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High sensitivity assays for docetaxel and paclitaxel in plasma using solid-phase extraction and high-performance liquid chromatography with UV detection
BMC Clinical Pharmacology
Technical advance High sensitivity assays for docetaxel and paclitaxel in plasma using solid-phase extraction and high-performance liquid chromatography with UV detection
Anders Andersen 2
David J Warren 2
Paal F Brunsvig 1
Steinar Aamdal 0
Gunnar B Kristensen 3
Harald Olsen 2
0 Department of Clinical Research, The Norwegian Radium Hospital , N-0310 Oslo , Norway
1 Department of Medical Oncology, The Norwegian Radium Hospital , N-0310 Oslo , Norway
2 Central Laboratory, The Norwegian Radium Hospital , N-0310 Oslo , Norway
3 Department of Gynecologic Oncology, The Norwegian Radium Hospital , N-0310 Oslo , Norway
Background: The taxanes paclitaxel and docetaxel have traditionally been used in high doses every third week in the treatment of cancer. Lately there has been a trend towards giving weekly low doses to improve the therapeutic index. This article describes the development of high performance liquid chromatographic (HPLC) methods suitable for monitoring taxane levels in patients, focusing on patients receiving low-dose therapy. Methods: Paclitaxel and docetaxel were extracted from human plasma by solid phase extraction, and detected by absorbance at 227 nm after separation by reversed phase high performance liquid chromatography. The methods were validated and their performance were tested using samples from patients receiving paclitaxel or docetaxel. Results: The limits of quantitation were 1 nM for docetaxel and 1.2 nM for paclitaxel. For both compounds linearity was confirmed from the limit of quantitation up to 1000 nM in plasma. The recoveries ranged between 92% and 118% for docetaxel and between 76% and 104% for paclitaxel. Accuracy and precision were within international acceptance criteria, that is within 15%, except at the limit of quantitation where values within 20% are acceptable. Low-dose patients included in an on going clinical trial had a median docetaxel concentration of 2.8 nM at 72 hours post infusion. Patients receiving 100 mg/m2 of paclitaxel had a mean paclitaxel concentration of 21 nM 48 hours after the end of infusion. Conclusion: We have developed an HPLC method using UV detection capable of quantifying 1 nM of docetaxel in plasma samples. The method should be useful for pharmacokinetic determinations at all relevant doses of docetaxel. Using a similar methodology paclitaxel can be quantified down to a concentration of 1.2 nM in plasma with acceptable accuracy and precision. We further demonstrate that the previously reported negative influence of Cremophor EL on assay performance may be overcome by degradation of the detergent by incubation with lipase.
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Background
Paclitaxel (figure 1a) was discovered in the early 1970's as
being the active cytotoxic constituent in extracts of the
bark of the yew Taxus Brevifolia. Paclitaxel had a unique
mechanism of action, but the supply was limited and
formulation of the compound for clinical use was difficult as
paclitaxel is practically insoluble in water. Docetaxel
(figure 1b), a semisynthetic analog of paclitaxel with higher
aqueous solubility, was constructed from 10-deacetyl
baccatin III about 10 years later. Several review articles on the
clinical and preclinical pharmacokinetics of paclitaxel and
docetaxel have been published [1-3].
Docetaxel for clinical use is currently formulated as a
concentrate in polysorbate 80, and about 4 ml of this
nonionic detergent is infused together with a standard dose
(173 mg) of docetaxel. The plasma concentration of
polysorbate 80 is about 0.1% during the infusion and the
compound is rapidly cleared from plasma [4,5]. This is in
contrast to Cremophor EL, the solvent for paclitaxel,
where about 27 ml is infused together with a standard
dose (303 mg), and the plasma concentration remains
high (above 0.1%) for days due to a low volume of
distribution (3 L/m2) and a long half-life (4080 h) [6-10].
Cremophor EL has been reported to have a negative
influence on the reproducibility of paclitaxel determinations
[11]. No such effects have been reported for polysorbate
80 and docetaxel.
Numerous analytical methods for the taxanes have been
published. Most of them rely on sample preparation by
solid phase extraction followed by separation by HPLC
and detection by absorbance at 225230 nm. The
majority of methods for both paclitaxel and docetaxel are based
on the principles for extraction and separation published
by Willey et al. [12] in 1993, but also methods based on
organic extraction are used [13-15]. Lately, mass
spectrometry (MS) based methods with simplified sample
preparations and limits of detections in the
sub-nanomolar range have appeared [4,16-19]. The improved
sensitivity has given new insight into the pharmacokinetics of the
taxanes [20,21].
The method development presented here was started to
support a dose escalation study with paclitaxel in patients
with ovarian cancer. As we lacked an MS-instrument, the
detection utilized UV absorbance. Later on the
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