Application Note 21498 The Hypersil GOLD VANQUISH range of UHPLC/HPLC VANQUISH The Hypersil GOLD and reliablecolumns was developed to give reproducible peak shape. chromatography analysis with excellent VANQUISH Hypersil GOLD Based on highly pure silica, even UHPLC columns provide very symmetrical peaks, when analyzing compounds that give notoriously poor The peak shape on traditional silica-based chemistries. UHPLC medium provides a VANQUISH Hypersil GOLD stationary phase with C18 selectivity and a predictable such as elution order but can provide new capabilities, and greater capacity, increased peak improved peak shape, especially for trace compound analysis. sensitivity, Hypersil ™ UHPLC system for
1.9 µm column and the Vanquish column band dispersion and allow users to preserve the The excellent peak efficiency provided by the column. allows the up to 200 Hz, very fast acquisition rate, even for the narrowest collection of sufficient data points, peaks obtained in high-throughput application. system allow for the best possible chromatographic high pressure By exploiting the 1500 bar performance. the flow rate UHPLC system, Vanquish capability of the UHPLC VANQUISH used with the Hypersil GOLD column can be increased while maintaining peak capacity, resulting in shorter method times and increased assay The system is optimized to reduce extra throughput. demonstration of a multi-analyte method withdemonstration of a multi-analyte method an efficientstructurally similar compounds that require separation to achieve good resolution. GOLDThe complementing technologies of the Hypersil UHPLC Vanquish UHPLC column and the VANQUISH Introduction analytes is moreCreating screening methods for multiple fewer analytes. cost-effective than dedicated methods for of data, Reduced analysis times allow quicker release sample and overall greater reduced costs per assay, provides a goodThe analysis of beta blockers throughput. Joanne Jones, Thermo Fisher Scientific, Runcorn,Joanne Jones, Thermo UK A High-Throughput Method for the Analysis Analysis the for Method A High-Throughput a UHPLC Using Beta Blockers 19 of andColumn Combination System ™ VANQUISH ™
the fast screening analysis of 19 beta blockers. the fast screening GOLD To demonstrate the advantages of using the Thermo Scientific To betaxolol, carvidilol, nebivolol, penbutalol Goal esmolol, celiprolol, oxprenolol, labetalol, bisoprolol, propranolol, alprenolol, alprenolol, propranolol, labetalol, bisoprolol, oxprenolol, esmolol, celiprolol, Hypersil GOLD, VANQUISH, Beta Blockers, Beta Antagonists, UHPLC, Beta Blockers, Hypersil GOLD, VANQUISH, timolol, acebutolol, metoprolol, atenolol, sotalol, carteolol, nadolol, pindolol, Key Words Key 2 Experimental UHPLC Conditions Consumables UHPLC Column Hypersil GOLD VANQUISH, 1.9 µm, • Hypersil GOLD VANQUISH, 1.9 µm UHPLC column, 100 × 2.1 mm 100 × 2.1 mm (P/N 25002-102130-V) Mobile Phase A Ammonium formate, 20 mM, pH 3.0 • LC-MS grade 18 MΩ∙cm water from Thermo Scientific™ Barnstead™ Smart2Pure™ system (P/N 50129845) Mobile Phase B Acetonitrile + 0.1% formic acid • Fisher Scientific™ HPLC grade acetonitrile Flow Rate See Table 1 (P/N 5A/0626/17) Column Temperature 40 °C, still air with eluent pre-heating • Fisher Scientific Analytical grade formic acid Injection Details 2 µL (P/N 10559570) UV Detection 270 nm • Fisher Scientific ammonium formate Acquisition Rate 100 Hz (P/N A/5080/53) Response Time 0.04 s • Thermo Scientific™ Virtuoso™ 9 mm wide opening, 2 mL screw thread vial and cap kit (P/N 60180-VT400) Table 1. LC gradient conditions. Instrumentation Flow Rate (mL/min) 0.8 1.7 Analyses were performed using a Vanquish UHPLC System consisting of: Gradient Time (min) %B Time (min) %B • System Base (P/N VH-S01-A) 0 10 0 10 1.50 10 0.71 10 • Binary Pump H (P/N VH-P10-A) 4.00 60 1.88 60 • Split Sampler HT (P/N VH-A10-A) 4.01 10 1.89 10 • Column Compartment H (P/N VH-C10-A) 6.00 10 2.82 10 • Active Pre-heater (P/N 6732.0110) Maximum 710 1365 • Diode Array Detector HL (P/N VH-D10-A) Backpressure (bar) • Thermo Scientific™ LightPipe™ flow cell, 10 mm (P/N 6083.0100) Thermo Scientific™ Virtuoso™ Vial Identification System (P/N 60180-VT-100) Software Thermo Scientific™ Dionex™ Chromeleon™ 7.2 SR2 MUa Chromatography Data System Sample Preparation Solutions of the 19 compounds shown in Figure 3 were prepared by dissolving 10 mg amounts in 10 mL of methanol or water to produce 1 mg/mL primary solutions. Dilutions were then made with water to produce a 25 µg/mL working solution. Vial labeling was supported by the Virtuoso Vial Identification System. Results and Discussion impact the resolution of two critical pairs (peaks 1/2 and 3 Full resolution of all components was achieved using a 14/15). However, as a screening method, sufficient 0.8 mL/min flow rate within a 4 minute detection resolution to allow identification prior to confirmation window. The maximum system pressure throughout the analysis is demonstrated (Figures 2 and 3). The advantage gradient was 710 bar (Figure 1). However, the Vanquish is that by increasing the flow rate to 1.7 mL/min (and UHPLC system and the Hypersil GOLD VANQUISH scaling the method appropriately), the detection window UHPLC column combination are able to routinely operate has now been halved from 4 to 2 minutes. Therefore, by at back pressures up to 1500 bar. Therefore, the method exploiting the maximum operating conditions of both the was adjusted to take advantage of the maximum operat- UHPLC system and column, we can double throughput. ing conditions to ultimately increase throughput. To modify the method, the UHPLC speed-up method transfer calculator, built in to the Chromeleon software, was used. Increasing the flow rate to 1.7 mL/min does
50.0 17
40.0 14
30.0 4 7 3 15
6 20.0
mAU 11 1 9 13 18 8 19 10 16 10.0 5 12 2 0.0
-10 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.5 2.50 2.75 3.00 3.25 3.50 3.75 4.00 Min
Figure 1. Chromatogram showing the separation of 19 beta blockers at a flow rate of 0.8 mL/min (710 bar back pressure), within a 4-minute detection window.
40.0
35.0 17
30.0 14 25.0 15 4 7 3 20.0
mAU 6 15.0 11 1 9 13 18 8 19 0.0 10 16
5.0 5 12 2
0.0
-5.0 0.000 0.250 0.500 0.750 1.000 1.250 1.500 1.750 2.000 Min
Figure 2. Chromatogram showing the separation of 19 beta blockers at a flow rate of 1.7 mL/min (back pressure 1365 bar), within a 2 minute detection window. 1.7 mL/min
Atenolol 0.8 mL/min Application Note 21498
Sotalol
Carteolol
Nadolol
Pindolol
Timolol
Acebutolol
Metoprolol
Esmolol
Celiprolol
Oxprenolol Peak Name in Retention Time Order Labetalol
Bisoprolol
Propranolol
Alprenolol
Betaxolol
Carvedilol
Nebivolol
Penbutalol
0510 15 20 25 USP Resolution (Rs)
Figure 3. Peak identification and resolution with baseline resolution indicator (Rs=1.5) for 19 beta blockers.
Conclusion Useful Links By exploiting the high pressure capabilities of the AppsLab Library Vanquish UHPLC system, in conjunction with the The eWorkflow and the Chromeleon Backup (cmbx) Hypersil GOLD VANQUISH UHPLC column and a file can be downloaded at AppsLab Library: simple binary gradient, it was demonstrated that a www.thermofisher.com/appslab screening method for 19 compounds within a 2 minute detection window (and a full method cycle time of 2.8 minutes) can be achieved. This application demonstrates the advantages of using the Hypersil GOLD VANQUISH 1.9 µm UHPLC column and the Vanquish UHPLC system. The performance of the Hypersil GOLD VANQUISH UHPLC column coupled with the low internal volume and fast acquisition of the Diode Array Detector deliver the following: • Full resolution UHPLC method for 19 beta blockers in a method time < 4 minutes • Rapid screening UHPLC method for 19 beta blockers in a method time < 2.8 minutes
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