TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2018 Waters Corporation CARRYOVER IMPROVEMENT ACHIEVED THROUGH INSTRUMENT DESIGN CHANGES AND NEEDLE WASH OPTIMIZATION FOR HPLC SYSTEMS Amanda B. Dlugasch, Jennifer Simeone, and Patricia R. McConville Waters Corporation, Milford, Massachusetts, USA INTRODUCTION Sample carryover is a common problem for analytical labs and may adversely affect chromatographic methods. Sample carryover occurs when material from an injection is present in subsequent injections. There are several factors that can influence carryover including the chemistry of the sample analyte as well as the injector design of the High Performance Liquid Chromatographic (HPLC) system. The injector design is important not only to minimize the potential for carryover, but also to properly clean the components of the needle that are vulnerable to contamination. A HPLC system based upon a flow through-needle design is capable of providing improved carryover performance due to the interior of the needle being washed by the mobile phase during the analysis and the exterior of the needle being washed with an appropriate wash solvent. Modifying the injector design provides a significant reduction of the sample carryover with optimal washing procedures of the needle. The needle wash, therefore, is an important factor in which depends on the sample. Optimally, the wash will be able to easily solubilize the analyte(s) of interest. In this study, we will examine the improved carryover obtained after design optimization of the HPLC system injector and the optimization of the needle wash solvent over a wide range of sample types. METHODS LC Systems: Alliance : Alliance e2695 Separations Module with 100 μL syringe 2998 PDA Detector and CH-30 equipped with passive column preheater. Firmware 3.03 2018 Alliance : Alliance e2695 Separations Module with 100 μL syringe 2998 PDA Detector, CH-30 equipped with passive column preheater and the e2695 Enhancement 1 Kit (p/n: 700011805) Firmware 3.04 Caffeine: Column: XBridge C 18 4.6 x 50 mm, 3.5 μm (p/n: 186003031) Needle Wash: Methanol Isocratic: 70:30 Water:Methanol Chlorhexidine: Column: CORTECS C 18 Column, 2.7 μm, 3 mm x 100 mm (p/n: 186007372) Needle Wash: 50:50 Water:Acetonitrile Isocratic: 67:33 0.1% TFA in Water: 0.1% TFA in Acetonitrile Coumarin: Column: CORTECS C 18 Column, 2.7 μm, 3 mm x 100 mm (p/n: 186007372) Needle Wash: 90:10 Water:Acetonitrile Isocratic: 90:10 Water:Acetonitrile Quetiapine: Quetiapine Fumarate Assay USP 40 NF35 S1 1 Column: XBridge BEH C 8 Column, 5 μm, 4.6 mm x 250 mm (p/n: 186003018) Needle Wash: 90:10 Water:Acetonitrile 70:30 Methanol:Water Isocratic: 54:7:39 Methanol:Acetonitrile:Buffer Buffer: 2.6 g/L of dibasic ammonium phosphate adjusted to pH 6.5 with phosphoric acid References 1. Quick Reference Guide: 2018 Alliance Enhancements. Waters User Manual 720006332EN. 2018 June. 2. Jenkins, Tanya, Michael Waite. Low Sample Carryover with Key Performance Indicators on the Alliance HPLC System. Waters Technology Brief 720004534EN. 2012 Dec. 3. Official Monographs, Quetiapine Fumarate USP 40 NF35 S1, United States Pharmacopeia and National Formulary (USP 40-NF35 S1) Baltimore, MD: United Book Press, Inc.; 2017. p. 5939. 4. Dolan, John. Autosampler Carryover. LCGC Europe. Volume 19, Issue 10, pg 522-529. http:// www.chromatographyonline.com/autosampler-carryover-3?id=&pageID=1&sk=&date= RESULTS CONCLUSION The 2018 Alliance HPLC System incorporates a newly designed seal pack 1 that improves needle wash flow over the exterior of the injector needle. The newly designed seal pack results in a significant reduction in carryover for a wide range of chemical compounds. The 2018 Alliance HPLC System can reduce the amount of carryover present regardless of the specific compounds being analyzed. The optimal needle wash setting and needle wash solvent composition is essential to decreasing sample carryover, therefore application specific. When combined, the needle wash mode and the needle wash composition significantly reduce carryover on the 2018 Alliance HPLC System. Figure 1. Structures of caffeine, chlorhexidine, coumarin and quetiapine fumarate. Table 1. Carryover results for caffeine, chlorhexidine, coumarin and quetiapine fumarate on the Alliance HPLC System and the 2018 Alli- ance HPLC System using the default ‘Normal’ wash mode setting. *The carryover instrument specification is based upon the compound caffeine under specific method conditions. Compound Alliance HPLC System 2018 Alliance HPLC System Carryover Improvement Caffeine 0.0011% 0.00016% 6.9x Chlorhexidine 0.010% 0.0005% 20x Coumarin 0.0002% 0.00006% 3.3x Quetiapine fumarate 0.028% 0.019% 1.5x System Specification* < 0.01% < 0.0025% NA Figure 3. The observed impact of the advanced features for the needle wash modes for coumarin and quetiapine fumarate on the 2018 Alliance HPLC System. Figure 4. Carryover results for the two different needle wash compositions on the 2018 Alliance HPLC System. Figure 2. The chromatographic results for the Alliance HPLC Sys- tem (red) and the 2018 Alliance HPLC System (blue) for the post- challenge blank are represented in the figure above for each of the compounds, caffeine, chlorhexidine, coumarin, and quetiapine fu- marate. The black chromatogram represents the challenge or stan- dard solution. Caffeine Chlorhexidine Coumarin Quetiapine Fumarate Caffeine Challenge Solution Alliance Carryover 2018 Alliance Carryover Chlorhexidine Standard Solution Alliance Carryover 2018 Alliance Carryover Coumarin Challenge Solution Alliance Carryover 2018 Alliance Carryover Quetiapine Standard Solution Alliance Carryover 2018 Alliance Carryover