Anal. Chem. 2005, 77, 4677-4683
Development and Validation of a Liquid Chromatography-Tandem Mass Spectrometry Assay for the Quantification of Docetaxel and Paclitaxel in Human Plasma and Oral Fluid Kjell A. Mortier,† Vincent Renard,‡ Alain G. Verstraete,§ Annie Van Gussem,‡ Simon Van Belle,‡ and Willy E. Lambert*,†
Laboratory of Toxicology, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium, and Department of Clinical Oncology and Department of Clinical Biology, Ghent University Hospital, De Pintelaan 185, B-9000 Ghent, Belgium
A quantitative method for the simultaneous determination of docetaxel (Taxotere), paclitaxel (Taxol), 6r-hydroxypaclitaxel, and p-3′-hydroxypaclitaxel in human plasma and oral fluid is developed and validated. Oral fluid (this term is now preferred to saliva) was sampled with a Salivette collection device. The procedure used a simple liquid/liquid extraction with methyl tert-butyl ether followed by LC-ESI-MS/MS. Gradient elution was applied and provided increased robustness to ion suppression by the drug formulation vehicle (polysorbate 80 and Cremophor EL). Adduct ion formation with sodium and potassium was noticed and controlled by mobile-phase optimization. The protonated analytes generated in the positive ion mode were monitored through multiple reaction monitoring. Calibration was performed by internal standardization with cephalomannine, and regression curves were constructed ranging between 2 and 1000 ng/mL in plasma and 0.125 and 62.5 ng/mL in oral fluid, using a weighing factor of 1/x2. The regression curves were quadratic for paclitaxel and docetaxel and linear for the paclitaxel metabolites. Accuracy varied from 91.3 to 103.6%, and imprecision did not exceed 12.7% for all analytes in plasma and oral fluid. In conclusion, a sensitive and robust method was obtained, which fulfilled all validation criteria. The taxanes docetaxel (Taxotere) and paclitaxel (Taxol) are used in the treatment of cancer (structures, see Figure 1). The naturally occurring paclitaxel was first isolated from the bark of the Pacific yew tree (Taxus brevifolia) in the 1960s and gained commercial approval in December 1992. Docetaxel was first synthesized starting from 10-deacetyl baccatin III, a nontoxic precursor found in the European yew (Taxus baccata) in 1986.1 Today, these drugs have contributed significantly to the treatment of a variety of malignancies, such as ovarian, breast, and non small * To whom correspondence should be addressed. Phone: 32 9 264 81 35. Fax: 32 9 264 81 83. E-mail:
[email protected]. † Ghent University. ‡ Department of Clinical Oncology, Ghent University Hospital. § Department of Clinical Biology, Ghent University Hospital. (1) Cortes, J. E.; Pazdur, R. J. Clin. Oncol. 1995, 13, 2643-2655. 10.1021/ac0500941 CCC: $30.25 Published on Web 06/01/2005
© 2005 American Chemical Society
Figure 1. Structures of paclitaxel, docetaxel, and cephalomannine. Hydroxylation sites of paclitaxel metabolites are indicated with their respective numbers.
cell lung cancers, as well as head and neck cancer and some cancers of the digestive system.2 Despite the major benefits of these products, patients receiving chemotherapeutic treatment can experience severe to lifethreatening side effects, primarily myelosuppression leading to neutropenia. On the other hand, underdosage might result in suboptimal treatment of the cancer. In addition to their narrow therapeutic range, these substances also display highly variable pharmacokinetics. Traditionally, the dosing of anticancer agents is calculated on the basis of the patient’s body surface area. It has been suggested that pharmacokinetically guided chemotherapy and dose individualization might lead to a better treatment outcome. Although this subject is still under discussion, it is clear that further clinical studies are necessary to reveal the optimal treatment schedule.3,4 For this purpose, validated analytical methods for the quantification of these compounds in plasma are a necessity. In plasma, paclitaxel and docetaxel are highly bound (2) Dubois, J.; Guenard, D.; Gueritte, F. Expert Opin. Ther. Pat. 2003, 13, 1809-1823. (3) Smorenburg, C. H.; Sparreboom, A.; Bontenbal, M.; Stoter, G.; Nooter, K.; Verweij, J. J. Clin. Oncol. 2003, 21, 197-202. (4) Egorin, M. J. J. Clin. Oncol. 2003, 21, 182-183.
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to proteins, with free fractions generally lower than 10%. This free, active, fraction is better related to the pharmacological and/or toxic effect, and measuring free fractions could therefore be superior to total plasma concentration. The determination of free fractions is however complicated and time-consuming, limiting its use in clinical practice. Oral fluid as a therapeutic drug monitoring matrix offers some interesting opportunities. This matrix can be viewed as a natural ultrafiltrate of plasma, and oral fluid concentrations often correlate to free drug levels in plasma. In addition, administration of taxanes is based on short infusion duration, and patients are often not hospitalized. Under these conditions, monitoring plasma concentrations, which requires blood sampling by medical personnel, is laborious and increases stress on patients and health care workers. The collection of oral fluid, with a collection device, could be performed by the patient, thus not requiring medical personnel or a hospital visit. To investigate the correlation between oral fluid and plasma concentrations, a method for the quantification of paclitaxel and docetaxel in oral fluid was developed. In the past, methods have already been developed to monitor docetaxel or paclitaxel concentrations in plasma or serum. Earlier methods using liquid chromatography with ultraviolet detection suffered from limited sensitivity and selectivity, due to their relatively low UV absorbance and nonselective UV maximum (227 nm). As a consequence, methods based on liquid chromatography coupled to mass spectrometry (LC-MS) were developed. Most published methods report the quantification of either docetaxel5-9 or paclitaxel.10-14 The simultaneous analysis was reported by only one paper.15 Most methods use isocratic elution, which minimizes the total run time but does not provide a column wash. In addition, previous methods did not evaluate ion suppression by the drug formulation vehicle. In a recent paper, our group reported ion suppression by the drug formulation of docetaxel (Tween 80) and paclitaxel (Cremophor EL) due to carryover in subsequent runs with an isocratic LC elution.16 To monitor paclitaxel and docetaxel levels in patient samples, a new, robust method needed to be developed, devoid of matrix effect. The development, optimization, validation, and evaluation of this new method are reported in the (5) Gustafson, D. L.; Long, M. E.; Zirrolli, J. A.; Duncan, M. W.; Holden, S. N.; Pierson, A. S.; Eckhardt, S. G. Cancer Chemother. Pharmacol. 2003, 52, 159-166. (6) Grozav, A. G.; Hutson, T. E.; Zhou, X.; Bukowski, R. M.; Ganapathi, R.; Xu, Y. J. Pharm. Biomed. Anal. 2004, 36, 125-131. (7) Hou, W. Y.; Watters, J. W.; McLeod, H. L. J. Chromatogr., B 2004, 804, 263-267. (8) Baker, S. D.; Zhao, M.; He, P.; Carducci, M. A.; Verweij, J.; Sparreboom, A. Anal. Biochem. 2004, 324, 276-284. (9) Wang, L. Z.; Goh, B. C.; Grigg, M. E.; Lee, S. C.; Khoo, Y. M.; Lee, H. S. J. Am. Soc. Mass Spectrom. 2003, 17, 1548-1552. (10) Alexander, M. S.; Kiser, M. M.; Culley, T.; Kern, J. R.; Dolan, J. W.; McChesney, J. D.; Zygmunt, J.; Bannister, S. J. J. Chromatogr., B 2003, 785, 253-261. (11) Guo, P.; Ma, J. G.; Li, S. L.; Gallo, J. M. J. Chromatogr., B 2003, 798, 7986. (12) Basileo, G.; Breda, M.; Fonte, G.; Pisano, R.; James, C. A. J. Pharm. Biomed. Anal. 2003, 32, 591-600. (13) Schellen, A.; Ooms, B.; van Gils, M.; Halmingh, O.; van der Vlis, E.; van de Lagemaat, D.; Verheij, E. J. Am. Soc. Mass Spectrom. 2000, 14, 230-233. (14) Sottani, C.; Minoia, C.; D’Incalci, M.; Paganini, M.; Zucchetti, M. J. Am. Soc. Mass Spectrom. 1998, 12, 251-255. (15) Parise, R. A.; Ramanathan, R. K.; Zamboni, W. C.; Egorin, M. J. J. Chromatogr., B 2003, 783, 231-236. (16) Mortier, K. A.; Verstraete, A. G.; Zhang, G. F.; Lambert, W. E. J. Chromatogr., A 2004, 1041, 235-238.
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present work. Determination of taxane concentrations in oral fluid has been reported by only one paper. By the development of an enzyme-linked immunosorbent assay (ELISA), paclitaxel has been demonstrated in oral fluid at low levels.17 The chromatographic analysis of paclitaxel or docetaxel in oral fluid has not yet been described. As oral fluid concentrations reflect the free concentration in blood, the anticipated concentrations are much lower than the total plasma concentration, further emphasizing the need for high sensitivity. This work combines the development and validation of the first method for the measurement of taxanes in oral fluid and plasma. Careful validation demonstrated that matrix suppression was controlled. High sensitivity was achieved through optimization of the chromatographic as well as the mass spectrometric parameters. The developed method will allow us to further study the usefulness of oral fluid for monitoring taxane chemotherapy. EXPERIMENTAL SECTION Instrumentation. All experiments were carried out on an API 4000 triple quadrupole mass spectrometer from Applied Biosystems (Foster City, CA) equipped with Turbo V source and TurboIonspray interface. The system was calibrated by continuous infusion of a mixture of poly(ethylene glycol)s, provided by the manufacturer. Ultrapure nitrogen was used as nebulization, curtain, and collision gas. The HPLC consisted of a fully equipped Agilent 1100 configuration (Agilent Technologies, Palo Alto, CA) with a temperature-controlled autosampler and column oven. The data were processed using Analist 1.4 software, and Microsoft Excel was used for further processing, if necessary. Evaporation under nitrogen was conducted in a TurboVap LV evaporator from Zymark (Hopkinton, MA). Chemicals and Reagents. Paclitaxel, docetaxel, Cremophor EL, and Tween 80 were purchased from Sigma (Bornem, Belgium). Cephalomannine (internal standard) was purchased from Hauser, Inc. (Denver, CO). Paclitaxel metabolites p-3′hydroxypaclitaxel and 6R-hydroxypaclitaxel were purchased from BD Biosciences (Erembodegem, Belgium). Methyl tert-butyl ether, ammonium acetate, and acetic acid were obtained from VWR (Leuven, Belgium). Methanol and water of LC-MS grade were obtained from Biosolve (Valkenswaard, The Netherlands). Oral fluid collection devices (Salivette) consisting of a cotton wool swab and a container for saliva collection by centrifugation were purchased from Sarstedt (Essen, Belgium). Drug free oral fluid was obtained from healthy volunteers. Drug-free EDTA plasma was obtained from the Ghent University Hospital and a healthy volunteer. Plasma samples were harvested after a 10-min centrifugation period at 1200g. Preparation of Standard Solutions and Calibrators. Individual stock solutions of paclitaxel, docetaxel, and cephalomannine (IS) were prepared by accurately weighing 10 mg and dissolving in 10 mL of 0.1% acetic acid in methanol (the dilution solvent). For the paclitaxel metabolites, the total amount of the standard powder (less than 25 µg) was dissolved in 1 mL of dilution solvent, of which the concentration was determined by UV absorbance (molar extinction coefficients are 26.2 and 35.8 mM-1 cm-1 for 6R-hydroxypaclitaxel and p-3′-hydroxypaclitaxel, respectively). (17) Svojanovsky, S. R.; Egodage, K. L.; Wu, J.; Slavik, M.; Wilson, G. S. J. Pharm. Biomed. Anal. 1999, 20, 549-555.
Table 1. LC-ESI-MS/MS Parametersa
a
compound
MRM transition (m/z)
DP (V)
CE (V)
Rt (min)
variation Rt (CV%, n ) 20)
docetaxel paclitaxel 6R-OH-pac p-3′-OH-pac cephalomannine (IS)
808.4 > 226.1, 526.9 854.4 > 509.3, 569.0 870.4 > 286.0, 525.3 870.4 > 302.2, 509.3 832.4 > 264.1
60 60 60 60 60
17, 15 17, 17 17, 15 20, 23 23
3.5 3.2 2.5 1.5 2.9
0.29 0.78 1.13 1.32 0.48
DP, declustering potential; CE, collision energy.
Acetic acid was added to the dilution solvent to improve the stability of the compounds as has been demonstrated by others.18 Stock solutions were stored in the dark at -20 °C. Working solutions (separate mixtures of paclitaxel/docetaxel and paclitaxel metabolites) were prepared with dilution solvent to obtain 10, 25, 100, 250, 1000, and 2500 ng/mL for paclitaxel and docetaxel and 5, 10, 25, 50, 100, and 250 ng/mL for p-3′-hydroxypaclitaxel and 6R-hydroxypaclitaxel. Dilution factors were calculated by weighing the amount of standard solution and dilution solvent added. The internal standard cephalomannine was diluted to a concentration of 100 ng/mL with the dilution solvent. For the preparation of plasma sample calibrators, 500 µL of water, 100 µL of working solutions, and 100 µL of internal standard working solution were added to 250 µL of blank plasma, resulting in calibrator levels at 4, 10, 40, 100, 400, and 1000 ng/mL for docetaxel and paclitaxel and 2, 4, 10, 20, 40, and 100 ng/mL for paclitaxel metabolites. These calibration levels reflect clinical concentrations found in plasma of patients receiving chemotherapeutic treatment.5,19 For oral fluid sample calibrators, 50 µL of the working standard solutions and 50 µL of the internal standard working solution were added to 2.0 mL of blank oral fluid, resulting in the following levels: 0.25, 0.625, 2.5, 6.25, 25, and 62.5 ng/mL for docetaxel and paclitaxel and 0.125, 0.25, 0.625, 1.25, 2.5, and 6.25 ng/mL for the paclitaxel metabolites. Cremophor EL and Tween 80 were dissolved in methanol/water (50/50 by volume) and further diluted with water to obtain concentrations of 0.5, 1, 2, 4, and 8 mg/mL for Cremophor EL and of 10, 20, 50, 150, and 300 µg/mL for Tween 80. Sample Preparation. For the preparation of plasma samples, 100 µL of the internal standard working solution and 0.5 mL of water were added to 250 µL of plasma. A liquid/liquid extraction with 3 mL of methyl tert-butyl ether was performed by placing the samples on a rotary device for 10 min. After centrifugation at 1500g for 5 min, the upper organic layer was transferred to a conical test tube and evaporated to dryness under nitrogen at 30 °C. The residue was redissolved in 1 mL of a mixture of water/ methanol/acetic acid (50/50/0.1, by volume) of which a 20-µL aliquot was injected in the LC-MS/MS system. For oral fluid samples, the Salivette collection device was centrifuged at 2000g for 8 min to obtain the oral fluid. To increase the recovery of analyte from the cotton swab, 1.5 mL of methyl tert-butyl ether was added to the centrifuged swab and the device was centrifuged again. The obtained oral fluid (determined by (18) Richheimer, S. L.; Tinnermeier, D. M.; Timmons, D. W. Anal. Chem. 1992, 64, 2323-2326. (19) Sparreboom A., de Bruijn, P.; Nooter, K.; Loos, W. J.; Stoter, G.; Verweij, J. J. Chromatogr., B 1998, 705, 159-164.
weighing, volume adjusted to 2.0 mL), the washing fluid, 3 mL of methyl tert-butyl ether, and 50 µL of internal standard working solution were collected in a test tube and placed on a rotary device for 10 min. After centrifugation (1500g, 5 min) and transfer to a conical test tube, the organic layer was evaporated to dryness under nitrogen and the residue was redissolved in 0.5 mL of a mixture of water/methanol/acetic acid (50/50/0.1, by volume) of which a 20-µL aliquot was injected into the LC-MS/MS system. Liquid Chromatography. Chromatographic separation was achieved on a Merck Purospher Star, RP-18 column (55 × 2.0 mm, 3-µm particle size) from VWR (Leuven, Belgium). The column oven was maintained at 25 °C. The samples were placed in the autosampler at a temperature of 10 °C, protected from light. Gradient elution with (A) 2 mM acetic acid/0.2 mM ammonium acetate in water and (B) 2 mM acetic acid/0.2 mM ammonium acetate in methanol at a flow rate of 0.4 mL/min, was applied. A linear gradient ran from 63 to 73% B in 1.8 min followed by a steeper gradient to 95% B in 0.2 min. This condition was maintained for 3 min to remove late-eluting substances from the column (column wash) after which the system returned to the initial conditions for a 6-min equilibration period. Hence, the total analysis time including column wash and equilibration was 11 min. Mass Spectrometry. Optimization of the mass spectrometric signal of all analytes was performed by continuous infusion, using the automatic quantitative optimization function provided by the manufacturer. Further optimization was performed with flow injection optimization, also controlled automatically by the software. Highest signal intensities were obtained with TurboIonspray (TIS) or pneumatically assisted electrospray in the positive ion mode. The following TIS settings were applied: nebulizer gas, 60 psi; turbo gas, 40 psi; curtain gas, 20 psi; temperature, 425 °C; declustering potential, 60 V; needle voltage, +5.5 kV. The signal of analytes and internal standard (IS) was monitored through multiple reaction monitoring (MRM). A complete overview of the MS/MS transitions, collision energy voltages, and retention times is compiled in Table 1. By evaluating the purity of the individual standards, no cross-talk was observed. Each transition was monitored with a 100-ms dwell time. Method Validation. The method was validated according to the guidelines on bioanalytical method validation, set by the Food and Drug Administration.20 The following validation parameters were evaluated: selectivity, accuracy, intra- and interbatch precision, recovery, matrix effect, stability, and linearity. (20) U.S. Department of Health and Human Services Food and Drug Administration-Center for Drug Evaluation and Research (CDER), Guidance for Industry, Bioanalytical Method Validation, 2001.
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Selectivity, defined as the ability of an analytical method to differentiate and quantify the analyte in the presence of other components in the sample, was evaluated by analyzing two sets of fortified blank samples at the lower limit of quantitation (LLOQ). The first set (n ) 3) contained only the target analytes, while the second set (n ) 3) contained target analytes as well as probable concomitant medication (dexamethasone, dimetindene, ranitidine, methylprednisolone, ondansetron, carboplatin, alizapride) at a therapeutic concentration. In addition, six blank samples from different individuals, and one zero sample (IS added) were analyzed. Accuracy, recovery, and precision were evaluated at three concentration levels, prepared by separate weighing from the stock solution, equally distributed over the calibration range (QC1, QC2, QC3: representing the LLOQ, a medium concentration equivalent to point four of the calibration curve, and the upper limit of quantitation, respectively). All validation experiments were performed with the blank matrix from different individuals, instead of using a blank matrix pool. Accuracy was determined by comparison of the mean result of five analyses to the nominal concentration. The result is expressed as the ratio (expressed in percentage) of the concentration calculated by regression analysis to the nominal concentration. Intrabatch precision was assessed by analyzing five samples, prepared by spiking blank samples, in the same batch, whereas interbatch precision reflects the precision when samples were prepared and analyzed on five different days. Recovery was measured by comparing the response of the respective analytes, spiked before and after sample preparation. Matrix effect (and ion suppression by the drug formulation) was assessed as follows: to the blank matrix from five different individuals, analytes were added at three concentrations (QC1, QC2, QC3), making a total of 15 samples. To each sample with equal analyte concentration, a different amount of drug formulation (0.5 mL of the drug formulation dilutions) was added. These samples were subjected to the analytical procedure and compared to samples not containing the drug formulation regarding precision and accuracy. Analyte stability determinations comprised freeze-and-thaw cycle stability (3 cycles), short-term temperature stability, longterm stability, autosampler stability, and stock solution stability. For evaluation of linearity, six-point calibration curves were constructed in quintuplicate. Calibrator samples were fortified blank matrix samples and were treated in a way similar to the unknowns. Regression type and most appropriate weighing factor were determined. The percent relative error (%RE), which is the regressed concentration minus the nominal standard concentration divided by the nominal standard concentration, was calculated. For evaluation, this %RE was plotted versus concentration.21 In addition, the sum of the squares of the %RE of all data points for a given curve estimation was calculated, to facilitate comparison. Calibration, using internal standardization, was done by calculation of the peak area ratios. Safety Considerations. When handling paclitaxel, docetaxel, or cephalomannine, gloves were always used. Docetaxel-, paclitaxel-, or cephalomannine-contaminated waste was subjected to a degradation procedure with sodium hypochlorite prior to disposal.
No further specific safety precautions are demanded. Universal precautions for the handling of chemicals and biofluids were applied.
(21) Almeida, A. M.; Castel-Branco, M. M.; Falcao, A. C. J. Chromatogr., B 2002, 774, 215-222.
(22) Mortier, K. A.; Zhang, G. F.; Van Peteghem, C. H.; Lambert, W. E. J. Am. Soc. Mass Spectrom. 2004, 15, 585-592.
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RESULTS AND DISCUSSION Method Development. During initial infusion experiments with docetaxel and paclitaxel, the presence of sodium adduct ions instead of protonated molecules [M + H]+ was noted. Additives such as acetic acid and ammonium acetate helped to promote the formation of [M + H]+. To enhance the reproducible formation of [M + H]+ and sensitivity, solvent type (methanol versus acetonitrile) and additives (ammonium acetate, ammonium formate, acetic acid, formic acid) in various concentrations and ratios were tested, with APCI as well as ESI. The use of additives proved to be a necessity to control the reproducibility, as was demonstrated in earlier work.22 Optimal sensitivity was achieved by the addition of 2 mM acetic acid/0.2 mM ammonium acetate and methanol as organic solvent. Although the amount of added ammonium acetate is low compared to the acetic acid level and has little effect on the pH, it resulted in a nearly 2-fold increase in sensitivity. The underlying mechanism of this phenomenon is presumably the additional generation of an intermediate ion [M + NH4]+, which may dissociate into [PAC + H]+ and ammonia during the electrospray process. Precursor and product ions for each analyte of interest were determined by direct infusion. Two product ions were monitored for each analyte, so that the product ion ratios could be used for confirmation of the identity (Table 1). Oral fluid was sampled with a collection device. The main advantages for this are ease of use and good hygienic circumstances. The Salivette device uses a cotton wool swab to obtain a clear fluid sample. As taxanes are hydrophobic, adsorption on the cotton swab was evaluated. Recoveries of the target compounds from the device ranged from 42.8 to 58.3%, indicating pronounced adsorption on the device. When the device was washed afterward with 1.5 mL of methyl tert-butyl ether, recoveries increased to acceptable values ranging from 84.5 to 103.4%, evaluated at three concentrations with 5-fold replication (data not shown). Therefore, this wash step was incorporated in the method. As a result of this procedure, the entire sample was used for extraction, and volume determination was assessed by weighing the Salivette device before and after sampling (101.4% accurate with CV of 0.13%, n ) 6). The marginal differences in relative density between water and the individual oral fluid samples were not taken into account for further calculations. Choosing the appropriate internal standard is an important aspect to achieve acceptable method performance, especially with LC-MS/MS, where matrix effects can lead to poor analytical results. Others used docetaxel as internal standard in paclitaxel assays or vice versa, which has the drawback that method validation and standard dilution preparation require more handling if quantification of both compounds is necessary. Ideally, isotopically labeled internal standards for all analytes should be used, but these are not commercially available. Therefore, we opted for cephalomannine, a natural compound with a taxoid structure found in the needles of T. baccata and commercially available. In
Table 2. Accuracy, Precision, and Recovery Data for Plasma and Oral Fluid Samples (n ) 5) plasma
oral fluid
docetaxel
paclitaxel
6R-OH-pac
docetaxel
paclitaxel
6R-OH-pac
p′3-OH-pac
QC1 QC2 QC3
98.9 98.5 96.9
101.2 98.6 100.2
93.3 96.4 100.9
Accuracy 103.6 93.9 99.9
97.8 94.9 98.9
91.3 96.4 99.0
99.6 98.8 98.3
103.2 99.3 98.7
QC1 QC2 QC3
4.1 3.6 5.7
7.1 6.3 11.4
8.1 4.1 7.0
Intrabatch Precision 10.8 3.2 5.9
2.9 7.3 3.2
4.2 6.1 8.1
3.4 3.0 3.1
9.3 4.7 3.3
QC1 QC2 QC3
7.5 2.2 7.9
4.8 5.8 6.7
6.4 6.3 10.8
Interbatch Precision 12.1 6.5 10.3
9.3 3.4 2.5
10.2 4.6 3.0
12.7 1.6 2.4
11.5 4.2 2.6
QC1 QC2 QC3
96.2 92.7 81.7
69.9 88.7 81.9
91.9 86.2 76.8
Recovery 87.0 85.6 101.0
94.9 90.0 93.1
92.8 86.4 87.4
86.0 86.8 89.6
99.1 89.0 90.1
QC1 QC2 QC3
101.5 98.6 95.3
96.0 96.5 97.0
96.7 100.7 90.2
Accuracy with Excipient 85.2 96.8 100.2 102.2 92.9 101.6
92.5 101.7 102.2
90.7 100.8 92.4
92.4 92.6 91.2
QC1 QC2 QC3
7.6 5.9 6.9
8.1 6.2 6.9
7.3 6.5 5.8
1.1 0.7 2.0
4.7 1.9 1.2
8.4 1.3 2.2
p′-3-OH-pac
Intrabatch Precision with Excipient 9.6 7.1 6.3 0.7 5.1 1.3
Table 3. Stability Data (% of Initial Value) at Low and High Concentration (n ) 3)a plasma
oral fluid
docetaxel
paclitaxel
6R-OH-pac
paclitaxel
6R-OH-pac
p′-3-OH-pac
low high
97.7 96.6
90.8 93.0
100.4 103.1
Freeze-Thaw Stability (3 Cycles) 74.9 87.3 74.8 87.4
92.5 91.9
98.0 104.2
104.7 88.7
low high
104.3 105.0
99.3 105.3
Short-Term Stability (5 h, Room Temperature)) 103.1 103.3 99.1 107.5 97.2 99.3
99.9 101.1
99.6 97.2
107.0 95.5
low high
108.2 107.2
107.0 109.0
Long-Term Stability (1 Month, -20 °C) 107.2 111.2 95.0 108.5 105.2 95.5
98.2 97.4
99.3 102.3
108.3 94.2
low high
101.8 104.0
94.1 105.4
99.1 103.0
102.1 99.9
97.8 100.7
106.2 100.6
p′-3-OH-pac
docetaxel
Autosampler Stability (5 h) 95.9 97.1 104.6 98.9
a Plasma low and high concentration: 10 and 100 ng/mL for paclitaxel and docetaxel and 5 and 20 ng/mL for paclitaxel metabolites. Oral fluid low and high concentration: 1 and 10 ng/mL for paclitaxel and docetaxel and 0.5 and 5 ng/mL for paclitaxel metabolites.
addition, cephalomannine is not used for administration to patients and its retention behavior is similar to that of the target analytes. Validation. Selectivity was demonstrated at the LLOQ for both plasma and saliva. A t-test could not demonstrate a significant difference (p ) 0.05) in peak area ratio between samples spiked with concomitant medication and samples not containing these drugs. The chromatogram obtained from the analysis of six blank samples from different origin and a zero sample did not show any peaks. Accuracy, expressed as the ratio of the calculated to the nominal concentration (%), was evaluated at three concentrations: QC1, QC2, and QC3. The accuracy ranged from 93.3 to 103.6% in plasma and from 91.3 to 103.2% in oral fluid. These results fall within the limits set by the guidelines, where deviations up to 20% at the LLOQ and 15% at other levels are allowed. Precision was also evaluated at QC1, QC2, and QC3, within a batch,
or on different batches on consecutive days. The precision data for both plasma and oral fluid never exceeded 12.7%, which is less than the widely accepted limit of 15% RSD. Recovery of the sample preparation was also determined at three concentrations. For plasma samples, recovery ranged from 69.9 to 101.0%, whereas for oral fluid, recoveries varied between 86.4 and 99.1% (Table 2). The importance of including the evaluation of matrix effect in any LC-MS/MS method is outlined in an excellent paper by Matuszewski and co-workers.23 Their data strongly emphasize the need to use a blank matrix from (at least five) different sources/ individuals instead of using one blank matrix pool to determine method precision and accuracy. Therefore, all validation experiments in this assay were performed with matrixes obtained from different individuals. As all data fall within the guidelines, we (23) Matuszewski, B. K.; Constanzer, M. L.; Chavez-Eng, C. M. Anal. Chem. 2003, 75, 3019-3030.
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Figure 2. Chromatogram of matrix-matched calibration plasma sample at LLOQ. 1, p-3′-hydroxypaclitaxel; 2, 6R-hydroxypaclitaxel; 3, IS, cephalomannine; 4, paclitaxel; 5, docetaxel; Y-scale normalized to the paclitaxel peak intensity. Table 4. Calibration Curve Characteristics compound
calibration range (ng/mL)
equation
weighing factor
R
1/x 1/x 1/x 1/x
0.999 0.999 0.997 0.997
1/x 1/x 1/x 1/x
0.999 0.999 0.998 0.997
docetaxel paclitaxel 6R-OH-paclitaxel p-3′-OH-paclitaxel
4-1000 4-1000 2-100 2-100
Plasma Y ) -3.5 × 10-7X + 0.013X + 0.0156 Y ) -4.6 × 10-7X + 0.016X + 0.0039 Y ) 0.00817X - 0.0016 Y ) 0.00436X + 0.0002
docetaxel paclitaxel 6R-OH-paclitaxel p-3′-OH-paclitaxel
0.25-62.5 0.25-62.5 0.125-6.25 0.125-6.25
Oral Fluid Y ) -1.9 × 10-7X + 0.009X + 0.0042 Y ) -2.1 × 10-7X + 0.010X + 0.0235 Y ) 0.00455X - 0.00066 Y ) 0.00238X - 0.00040
conclude that the degree of matrix effect was sufficiently low to produce acceptable data, and the method can be considered as valid. Another important, but rarely explored, aspect of matrix effect in bioanalytical drug analysis is suppression by the drug formulation (vehicle). These vehicles are normally not present in a blank matrix and thus are omitted in a typical method validation. However, they can be present in patient plasma at high levels, and suppression has been reported for PEG 400.24 The dosing vehicle of commercially available paclitaxel for infusion (Taxol) contains Cremophor EL, which is castor oil reacted with ethylene oxide, and it remains at high concentrations in the plasma after infusion.25 The typical dosing vehicle for docetaxel (Taxotere), Tween 80, or polysorbate 80 (major component: polyoxyethylene-20-sorbitan monooleate), is also detected in plasma.25 For the method to be valid, we considered it to be essential to examine whether the analytes could be accurately quantified in the presence of their dosing vehicle. Already during method development it was noticed that an
isocratic LC method suffered from matrix effects due to late elution of the vehicle in subsequent runs.16 In an effort to solve this carryover problem, a gradient run with a column wash was introduced. Analysis of samples without vehicle or with different amounts of these vehicles revealed comparable area ratios and RSD (see Table 2, accuracy and intrabatch precision). Abraxane, paclitaxel-protein-bound particles for injectable suspension is another approved taxane formulation. The method was not developed or validated for the evaluation of paclitaxel concentrations after Abraxane administration, and thus, additional validation experiments should be performed prior to analysis of these samples. Analyte stability was assessed at a low and a high concentration in plasma and oral fluid (Table 3). All stability data fall within a 15% deviation range, except for the freeze-thaw experiment with p′-3-hydroxypaclitaxel in plasma showing 25% degradation. As a result, samples were kept frozen at -20 °C until analysis. Stock solutions remained stable for at least one month (data not shown).
(24) Schuhmacher, J.; Zimmer, D.; Tesche, F.; Pickard, V. Rapid Commun. Mass Spectrom. 2003, 17, 1950-1957.
(25) ten Tije, A. J.; Verweij, J.; Loos, W. J.; Sparreboom, A. Clin. Pharmacokinet. 2003, 42, 665-685
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the electrospray process at high concentrations. Although deviations from linearity were small, as can be derived from the low quadratic slope representing the bending of the curve, and acceptable data could be generated with linear curves, quadratic regression resulted in a lower %RE. The incorporation of a weighing factor 1/x2 further ameliorated the regression result. The method will be applied in a larger clinical study to evaluate the correlation between oral fluid and plasma concentrations of these drugs. A set of results is presented in Figure 3 displaying the concentration - time profile of individuals following administration of Taxol (Figure 3a, data for plasma) and of Taxotere (Figure 3b, data for oral fluid). The method fulfills all typical validation criteria, including the assessment of adduct formation and matrix effects, after the introduction of LC gradient elution. In addition, the very high sensitivity of the presented method offers possibilities for the determination of the unbound fraction.
Figure 3. Concentration-time profile (a) in plasma of human subject following administration of a 3-h infusion of Taxol (175 mg/m2) and (b) in oral fluid of a human subject following administration of a 1-h infusion of Taxotere (100 mg/m2).
A typical chromatogram obtained from the analysis of a calibrator plasma sample at the LLOQ is displayed in Figure 2. The calibration curve equation was estimated by calculating the percent relative error at each calibrator level. Quadratic calibration curves were obtained for paclitaxel and docetaxel, while for the paclitaxel metabolites, a linear regression provided better results (Table 4). The reason for this discrepancy can be found in the different concentration range used for the metabolites (factor 50) and the main compounds (factor 250), and in the saturation of
CONCLUSION A method for the determination of docetaxel, paclitaxel, and its metabolites in human plasma and oral fluid has been developed with LC-ESI-MS/MS. Comprehensive method development with optimization of the amounts and the nature of the additives led to a highly sensitive assay. This was a prerequisite for the monitoring of taxanes in oral fluid, present only at low levels similar to the free fraction in plasma. This is the first chromatographic method monitoring taxanes in oral fluid ever reported. Adduct formation was controlled through the optimization of the mobile phase. Validation was successfully performed: precision, accuracy, recovery, and calibration curves fulfilled analytical validation criteria. A gradient LC method was developed to avoid carryover and suppression by the drug formulation. In future work, the method will enable us to evaluate the correlation between plasma and oral fluid concentrations in a clinical study. Received for review January 17, 2005. Accepted April 26, 2005. AC0500941
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