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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 Enhancement1 Kit (p/n: 700011805)

Firmware 3.04

Caffeine:

Column: XBridge C18 4.6 x 50 mm, 3.5 µm

(p/n: 186003031)

Needle Wash: Methanol

Isocratic: 70:30 Water:Methanol

Chlorhexidine:

Column: CORTECS C18 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 C18 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 S11

Column: XBridge BEH C8 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 pack1 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

SolutionAlliance

Carryover

2018 Alliance

Carryover

Quetiapine

Standard Solution

Alliance

Carryover

2018 Alliance

Carryover