Application Note 21178 method times and increased assay throughput. manufacturing processes, results in a column that delivers manufacturing processes, required forthe increased chromatographic performance rapid screening methods. UHPLC column The Accucore Vanquish and Vanquish UHPLC system were designed in combination to provide The the best possible chromatographic performance. system is optimized to reduce extra column Vanquish band dispersion and allow users to significantly improve By the separation power in their analytical assays. exploiting the 1500 bar high pressure capability of the used with the the flow rate UHPLC system, Vanquish be increased UHPLC column can Vanquish Accucore resulting in shorter while maintaining peak capacity,
™ Accucore ™ to facilitate fast and highly ™ Vanquish particles, in combination with controlled in combination with particles, Vanquish 1.5 µm solid core particles that are not totally porous, but 1.5 µm solid core particles that are not totally porous, The instead have a solid core and a porous outer layer. robust optimized phase bonding creates a high-coverage, This coverage results in a significant reduction in phase. secondary interactions and delivers highly efficient peaks. Accucore The tightly controlled 1.5 µm diameter of methods for fewer analytes. Reduced analysis times Reduced analysis methods for fewer analytes. costs per reduced provide for quicker release of data, and overall greater sample throughput. assay, use Core C18+ UHPLC columns Vanquish Accucore Enhanced Technology This next-generation column features efficient separations. demonstration of a multiple analyte method, as the demonstration of a multiple analyte method, similar structures of the compounds require an efficient good In addition, separation to achieve good resolution. peak shape for these basic analytes will highlight low methods for Creating screening secondary interactions. described here for such as the method multiple analytes, dedicated is more cost effective than beta blockers, hypertension, angina, heart failure, and heart attacks. Beta and heart attacks. heart failure, angina, hypertension, of ablockers are designed to stop the functioning which is a noradrenaline, naturally occurring compound, the arterieschemical released in the body that can cause to narrow and the heart rate to increase. The analysis of beta blockers provides a good Introduction category of drugsBeta blockers (or beta antagonists) are a such as used to treat a number of medical complaints, Ken Meadows, Thermo Fisher Scientific, Runcorn,Ken Meadows, Thermo UK Rapid Screening Method for Beta Blockers Beta Blockers for Method Screening Rapid UHPLC Core Solid an Advanced Using andColumn Combination System C18+, 1.5 µm column and Thermo Scientific Vanquish UHPLC C18+, 1.5 µm column and Thermo Scientific ™
methods while maintaining high performance. methods while maintaining high performance. be operated at high flow rates that enable development of rapid analyticalbe operated at high flow rates that enable system for the fast analysis of beta blockers. The advanced capabilitiessystem for the fast analysis of beta blockers. columns to Vanquish UHPLC system allow the Accucore of the Vanquish Vanquish To demonstrate the advantages of using the Thermo Scientific To penbutalol Goal celiprolol, oxprenolol, labetalol, propranolol, betaxolol, carvidilol, nebivolol, labetalol, propranolol, oxprenolol, celiprolol, Vanquish, Accucore, beta blockers, beta antagonists, UHPLC, solid core, beta antagonists, UHPLC, solid core, beta blockers, Accucore, Vanquish, esmolol, timolol, acebutolol, metoprolol, sotalol, pindolol, fused core, Key Words Key 2 Experimental Software Consumables and Apparatus The Thermo Scientific™ Dionex™ Chromeleon™ 7.2 SR2 • Accucore Vanquish C18+, 1.5 µm UHPLC column, Chromatography Data System was used for data 100 × 2.1 mm (P/N 27101-102130) acquisition and analysis. • Thermo Scientific™ Virtuoso™ Vial Identification System Results and Discussion (P/N 60180-VT-100) By exploiting the high pressure capabilities of the • Thermo Scientific™ Virtuoso™ 9 mm wide opening screw Vanquish UHPLC system, in conjunction with the thread vial, 2 mL, clear glass vial with V-patch and red Accucore Vanquish UHPLC column and a simple binary PTFE/white silicone/red PTFE septum gradient, it was demonstrated that a screening method for (P/N 60180-VT400) 14 compounds within a 4.5 minute detection window • LC–MS grade 18 MΩ water from Thermo Scientific™ (and a full method cycle time of six minutes) can be Smart2Pure™ System (P/N 50129845) achieved (Figure 1). The performance of the Accucore Vanquish UHPLC column has ensured that even with the • Fisher Scientific™ HPLC grade acetonitrile fast method time, good separation of critical pairs has (P/N A/0626/17) been achieved with resolution > 2.00 for all pairs except • Fisher Scientific analytical grade formic acid celiprolol and oxprenolol at 1.53 (Table 2 and Figures 2 (P/N 10559570) and 3). Replicate injections showed that the Accucore • Fisher Scientific ammonium formate (P/N A/5080/53) Vanquish UHPLC column produced stable and Sample Preparation reproducible results (Table 2). Solutions of the fourteen compounds shown in Table 1 Using a 500 µL/min flow rate, the system pressure at the were prepared by dissolving 10 mg amounts in 10 mL start of the gradient was 967 bar and rose to a maximum of methanol to produce 1 mg/mL primary solutions. of 1024 bar during the gradient cycle. The Vanquish Dilutions were then made with water to produce UHPLC system and column combination are able to 100 µg/mL working solutions. routinely operate at these pressure conditions.
Vial labeling was supported by the Thermo Scientific 129 Virtuoso Vial Identification System. 120 12 110 Instrumentation 2 100 Analyses were performed using a Vanquish UHPLC System consisting 90 of: 80 • System base (P/N VH-S01-A) 70 60 mAU • Binary pump H (P/N VH-P10-A) 50 4 10 13 40 • Split sampler HT (P/N VH-A10-A) 3 30 14 • Column compartment H (P/N VH-C10-A) 8 20 5 6 11 7 • Diode Array Detector HL (P/N VH-D10-A) 10 1 9 UHPLC Conditions 0 -11 UHPLC column Accucore Vanquish C18+, 1.5 µm, 100 × 2.1 mm 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 Mobile phase A Ammonium formate, 20 mM, pH 3.0 Time (Mins) Mobile phase B Acetonitrile + 0.1% formic acid Figure 1. Chromatogram showing separation of 14 beta blockers within a 4.5 minute detection window. Flow rate 500 µL/min Column temp. 40 °C, still air with eluent pre-heating Injection details 2 µL standard needle in loop UV detection 270 nm
Table 1. LC gradient conditions.
Time (min) % B 0.00 5 3.00 30 4.50 100 5.00 100 5.00 5 Application Note 21177 4.315 4.250 0.19% 0.22% 0.23% 0.23% 0.18% 0.16% 0.14% 0.070% 0.030% 0.00%* 0.0099% 0.00%* 0.10% 0.20% Time (n= 6) 4.200 % RSD of Retention 14 4.150 13 Time (Mins) 4.100
1.53 n.a. 4.18 4.92 2.09 2.41 5.88 5.72 8.45 8.72 3.37 12 27.18 10.59 20.34 4.050
Resolution (USP) 4.000 3.968 Technical Support For advice and support, please visit our website: www.thermoscientific.com/chromexpert -2 10 90 80 70 60 50 40 30 20
114 100
Rapid screening UHPLC method for 14 beta blockers Method time less than 6 minutes Excellent retention time reproducibility Resolution of critical pairs mAU • • • • Figure Chromatogram 3. showing separation of carvidilol, nebivolol, penbutalol. and 1.10 1.95 4.06 4.14 4.19 2.55 2.61 2.68 2.96 3.34 3.67 3.85 3.14 3.19
3.311 (free call domestic) Retention Time (min) 8 (fax) (free call domestic) 3.200 7 +44 (0) 1928 534 050 +44 (0) 1928 534 110 3.100 0800 933 966 +65 6289 1190 0120 753 670 0120 753 671 3.000 6 +82 2 3420 8600 2.900 Korea United Kingdom New Zealand Singapore All Other Enquiries Japan Sotalol Timolol Esmolol Pindolol Nebivolol Labetalol Betaxolol Carvidilol Celiprolol Acebutolol Penbutalol Metoprolol Oxprenolol Propranolol Peak Name Time (Mins)
2.800 2.700 5 (free call domestic)
4 2.600 (free call domestic) 3 +1 800 332 3331 2.500 7 8 9 4 5 6 2 3 1 11 12 13 14 10 +49 (0) 2423 9431 20 or 21 1300 735 292 2.406 800 810 5118 400 650 5118 +33 (0)1 60 92 48 34 +91 22 6742 9494 +91 27 1766 2352
0.0
-3.7 10.0 30.0 20.0 50.0 40.0 70.0 60.0 77.7 Peak Number Peak
mAU Germany India China France USA and Canada Australia these products in any manners that might infringe the intellectualproperty rights of others.Not Specifications, all products are available terms in alland countries.pricing are subject Please to change. consult your local sales representative for details. ©2015 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the propertyThis information of Thermo Fisher is presented Scientific as an example and of theits capabilitiessubsidiaries. of Thermo Fisher Scientific products. It is not intended to encourage use of thermoscientific.com/Accucore For Research Use Only the following: UHPLC column coupled with the low internal volume UHPLC deliver Vanquish and advanced capabilities of the The performance of the Accucore Vanquish C18+ 1.5 µm Vanquish Accucore The performance of the Conclusion * No retention time variation seen for replicate injections measured to three decimal places. Table 2.Table Peak identification, resolution, and retention time variability. Figure 2. Chromatogram showing separation of timolol, acebutolol, oxprenolol. celiprolol, and AN21178-EN 0415S