• Application Note

A Novel Extraction Procedure Using Micro Elution Plates for the Estimation of Docetaxel Using UPLC and ACQUITY TQD

A Novel Extraction Procedure Using Micro Elution Plates for the Estimation of Docetaxel Using UPLC and ACQUITY TQD

  • Veeranjaneyulu P.
  • Tirupatewara B. Rao
  • Sudarshan Mantha
  • Gopal Vaidyanathan
  • Waters Corporation

Abstract

This application note focuses on the use and benefits of micro-elution extraction methods and also the use of the ACQUITY TQ Detector in obtaining a signal-to-noise (S/N) ratio of ~225 at the LLOQ level for docetaxel.

Benefits

  • Demonstrates the benefits of ACQUITY TQD for monitoring multiple transitions in compounds
  • Oasis Micro-elution Plates ensure high-throughput sample extraction of compounds
  • Addresses several key challenges faced by today’s bioanalytical scientist in acquiring fast, high, accurate, and robust LC-MS results
  • Maintains high throughput capability.

Introduction

Docetaxel is a clinically well-established anti-mitotic chemotherapy medication used mainly for the treatment of breast, ovarian, and non-small cell lung cancer. It belongs to the chemotherapy drug class of “Taxane”, and is a semi-synthetic analogue of Paclitaxel (Taxol). Docetaxel binds to microtubules reversibly with high affinity and has a maximum stoichiometry of 1 mole docetaxel per mole tubulin in microtubules.

Figure 1. Molecular structure of docetaxel.

This molecule also has high protein binding nature, and it has been reported that protein disruption results in improper assay results. Hence, it is highly challenging to extract such types of molecules. Micro-elution offers the advantage of using less plasma volume, coupled with the fact that the sample cleanup can be done effectively. This application note focuses on the use and benefits of micro-elution extraction methods and also the use of the ACQUITY TQ Detector in obtaining a signal-to-noise (S/N) ratio of ~225 at the LLOQ level for docetaxel.

Experimental

LC conditions

LC system:

ACQUITY UPLC

Column:

ACQUITY UPLC BEH C8 1.7-μm, 2.1 x 100 mm

LC column elution:

70% aqueous buffer over 2.0 min followed by a 90% organic elution until 4.2 min; then change back to initial conditions.

Column temp.:

40 °C

Flow rate:

0.300 mL/min

Injection volume:

20 μL

MS conditions

MS system:

ACQUITY TQD

MS mode:

ESI positive MS/MS method

MRM transition:

808.7 → 226.3 and 808.7 → 282.3

The analyte from the spiked plasma samples was isolated using solid phase micro-elution extraction employing Waters Oasis HLB Micro-Elution Plates. A 300-µL aliquot of plasma was diluted with water, centrifuged at 12,000 rpm in a micro-centrifuge, and loaded onto SPE cartridges previously conditioned with organic solvent and water. The SPE cartridges were then washed with water twice followed by an organo-aqueous wash, and the samples were then eluted with the elution solvent. The eluted samples were injected on to the system directly.

Results and Discussion

Docetaxel eluted with a retention time of 2.67 mins and with a peak width of 10 s at the base. The data shown below illustrates the blank signal, shown in Figure 3, as well as the signal obtained from the lower limit of quantification (LLOQ) of docetaxel in human plasma. It can be observed that the analyte was well resolved from co-eluting peaks coming from the endogenous plasma components, shown in Figure 2. Figure 2 also shows chromatograms of six LLOQ samples and their respective signal-to-noise (S/N) ratios. The average of the S/N ratios was found to be 196.33.

Figure 2. Chromatogram of docetaxel at the LLOQ concentration of 200 pg/mL, along with the S/N ratios and the average S/N ratio obtained for LLOQs of six samples.
Figure 3. Chromatogram of  blank and LLOQ concentration (200 pg/mL) of docetaxel.

The assay in this report showed a linear calibration over the range of 200 pg/mL to 100 ng/mL with an excellent r2 value of 0.9994, shown in Table 1 and Figures 3.1 and 3.2. The back-calculated concentration of the standard was found to be within 12% of the nominal concentration, shown in Table 1.

Table 1. Calibration data of docetaxel over the range of 200 pg/mL to 100 ng/mL.
Figure 3.1. Comparison of area under curve for docetaxel (analyte) and IS for the concentration range of 200.000 pg/mL to 100.000 ng/mL.
Figure 3.2. Calibration curve of docetaxel.

Recovery of the analyte and internal standard (IS) was performed by comparison of the extracted QC samples against six post-extracted samples, which was found to be approximately 48% at LLOQQC, LQC, MQC, and HQC levels for both analyte and the internal standard, as shown in Figures 4.1, 4.2, 4.3, and 4.4, and Table 2. The %CV for repeat batches was found to be within 10% of LLOQQC and varied between 1% to 3% for all QC levels.

Figure 4.1. Analyte recoveries (area under the curve) from six samples of docetaxel at LLOQQC, concentrations.
Figure 4.2. Analyte recoveries (area under the curve) from six samples of docetaxel at LQC.
Figure 4.3. Analyte recoveries (area under the curve) from six samples of docetaxel at MQC, concentrations.
Figure 4.4. Analyte recoveries (area under the curve) from six samples of docetaxel at HQC, concentrations.
Table 2. Mean analyte recovery (%) of docetaxel at LQC, MQC, and HQC levels.

Data shown in Figures 4.1, 4.2, 4.3, and 4.4 shows that the analyte recovery values for the six samples did not vary significantly for any of the four concentration levels (LLOQQC, LQC, MQC, and HQC). In addition, as detailed in Table 2, the mean analyte recovery for the three concentration ranges was 48%.

For a comparison of samples within the global batches, three separate batches were prepared with six samples in each batch for LLOQQC, LQC, MQC, and HQC concentration levels. The data showed excellent agreement between the six samples for all three batches, as shown in Table 3. The mean accuracy obtained for all the samples levels was found to be > 93% for every concentration, shown in Table 3. 

Table 3. Comparison of the three separate batches, each containing six docetaxel samples at the LLOQQC, LQC, MQC, and HQC concentrations.

Conclusion

Micro-elution offered a better solution for extraction, which employed low plasma volumes, thereby reducing matrix effect, followed by enrichment of the samples for proper extraction. The sample cleanup was found to be very effective, which can be seen with the S/N ratio obtained for LLOQ (200 pg/mL). The analyte recovery varied very little through the entire range of the calibration including the LLOQ. Excellent reproducibility was observed after summing up of two traces. Thus the above method can be used for the estimation of docetaxel in human plasma with the ACQUITY UPLC System and ACQUITY TQD.

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720004408, June 2012

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