Extraction of Teicoplanin from plasma using EVOLUTE® EXPRESS ABN prior to HPLC-DAD analysis

Introduction

This application note describes a solid phase extraction (SPE) protocol for the extraction of teicoplanin from plasma prior to HPLC-DAD analysis.

Teicoplanin (Figure 1) is a glycopeptide antibiotic used in the treatment of serious infections including methicillin-resistant Staphylococcus aureus (MRSA) as it has an activity spectrum similar to Vancomycin, inhibiting peptidoglycan (cell wall) synthesis. Trough concentrations can be between 15 mg L-1 and 60 mg L-1. Teicoplanin has a UV chromophore at 279 nm (Figure 2), this was used for quantitation.

Figure 1: Teicoplanin A2-2 (Major side-chain variants shown in parenthesis)

Figure 2: Teicoplanin A2 UV Spectrum

The method described in this application demonstrates high, reproducible recovery of teicoplanin from human plasma.

Samples were extracted using an EVOLUTE® EXPRESS ABN 30 mg 96-well plate using 200 µL plasma volumes. EVOLUTE® EXPRESS ABN products provide clean, rapid, robust, efficient, high throughput and automatable extraction solutions for this analyte.

Analytes

Teicoplanin (as a mixture of A2 variants)

Sample preparation procedure

Format:

EVOLUTE® EXPRESS ABN 30 mg plate, part number 600-0030-PX01

Sample pre-treatment:

Dilute 200 µL plasma in a 1:3 ratio using 2% formic acid (aq).

Condition:

Condition each well with methanol (1 mL).

Equilibration:

Equilibrate each well with 0.1% HCOOH (aq) (1 mL).

Sample loading:

Load pre-treated sample (800 µL) at a flow rate of approximately 1 mL/min.

Wash:

Elute interferences with water (1 mL).

Elution:

Elute analyte with methanol/water (70/30, v/v, 500 µL).

Post-elution:

Dry in a stream of air or nitrogen at 40° C using a Biotage® SPE Dry 96 Sample Concentrator System.

Reconstitution:

Acetonitrile : 10 mM ammonium acetate pH 4.4 (10/90, v/v, 250 µL).

HPLC conditions

Instrument

Waters Alliance 2795 Separations Module

Cartridge

Shimadzu ShimPack ODS-XR 50 x 3.0 mm, 2.2 µm

Flow Rate

0.8 mL min-1

Cartridge temperature

Room Temperature

Sample temperature

12 °C

Injection volume

25 µL partial (50 µL loop)

Mobile phase

A: 10 mM ammonium acetate pH 4.4 B: Acetonitrile

Detector

Waters 996 PDA

Detection λ

279 nm

Resolution

1.2 nm

Frequency

2 Hz

Results

Assay performance is summarized below

Linear range was determined from a ten-point calibration curve of spiked pooled plasma (n=4) where replicate means were within 80% to 120% of the stated value and RSD were ≤15%.
Assay linearity was estimated using r². An example calibration curve is shown in Figure 3

Figure 3: Teicoplanin Calibration Curve (0.1 to 100 µg/mL)

LOQ was estimated as the lowest concentration spiked standard demonstrating 80% to 120% accuracy and repeatability ≤ 15% RSD. Extraction recovery was determined at a spike level of 20 µg mL-1 teicoplanin A2 in pooled human plasma (Welsh Blood Service, Pontyclun). Extraction repeatability was estimated using the % RSD of the post extraction spike samples (n=6).

Example spike and blank chromatograms are shown in Figure 4(a) and Figure 4(b).

Figure 4(a): Teicoplanin Pre-Extraction Spike in Pooled Human Plasma, 20 µg mL–1, λ279nm.

Figure 4(b): Blank Pooled Human Plasma, λ279nm.

Additional information

Eluent preparation

10 mM ammonium acetate pH 4: dilute 360 µL of glacial acetic acid and 252 mg of ammonium acetate in 1 L of LC-MS grade water, titrate to pH 4.4 using monovalent strong base or acid as required.

Reconstitution solvents

Reconstitution solvents should be fully evaluated depending on collection vessel type in order to avoid issues with reproducibility associated with non-specific binding effects.

Ordering information

Literature number: AN869