] (IMS), column technology, supercritical fluid chromatography (SFC), and Website: http://molnar-institute.com/ modelling software and their application in profiling and characterization fileadmin/user_upload/Training/ applications, specifically for complex samples. SeminarRegistrationForm.pdf The day will provide a unique means of sharing real experiences with other professionals and will offer excellent networking opportunities. The programme 12–17 May 2019 is being designed to give attendees a range of interesting topics from highly International Symposium on regarded international speakers and new emerging professionals. Capillary Chromatography (ISCC) × The programme has been separated into four separate sessions spread and the GC GC Symposium across the day. Peter Schoenmakers from the University of Amsterdam Fort Worth, Texas, USA (Netherlands), a previous ChromSoc Martin medal recipient, will begin the E-mail: [email protected] day with a presentation on the current status and novel developments in Website: www.isccgcxgc.com multidimensional chromatography. Peter is working at the cutting-edge of 22–23 May 2019 research in this area and brings an important perspective on academic research 3rd International Conference and and its application to separation challenges posed by complex samples as part Exhibition on Petroleum, Refi ning, of the STAMP (Separation Technology for A Million Peaks) project. There are and Environmental Technologies numerous challenges to overcome and this major project will look at multiple (PEFTEC 2019) aspects to address these, including developments in stationary materials, Rotterdam, Netherlands orthogonal mechanisms, detection systems, and ways to control the flows during E-mail: [email protected] the multiple separation stages. This will be followed by another complementary Website: www.ilmexhibitions.com/peftec/ talk by Noor Abdulhussain (as part of the STAMP project) on the development of 3D-printed devices, which is an interesting and novel approach. 16–20 June 2019 The second and third sessions will discuss the challenges of increasingly HPLC 2019 complex pharmaceuticals. This will include an industrial presentation from Eivor Milano-Bicocca University, Milan, Italy Ornskov from AstraZeneca (Gothenburg, Sweden) on various separation-based E-mail: [email protected] approaches for the analysis of polynucleotides, such as the development of Website: www.hplc2019-milan.org novel methods for the analysis of modified mRNA. Further talks will discuss the latest advances in column technology and column characterization for 18–20 June 2019 increasingly complex molecules and the separation challenges involved. These LABWorld China 2019 sessions will include several vendor-sponsored short talks covering a wide range Shanghai New International Exhibition of topics including advances in instrumentation and new detectors for improved Center (SNIEC), Shanghai, China selectivity and identification capabilities. E-mail: [email protected] Josephine Bunch from NPL (UK) will then highlight the current status and Website: www.pmecchina.com/ latest developments in IMS-based technologies and their increasing application labworld/en in imaging applications in life science fields, including new modalities. A further industrial talk will discuss approaches for the characterization and analysis 7–10 July 2019 of advanced drug delivery systems and formulations, including nanoparticle International Symposium formulation material characterization and methods for the determination of the on Preparative and Process release of the active pharmaceutical ingredient (API) from these drug–polymer Chromatography (PREP 2019) conjugates. Baltimore, USA ChromSoc student bursaries are also available for this event. For further E-mail: [email protected] details, please visit: www.chromsoc.com/academic-support.aspx Website: www.prepsymposium.org/ Further information is also available at: www.chromsoc.com/ ChromsocEvents.aspx Please send any upcoming event For further information on the meeting, submission of abstracts, sponsorship, information to Lewis Botcherby at or payment details, please e-mail the main organizers: adrian.clarke@novartis. [email protected] com or [email protected]

www.chromatographyonline.com 101 THE ESSENTIALS

The Essential Guide to Optimizing Sensitivity in GC–FID Analysis

Tips to improve signal-to-noise ratio when using standard gas chromatography (GC) equipment

It is often possible to achieve better After a predetermined length of time sample dependant. One should chose sensitivity and lower limits of detection (the splitless time), the split vent value the least polar column with the thinnest and quantitation using standard gas is opened, and the remaining inlet film, which results in a satisfactory chromatography (GC) equipment, such contents are vented to the atmosphere. separation, in order to reduce column as a standard split–splitless injection This helps to prevent wide and badly bleed to the minimum amount possible. port and a flame ionization detector tailing solvent peaks, increased Carrier Gas Operating Mode: Ensure (FID). Paying attention to some of the baseline noise, and a rising baseline that the carrier gas operating mode fundamental variables can lead to during the analysis. If the split vent is set to constant flow, as opposed to much improved method performance, is opened too early, there is a risk of constant pressure, to ensure that the and here we consider some important analyte loss, and, if opened too early, carrier flows at the same linear velocity aspects necessary to optimize GC–FID the broad and tailing solvent peak may during the whole temperature program. performance. reduce the sensitivity of early eluting This will avoid the carrier slowing as the Sample Solvent: By choosing a analytes; and noisier baselines will temperature increases, which can lead sample solvent that matches as reduce the signal-to-noise ratio for all to broadening of later eluting peaks, closely as possible the stationary analytes. One must experimentally and suboptimal detector response. phase polarity, the peak shape will be determine the optimum splitless time in FID Optimization: Optimize the fuel optimized with no tailing or broadening, order to achieve the best reproducibility (hydrogen) to oxidizer (air) ratio of the which might otherwise reduce the and sensitivity from the splitless FID to ensure the best response for signal-to-noise ratio. Here we think injection. In order for proper solvent your analytes (typically start with a 10:1 of choosing nonpolar solvents such focusing to occur, the initial oven ratio and adjust the fuel gas in steps as n-hexane with nonpolar stationary temperature must be maintained at of +/- 5 mL/min). Also, ensure that phase such as 1% polydimethyl around 20 °C below the boiling point of the make-up gas flow rate (nitrogen is siloxane (DB1, RTX-1), or methanol with the sample solvent during the injection recommended as the most effective polar stationary phases such as waxes. phase of the analysis. make-up gas by many manufacturers) High Head-Pressure (Pressure Initial Oven Temperature Hold: is optimized, and note that the make-up Pulsed) Injection: The inlet liner is On column sample focusing during gas flow rate can have a pronounced designed to hold a certain volume of splitless injection takes a finite amount effect on analyte sensitivity. Start with gas (typically 500 to 950 mL), and we of time, and, during this time, the oven a 1:1 ratio of make-up gas to fuel gas need to avoid overfilling the liner with temperature must be held constant. (typically hydrogen), and adjust in sample gas, or problems will occur with However, if this initial hold time is +/- 5 mL/min steps to investigate the poor quantitative reproducibility and too long, some degree of analyte optimum range. carryover. By choosing a sample solvent dispersion may occur, reducing the Thermal Gradients: Ballistic (short with a lower expansion coefficient, and peak efficiency, and, hence, the and fast) thermal gradients will increasing the pressure within the liner signal-to-noise ratio. The initial hold produce the sharpest peaks and the during the injection, in order to constrain time should be carefully evaluated if best signal-to-noise ratios, but ensure the volume of sample gas created, optimum sensitivity is to be achieved. the oven heater motor can follow larger injection volumes can be made. GC Column Choice: Shorter columns the required temperature profile in a Calculators, typically known as backflash (10–15 m) with narrow internal diameters reproducible way. Of course, the use of or vapour volume calculators, can be (i.d.) (0.18–0.25 mm) and thin films ballistic thermal gradients will depend used to determine the maximum sample (< 0.3 mm) will give the best peak upon the separation requirements and volume that can be injected before liner efficiencies, and therefore the optimum the complexity of the sample. overfilling occurs. signal-to-noise ratio. Further, less polar More Online: Optimize Splitless Time: In splitless stationary phase chemistries will show injection mode, the split vent valve is less inherent bleed, again improving the Get the full tutorial at www.CHROMacademy.com/Essentials closed and all of the contents of the signal-to-noise ratio, although the choice (free until 20 March). inlet are directed into the GC column. of stationary phase chemistry will be

102 LC•GC Europe February 2019

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