Paclitaxel In Plasma Research Articles

Sensitive HPLC method for quantitation of paclitaxel (Genexol ®) in biological samples with application to preclinical pharmacokinetics and biodistribution

A sensitive, specific and reproducible HPLC method has been developed and validated for the quantitative determination of paclitaxel in plasma, tissues and tumor of mice. Tissue specimens including liver, kidneys, spleen, lungs, heart and tumor were separately homogenized in bovine serum albumin (BSA, 40 g/l) in water. Plasma or tissue homogenates (0.1 ml) containing paclitaxel and internal standard (dimethyl-4,4′-dimethoxy-5,6,5′,6′-dimethylene dioxy biphenyl-2′,2′ dicarboxylate (DDB), I.S.) were extracted by ethyl acetate (10 ml). A 4.6 mm × 250 mm ODS column was used to separate the components in biological samples with UV detection at 227 nm and gradient system was applied to a quantitation of paclitaxel consisting of acetonitrile-deionized water. The I.S. and paclitaxel were eluted at 13.7 and 18.0 min, respectively, and no interfering peaks were observed. Linear relationships ( r 2 > 0.999) were obtained between the peak height ratios and the corresponding biological sample concentrations over the range of 0.1–20 μg/ml. The average intra- and inter-day variations (% R.S.D.s and % deviations) of the assay for biological samples were less than 10%. The LOD and LOQ were 5 and 10 ng/ml, respectively, for paclitaxel using a microsample volume (100 μl) of plasma sample. This HPLC method has been successfully applied for the determination of paclitaxel in pharmacokinetic and biodistribution study in after administration of 50 mg equivalent paclitaxel/kg dose of paclitaxel-loaded polymeric micelle and 20 mg equivalent paclitaxel/kg dose of Taxol ® to female SPF C57BL/6 mice.

A simple and sensitive assay for the quantitative analysis of paclitaxel and metabolites in human plasma using liquid chromatography/tandem mass spectrometry

A sensitive and specific LC-MS/MS assay for the determination of paclitaxel and its 3'p- and 6-alpha-hydroxy metabolites is presented. A 200 microL plasma aliquot was spiked with a 13C6-labeled paclitaxel internal standard and extracted with 1.0 mL tert-butylmethylether. Dried extracts were reconstituted in 0.1 M ammonium acetate-acetonitrile (1:1, v/v) and 25 microL volumes were injected onto the HPLC system. Separation was performed on a 150 x 2.1 mm C18 column using an alkaline eluent (10 mm ammonium hydroxide-methanol, 30:70, v/v). Detection was performed by positive ion electrospray followed by tandem mass spectrometry. The assay quantifies a range for paclitaxel from 0.25 to 1000 ng/mL and metabolites from 0.25 to 100 ng/mL using 200 microL human plasma samples. Validation results demonstrate that paclitaxel and metabolite concentrations can be accurately and precisely quantified in human plasma. This assay is now used to support clinical pharmacologic studies with paclitaxel.

A simple and sensitive assay for the quantitative analysis of paclitaxel in human and mouse plasma and brain tumor tissue using coupled liquid chromatography and tandem mass spectrometry

The development and validation of an assay for the determination of paclitaxel in human plasma, human brain tumor tissue, mouse plasma and mouse brain tumor tissue is described. Paclitaxel was extracted from the matrices using liquid-liquid extraction with tert-butyl methyl ether, followed by chromatographic analysis using an alkaline eluent. Positive ionization electrospray tandem mass spectrometry was performed for selective and sensitive detection. The method was validated according to the FDA guidelines on bioanalytical method validation. Validation results indicate that calibration standards in human plasma can be used to quantify paclitaxel in all tested matrices. In human samples, the validated range for paclitaxel was from 0.25-1000 ng ml(-1) using 200 microl plasma aliquots and from 5 to 5000 ng g(-1) using 50 microl tumor homogenate aliquots (0.2 g tissue ml(-1) control human plasma). In mice, the ranges were 1-1000 ng ml(-1) and 5-5000 ng g(-1) using 50 microl of mouse plasma and 50 microl of tumor homogenate aliquots (0.2 g tissue ml(-1) control human plasma), respectively. The method can be applied to studies generating only small sample volumes (e.g. mouse plasma and tumor tissue), but also to studies in human plasma requiring a lower limit of quantitation. The assay was applied successfully to several studies with both human and mouse samples.

Paclitaxel quantification in mouse plasma and tissues containing liposome-entrapped paclitaxel by liquid chromatography-tandem mass spectrometry: application to a pharmacokinetics study

A liquid chromatography-tandem mass spectrometry assay to quantify total paclitaxel in mouse plasma and tissue homogenates containing paclitaxel, Taxol, or liposome-entrapped paclitaxel-easy to use (LEP-ETU) was developed and validated. Docetaxel was used as the internal standard (IS). Liquid–liquid extraction with tert-butyl methyl ether was used for plasma sample preparation, and a one-step protein precipitation with acetonitrile containing 0.1% acetic acid was developed for tissue homogenates. Paclitaxel and IS are separated on a 50 × 2.1-mm C18 column and quantified using a triple-quadrupole mass spectrometer operating in positive ion electrospray multiple reaction monitoring mode, with a total run time of 3.5 min. The peak area of the m/ z 854.4 → 286.2 transition of paclitaxel is measured versus that of the m/ z 808.5 → 527.5 transition of IS to generate the standard curve. In plasma, the linear range is 0.2–500 ng/mL and could be extended by dilution to 100,000 ng/mL with acceptable precision and accuracy (⩽15%). The lower limit of quantification is 0.5 ng/mL in tissue homogenates (10 ng/g tissue), and the standard curve is linear up to 1000 ng/mL, with precision and accuracy ⩽15%. This assay was used to support a pharmacokinetics and tissue distribution study of LEP-ETU in mice.

Pharmacoproteomic analysis of pre-and post-chemotherapy plasma samples from patients receiving neoadjuvant or adjuvant chemotherapy for breast cancer

2109 Background: This study examined proteomic changes in response to paclitaxel or FAC chemotherapy in the plasma of patients with stage I-III breast cancer. We also compared plasma profiles of cancer patients with profiles of healthy women to identify breast cancer associated protein markers. Methods: Sixty-nine patients and 15 healthy volunteers participated in the study. Plasma was sampled on day-0, before chemotherapy, and on day-3 post-treatment, or 3 days apart in the case of healthy women. Twenty-nine patients received preoperative and 40 received postoperative chemotherapy. SELDI-MS was used to generate protein mass profiles. Results: We observed few changes in the plasma during treatment. We identified only 1 protein peak at m/z 2790 that was induced by paclitaxel and to a lesser extent by FAC chemotherapy. This proteomic response was detectable in 80% of patients treated preoperatively but was also present with lesser intensity in about 40% of postoperative cases. There was no clear correlation...

Development and validation of a method to determine the unbound paclitaxel fraction in human plasma

Paclitaxel is pharmaceutically formulated in a mixture of Cremophor EL and ethanol (1:1, v/v). The unbound fraction of the anticancer drug paclitaxel in plasma is dependent on both plasma protein binding and entrapment in Cremophor EL micelles. We have developed a simple and reproducible method for the quantification of the unbound paclitaxel fraction in human plasma. Human plasma was spiked with [ 3H]paclitaxel and [ 14C]glucose (unbound reference) and incubated at 37 °C for 30 min. Plasma ultrafiltrate was prepared by a micropartition system (MPS-1) and collected in a sample cup containing 100 μl of plasma to prevent the loss of paclitaxel due to adsorption. The radionuclides were separated after combustion of the biological samples using a sample oxidizer and the radioactivity was determined by liquid scintillation counting. The unbound fraction of paclitaxel was calculated by dividing the ratios of 3H and 14C in plasma ultrafiltrate and in plasma. The method was thoroughly validated using human plasma spiked with pharmacologically relevant concentrations of paclitaxel (10–1000 ng/ml) and Cremophor EL (0.25–2.0%). The method was precise, with a within-day precision ranging from 3.9 to 11.0% and a between-day precision ranging from 5.8 to 13.1%. In patient plasma with low serum albumin values containing 1% of Cremophor EL, the unbound fraction appeared to be significantly higher than that in plasma with normal albumin values. The determination of the unbound fraction of paclitaxel proved to be stable during a 10-week storage at −20 °C. Furthermore, the assay was applicable in patient samples. This assay can be used to determine the unbound fraction of paclitaxel in plasma. Moreover, its design should allow the determination of the unbound concentrations of other hydrophobic drugs.

The influence of the P-glycoprotein inhibitor zosuquidar trihydrochloride (LY335979) on the brain penetration of paclitaxel in mice.

We determined the effect of zosuquidar.3HCl, an inhibitor of P-gp, on the penetration of the anticancer drug paclitaxel into the brain. Zosuquidar.3HCl was administered orally at 25 and 80 mg/kg 1 h before i.v. paclitaxel and i.v. at 20 mg/kg 10 min and 1 h before paclitaxel. The concentrations of paclitaxel in plasma and tissues and of zosuquidar.3HCl in plasma were quantified by high-performance liquid chromatography. The results revealed 3.5-fold and 5-fold higher paclitaxel levels in the brain of wild-type mice treated orally with 25 and 80 mg/kg zosuquidar.3HCl, respectively. However, complete inhibition as in P-gp knockout mice (11-fold increase) was not achieved. Zosuquidar.3HCl also increased the paclitaxel concentrations in plasma and tissues to levels similar to those observed in P-gp knockout mice, suggesting selective P-gp inhibition of zosuquidar.3HCl. When zosuquidar.3HCl was administered i.v. 10 min before paclitaxel, the paclitaxel levels in the brain of wild-type mice increased by 5.6-fold, whereas the increase was only 2.1-fold when zosuquidar.3HCl was administered 1 h before paclitaxel. This suggests that the inhibition of P-gp at the blood-brain barrier by zosuquidar.3HCl is rapidly reversible and that the concentrations of zosuquidar.3HCl in the plasma have already declined to levels insufficient to inhibit P-gp at the blood-brain barrier. In conclusion, zosuquidar.3HCl is only moderately active as an inhibitor of P-gp at the blood-brain barrier.

Determination of paclitaxel in mouse plasma and brain tissue by liquid chromatography–mass spectrometry

Paclitaxel is an anticancer agent extracted from the bark of the yew tree and is widely used in chemotherapy for solid tumors, including non-small cell lung cancer and ovarian carcinoma. Most assays to measure paclitaxel in plasma require a large amount of sample (0.4–1 ml) to achieve the necessary sensitivity, and are not suitable when only small sample sizes are available. To circumvent this latter limitation, we developed a sensitive liquid chromatography–mass spectrometry (LC–MS) method for the determination of paclitaxel in plasma based on the use of small sample volumes (50 μl plasma). A solid phase extraction procedure was employed that enabled the eluent to be directly injected onto a reversed phase chromatographic HPLC system using positive electrospray ionization followed by mass spectrometric detection. The extraction recoveries of paclitaxel were 98 and 83% from plasma and brain tissues, respectively. The mobile phase consisted of 50% acetonitrile in 0.1% formic acid that was pumped at 0.2 ml/min to yield a retention time for paclitaxel of 6.2 and 5.4 min for cephalomannine, the internal standard. The method has been validated at paclitaxel plasma concentrations from 0.036 to 9.9 μg/ml, and from 0.054 to 1.96 μg/ml in brain homogenates. A sensitive and specific assay for paclitaxel has been developed that has the advantages of using small sample sizes, and a single extraction step without solvent evaporation.

A tubulin-based fluorescent polarization assay for paclitaxel

This paper reports the development, characterization, and application of a separation-free, homogeneous, and simple to perform fluorescence polarization bioassay for paclitaxel. The bioassay is based on the binding interaction of paclitaxel to tubulin, the receptor protein on which this drug acts. The bioassay was carried out in a competitive format where the paclitaxel and a synthetic fluorescent-labeled paclitaxel (Rh-Tx) competed for tubulin binding, causing a change in fluorescence polarization, which was an inverse function of the paclitaxel concentration. The bioassay had a dynamic range from 0.03 to 0.35 μM, which falls in the therapeutic range (0.01–10 μM), and a limit of detection of 23 nM. The bioassay was selective against other naturally occurring taxanes such as baccatin III and 10-deacetylbaccatin III. However, there was interference from cephalomannine. The excellent sensitivity, accuracy, reproducibility, and simplicity make this analytical technique suitable for routine and high-throughput analyses and might be helpful in providing better care for patients. The bioassay was successfully applied to the determination of paclitaxel in human plasma.

Locoregional opening of the rodent blood-brain barrier for paclitaxel using Nd:YAG laser-induced thermo therapy: a new concept of adjuvant glioma therapy?

Nd:YAG laser-induced thermo therapy (LITT) of rat brains is associated with blood-brain barrier (BBB) permeability changes. We address the question of whether LITT-induced locoregional disruption of the BBB could possibly allow a locoregional passage of chemotherapeutic agents into brain tissue to treat malignant glioma. CD Fischer rats were subject to LITT of the left forebrain. Disruption of the BBB was analyzed using Evans blue and immunohistochemistry (IH). Animals were perfused with paclitaxel, and high-pressure liquid chromatography (HPLC) was employed to analyze the content of paclitaxel in brain and plasma samples. LITT induces an opening of the BBB as demonstrated by locoregional extravasation of Evans blue, C3C, fibrinogen, and IgM. HPLC proved the passage of paclitaxel across the disrupted BBB. LITT induces a locoregional passage of chemotherapeutic agents into the brain tissue. This is of potential interest for the treatment of brain tumors.

Quantitative determination of paclitaxel in human plasma using semi-automated liquid–liquid extraction in conjunction with liquid chromatography/tandem mass spectrometry

This paper describes a high-throughput sample preparation procedure combined with LC–MS/MS analysis to measure paclitaxel in human plasma. Paclitaxel and an internal standard were extracted from plasma by a semi-automated robotic method using liquid–liquid extraction. Thereafter compounds were separated on a RP C18 column. Detection was by a PE Sciex API 3000 mass spectrometer equipped with a TurboIonSpray™ interface. The compounds were detected in positive ion mode using the mass transition m/ z 854.6→286.2 and m/ z 831.6→263.2 for paclitaxel and the internal standard, respectively. The limit of quantitation for paclitaxel was 1 ng/ml with an imprecision of 5.2% following extraction of 0.1 ml of plasma. Linearity was confirmed over the whole calibration range (1–1000 ng/ml) with correlation coefficients higher than 0.99 indicating good fits of the regression models. The inter and intra-day precision was better than 9.5% and the accuracy ranged from 90.3 to 104.4%. The assay was simple, fast, specific and exhibited excellent ruggedness.

Quantitation of paclitaxel in micro-sample rat plasma by a sensitive reversed-phase HPLC assay

A sensitive high-performance liquid chromatographic (HPLC) method was developed for the determination of paclitaxel in micro-samples of rat plasma in order to study the mechanism of enhanced systemic exposure of paclitaxel co-administered with P-glycoprotein inhibitors. The assay involved solid-phase extraction procedures using 2′-methylpaclitaxel as the internal standard. Chromatographic separations were achieved using a ZORBAX ODS C18 column and mobile phase consisting of acetonitrile, methanol and ammonium acetate buffer (10 mM, pH 5.0) (48.5:16.5:35) pumped at 0.8 ml/min. The effluents were measured for UV absorption at 227 nm, with retention times of 8.5 and 11.0 min for paclitaxel and 2′-methylpaclitaxel, respectively. The chromatographic separation was excellent, with no endogenous interference. The standard curves showed a good linearity ( r=0.9994) over the concentration ranges of 10–1000 ng/ml. At 1000 ng/ml, the absolute recoveries of paclitaxel and 2′-methylpaclitaxel are 89 and 90%, respectively. The intra- and inter-day variabilities of paclitaxel were both less than 15%. This validated method for the assay of paclitaxel in micro-sample rat plasma made it feasible to study the pharmacokinetics of the drug in a single rat.

Measurement of paclitaxel in biological matrices: high-throughput liquid chromatographic–tandem mass spectrometric quantification of paclitaxel and metabolites in human and dog plasma

A GLP-validated, sensitive and specific LC–MS–MS method for the quantification of paclitaxel and its 6-α- and 3′- p-hydroxy metabolites is presented. A 0.400 ml plasma aliquot is spiked with a 13C 6-labeled paclitaxel internal standard and extracted with 1 ml methyl- tert.-butyl ether. The ether is evaporated and the residue is reconstituted in 130 μl of 30% aqueous acetonitrile (ACN) containing 0.1% trifluoroacetic acid. Isocratic HPLC analysis is performed by injecting 50 μl of the reconstituted material onto a 50×2.1 mm C 18 column with an ACN–water–acetic acid (50:50:0.1) mobile phase at 200 μl/min flow. Detection is by positive ion electrospray followed by multiple reaction monitoring of the following transitions: paclitaxel (854>509 u), 6-α-hydroxy paclitaxel (870>525 u), 3′- p-hydroxy paclitaxel (870>509 u) and internal standard (860>509 u). Quantification is by peak area ratio against the 13C 6 internal standard. The method range is 0.117–117 n M (0.1–100 ng/ml) for paclitaxel and both metabolites using a 0.400 ml human or dog plasma sample. Analysis time per sample is less than 5 min.

Sensitive liquid chromatography–mass spectrometry assay for quantitation of docetaxel and paclitaxel in human plasma

We have developed a high-performance liquid chromatography–electrospray ionization mass spectrometry (LC–MS) method for quantifying docetaxel and paclitaxel in human plasma. The assay fulfills the need for defining the lower plasma concentrations of these antineoplastic agents that result from a number of changes in how these agents are used clinically. The assay uses paclitaxel as the internal standard for docetaxel, and vice versa; solid-phase extraction; a Phenomenex Hypersil ODS (5 μm, 100×2 mm) reversed-phase analytical column; an isocratic mobile phase of 0.1% formic acid in methanol–water (70:30, v/v); and mass spectrometric detection using electrospray positive mode electron ionization. The assay has a lower limit of quantitation (LLOQ) of 0.3 n M and is linear between 0.3 n M and 1 μ M for docetaxel. For paclitaxel, the LLOQ was 1 n M, and the assay is linear between 1 n M and 1 μ M. We demonstrated the suitability of this assay for docetaxel by using it to quantify the docetaxel concentrations in plasma of a patient given 40 mg/m 2 of docetaxel and comparing those results to results produced when the same samples were assayed with an HPLC assay using absorbance detection. In a similar manner, the suitability of the assay for paclitaxel was demonstrated by using it to quantify the concentrations of paclitaxel in the plasma of a patient given 15 mg/m 2 of paclitaxel and comparing those results to results produced when the same samples were assayed with an HPLC assay using absorbance detection. The LC–MS assay, which proved superior because of its greater sensitivity and relatively short (7 min) run time, should be an important tool for future pharmacokinetic analyses of docetaxel and paclitaxel.

On-line solid-phase extraction and determination of paclitaxel in human plasma

The application of coupled-column liquid chromatographic analysis to pharmacokinetic studies eliminates the need for sample clean-up from plasma. Considering lipophilic antineoplastic agents, we tested this approach to analyze paclitaxel under unfavourable circumstances (i.e., weekly low-dose regimen, plasma protein binding >90%, UV detection at 229 nm). The excellent quality control data (recovery: 95.6–100.7%, inter-assay relative standard deviation on 5 days: 1.3–3.2%, accuracy: 0.9–2.7%) and the detection limit of 19 n M indicates the usefulness of this method for the analysis of paclitaxel in plasma using on-line solid-phase extraction.

P-glycoprotein inhibition by the multidrug resistance-reversing agent MS-209 enhances bioavailability and antitumor efficacy of orally administered paclitaxel.

Recent studies in humans and mice have demonstrated that intestinal P-glycoprotein plays a causative role in the limited absorption of orally administered paclitaxel. Multidrug resistance (MDR)-reversing agents, such as cyclosporin A and PSC 833, are known to increase the systemic exposure to orally administered paclitaxel by enhancing absorption in the intestinal tract and decreasing elimination via the biliary tract. In this study, we demonstrated that coadministration of the MDR-reversing agent MS-209, which is known to inhibit P-glycoprotein function by direct interaction, improved the bioavailability of orally administered paclitaxel and consequently enhanced its antitumor activity. The pharmacokinetics of paclitaxel were examined by measuring [(3)H]paclitaxel in plasma drawn from rats and mice given the drug with or without MS-209. The influence of MS-209 on the intestinal transport of [(3)H]paclitaxel was studied using a human colorectal cancer cell line, Caco-2. The in vivo efficacy of orally administered paclitaxel in combination with MS-209 was further evaluated in B16 melanoma-bearing mice. The plasma concentration of [(3)H]paclitaxel following oral administration was significantly increased by coadministration of MS-209 at 100 mg/kg in both rats and mice. In rats, the AUC of [(3)H]paclitaxel following oral administration was strikingly increased (1.9-fold) by coadministration of MS-209, whereas the AUC of [(3)H]paclitaxel following i.v. injection was slightly increased (1.3-fold) by MS-209. The increase in apparent bioavailability of oral paclitaxel due to MS-209 was 1.4-fold. To demonstrate this enhancing action in vitro, we studied the influence of MS-209 on the transport of [(3)H]paclitaxel using Caco-2 cells, which is a well-known model of intestinal efflux. The transport of [(3)H]paclitaxel across the Caco-2 monolayer was markedly inhibited in the presence of MS-209, and the apparent K(i)of MS-209 for the active transport of [(3)H]paclitaxel was 0.4 microM. Moreover, paclitaxel administered orally at 100 mg/kg per day with MS-209 at 100 mg/kg per day showed significant antitumor activity in B16 melanoma-bearing mice, whereas paclitaxel administered orally alone at the same dose showed no antitumor activity. These results suggest that the coadministration of MS-209 improved low systemic exposure to paclitaxel through inhibition of P-glycoprotein, which is involved in drug excretion via the intestinal tract, resulting in a clear antitumor activity of paclitaxel administered orally. The present study suggests that coadministration of MS-209 may be a useful way to improve the bioavailability of drugs not suitable for oral administration due to elimination via the intestinal tract.

Continuous-flow fluoro-immunosensor for paclitaxel measurement

A fluorescence-based continuous-flow immunosensor for sensitive, precise, accurate and fast determination of paclitaxel was developed. The sensor utilizes anti-paclitaxel antibody immobilized through its Fc region and crosslinked by dimethylpimelimidate to protein A attached covalently onto the silanized inner walls of a glass capillary column followed by saturation of the paclitaxel-binding sites with rhodamine-labeled paclitaxel. The assay is based on the displacement and detection downstream of the rhodamine-labeled paclitaxel, by a flow-through spectrofluorometer, as a result of the competition with paclitaxel introduced as a pulse into the stream of carrier buffer flowing through the system. The peak height of the fluorescence intensity profile of the displaced rhodamine-labeled paclitaxel was directly proportional to the concentration of paclitaxel applied and was a function of the carrier buffer flow rate. The sensitivity of the immunosensor response ranged from 0.31 relative fluorescence units (RFU)/ng/ml at a flow rate 0.1 ml/min to 0.52 RFU/ng/ml at 1 ml/min, while the lower detection limit ranged from 1 ng/ml at 0.1 ml/min to 4 ng/ml at 1 ml/min. The immunosensor response was very reproducible (RSD=4.8%; n=10) and linear up to 100 ng/ml. The assay time ranged from 2 min at 1 ml/min to 8 min at 0.1 ml/min. A technique developed to resaturate the antigen binding sites of the immobilized antibody with rhodamine-labeled paclitaxel was successful in regenerating the capillary column without affecting its performance, thus enhancing the economic viability of the immunosensor. The immunosensor was successfully applied for the determination of paclitaxel in human plasma.

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC METHODS FOR THE DETERMINATION OF A NOVEL POLYMER-BOUND PACLITAXEL DERIVATIVE AND FREE PACLITAXEL IN HUMAN PLASMA

A high-performance liquid chromatographic (HPLC) assay has been designed for the quantitative determination of polymer-bound paclitaxel (after hydrolytic release of paclitaxel from the polymer) and free paclitaxel in human plasma after intravenous administration of the antitumor polymer-drug conjugate PNU166945. Chemical stabilization with 0.2 M ammonium acetate and solid-phase extraction (SPE) were required as sample pretreatment prior to the HPLC analysis. Separation was performed on an APEX Octyl analytical column and a mobile phase of acetonitrile-methanol-0.02 M ammonium acetate buffer pH 5.0 (4:1:5, v/v/v) and paclitaxel was detected at 227 nm. Total paclitaxel (polymer-bound pus free) levels were determined after chemical hydrolysis of the clinical samples with a mixture of 0.1 M KH2PO4 and methanol (1:1 v/v, pH 7.5) during 48 hours at room temperature. Concentrations of polymer-bound paclitaxel were calculated by subtraction of free from total drug levels. Plasma samples containing paclitaxel were stable for at least 10 months and hydrolyzed plasma samples were stable for at least 3 months at −30°C. Within-run and between-run precisions were less than 10.9% and the accuracy of the assay ranged from 94–102%. The limit of quantification for paclitaxel in plasma was established at 10 ng/mL using a 500 μL sample volume. The presented method was successfully applied in a clinical pharmacokinetic study in our Institute.

Adaptation of solid phase extraction to an automated column switching method for online sample cleanup as the basis of a facile and sensitive high-performance liquid chromatographic assay for paclitaxel in human plasma

An improved method for assaying paclitaxel in human plasma by high-performance liquid chromatography (HPLC) with UV detection at 227 nm has been developed by adapting previously reported sample preparation methods and chromatographic conditions to facilitate semi-automated sample cleanup using a column switching technique. Manual sample manipulations were limited to isolating the drug and internal standard from plasma (1.0 ml) by liquid–liquid extraction using tert-butyl methyl ether. The sample extract was initially loaded onto a short cartridge column containing a cyanopropyl stationary phase. During the predetermined time interval that the drug and internal standard eluted from the cartridge, 1.50–2.20 min, a fully automated 6-position switching valve was used to direct the effluent onto an octylsilica analytical column. The same mobile phase, composed of acetonitrile–methanol–ammonium acetate buffer (pH 5.0; 20 mM) (76:19:105, v/v/v) and delivered at flow rate of 1.0 ml/min, was used for both separations. The overall retention times of paclitaxel and the internal standard were 10.9 and 18.1 min, respectively. The analytical method was thoroughly validated for quantitating paclitaxel in plasma at concentrations ranging from 6 to 586 nM (5–500 ng/ml). The lowest concentration of paclitaxel measured with acceptable day-to-day accuracy (100.2%) and precision (RSD 11.7%, n=21, 5 months) was 6 nM (5 ng/ml). The sensitivity and selectivity of the assay proved to be more than adequate for monitoring steady-state plasma concentrations of the drug when administered to cancer patients as a 96 h continuous intravenous infusion in combination with other anticancer agents, such as doxorubicin and topotecan. Moreover, the heart-cutting procedure prevented the problematic introduction of interfering nonpolar plasma components onto the analytical column, thereby enhancing sample throughput while decreasing the technical demands of the assay. The method was found to be extremely reproducible and robust during extended use for the routine analysis of plasma specimens acquired from several clinical trials.

Determination of Paclitaxel in Human Plasma by HPLC

The aim of this study was to develop and validate an HPLC method for the determination of paclitaxel in plasma from cancer patients in the presence of paclitaxel metabolites and premedication drugs. The HPLC system involved a symmetry C18 column with a mobile phase of methanol, ammonium acetate buffer and isopropanol and UV detection at 230 nm. Paclitaxel and docetaxel were well separated and resolved within a run time of 25 min. Cyanopropyl solid-phase extraction (SPE) cartridges were used in the extraction procedure. The proposed method was successfully used to determine the concentration of paclitaxel in plasma from a cancer patient.