Abstract
We have developed a high performance liquid chromatography mass spectrometry method for quantitating paclitaxel and its 6-alpha-OH and 3-para-OH metabolites in 0.1 mL human plasma. After MTBE liquid-liquid extraction, chromatographic separation was achieved with a Phenomenex synergy polar reverse phase (4 μm, 2 mm × 50 mm) column and a gradient of 0.1% formic acid in acetonitrile and water over an 8 min run time. Mass spectrometric detection was performed on an ABI SCIEX 4000Q with electrospray, positive-mode ionization. The assay was linear from 10–10,000 ng/mL for paclitaxel and 1–1,000 ng/mL for both metabolites and proved to be accurate (94.3–110.4%) and precise (<11.3%CV). Recovery from plasma was 59.3–91.3% and matrix effect was negligible (−3.5 to 6.2%). Plasma freeze thaw stability (90.2–107.0%), stability for 37 months at −80 °C (89.4–112.6%), and stability for 4 h at room temperature (87.7–100.0%) were all acceptable. This assay will be an essential tool to further define the metabolism and pharmacology of paclitaxel and metabolites in the clinical setting. The assay may be utilized for therapeutic drug monitoring of paclitaxel and may also reveal the CYP2C8 and CYP3A4 activity phenotype of patients.
Keywords: paclitaxel, tandem mass spectrometry, metabolites, assay, validation
Graphical abstract
1. Introduction
Paclitaxel (PTX) is a widely used cytotoxic with efficacy in ovarian, breast, and lung cancer, and Kaposi’s sarcoma [1–3]. The pharmacokinetics are non-linear, and clearance is mostly dependent on metabolism, with CYP2C8 producing the 6-α-hydroxy PTX metabolite (6-α-OH- PTX), and CYP3A4 producing the 3-para-hydroxy PTX metabolite (3-p-OH-PTX) [4–6], see Fig.1.
Fig. 1.
Structures of PTX and its metabolites 3-p-OH-PTX (through CYP3A4), and 6-α-OH-PTX (through CYP2C8)[5, 6].
Previously reported assays for PTX and metabolites have been developed, yet the concentration ranges commonly do not cover the clinically relevant range, see Suppl.Table 4.
To support the clinical development of a combination regimen of PTX in the organ dysfunction setting (ClinicalTrials.gov Identifier: NCT01366144), we developed and validated an LC-MS/MS assay to quantitate PTX and its metabolites 6-α-OH-PTX, and 3-p-OH-PTX in human plasma.
2. Experimental
2.1. Chemicals and reagents
PTX (99.5% purity) was purchased from LC Labs, (Woburn, MA 01801), [D5]-6-α-OH-PTX (IS for both metabolites), and 3-p-OH PTX (95.0% and 98.0% purity, respectively) were purchased from Toronto Research Chemicals, (Ontario, Canada), while [13C6]-PTX, and 6-α-OH-PTX (99.0% and 98.0% purity, respectively) were purchased from ALSACHIM (Graffenstaden, France) Acetonitrile, Methanol, and water (all HPLC grade) were purchased from Fisher Scientific (Fairlawn, NJ, USA). Formic acid was purchased from Sigma-Aldrich (St. Louis, MO, USA). Methyl-tert-butyl ether was purchased from VWR Scientific Products, (West Chester, PA, USA). Control heparinized human plasma was purchased from Sera Care (Milford, MA, USA). Nitrogen for evaporation of samples was purchased from Valley National Gases, Inc. (Pittsburgh, PA, USA). Nitrogen for mass spectrometric applications was purified with a Nitrogen Generator (Parker Balston, Haverhill, MA, USA).
2.2. Chromatography
The LC system consisted of an Agilent (Palo Alto, CA, USA) 1200 SL autosampler and binary pump, a Phenomenex (Torrance, CA USA) Synergi Polar-RP 80Å (4 μm, 50 × 2 mm) column, and a gradient mobile phase with a gradient delay volume of 320 μL. Mobile phase solvent A was 0.1% (v/v) formic acid in water, and mobile phase solvent B was 0.1 % (v/v) formic acid in acetonitrile. The initial mobile phase composition is 45% solvent B pumped at 0.3 mL/min. The percentage of B was increased to 65% over 4.0 min. and held at 65% B from 4 to 5 min. Between 5.0 and 5.1, the percentage of solvent B was decreased back to 45%, while increasing the flow rate to 0.6 mL/min, which was held till 8 min when flow was reduced to 0.3 mL/min, resulting in a total run time of 8 min.
2.3. Mass spectrometry
Mass spectrometric detection was carried out using an ABI SCIEX (Concord, ON, Canada) 4000Q hybrid linear ion trap tandem mass spectrometer with electrospray ionization in positive multiple reaction monitoring (MRM) mode. The settings of the mass spectrometer for all analytes were as follows: curtain gas 40, CAD 10 Ion transfer voltage 5000 V, probe temperature 500°C, GS1 40, GS2 40, declustering potential 60 V, entrance potential 5 V collision energy 20 V, collision cell exit potential 15 V, and dwell time 0.1 s. The MRM m/z transitions monitored were: 854.5>286.0 for PTX; 860.5>292.0 for [13C6]-PTX; 870.5>286.0 for 6-alpha-OH; 875.51>291.0 for [D5]-6-α-OH-PTX; and 870.5>569.5 for 3-p-OH-PTX. The LC system and mass spectrometer were operated as previously reported with 1/y2 weighted linear regression [7].
2.4. Preparation of calibration standards and quality control samples
Stock solutions of PTX were prepared independently at 10 mg/mL in methanol and stored at −80 °C. Stock solutions of analytes 6-α-OH-PTX, and 3-p-OH-PTX, and internal standards [13C6]- PTX and [D5]-6-v-OH-PTX were prepared independently at 1 mg/mL in methanol and stored at - 80 °C. PTX, 6-α-OH-PTX, and 3-p-OH-PTX were diluted 10-fold in methanol to obtain a mixture working stock of 1 mg/ml, 0.1 mg/ml and 0.1 mg/ml, respectively, and also stored at −80 °C. On the day of assay, these solutions were serially diluted (in 10-fold steps) in methanol to obtain the lower calibration working solutions. In a final step, these calibration working solutions were diluted in human plasma to produce the following PTX, 6-α-OH-PTX, and 3-p-OH-PTX concentrations: 10/1/1, 30/3/3, 100/10/10, 300/30/30, 500/50/50, 1,000/100/100, 3,000/300/300, 10,000/1,000/1,000 ng/mL. For each calibration series, zero and blank samples were also prepared from 100 μL of control plasma. Quality control (QC) stock solutions were prepared independently and stored at −80 °C. These solutions were diluted in human plasma to produce the following QC samples of either: Lower Limit of Quantitation (LLOQ) 10/1/1 ng/mL, QC Low (QCL) 20/2/2 ng/mL; QC Mid (QCM) 500/50/50 ng/mL, and QC High (QCH) 8,000/800/800 ng/mL. Stock solutions were verified by duplicate independent preparation from powder. The final concentration of methanol was 5% or less for each calibration point and QC level.
2.5. Sample preparation
A volume of 100 μL of the standard, QC, or sample plasma was pipetted into a microfuge tube and 10 μL of internal standard mix (500 ng/mL for each internal standard; 50 ng/mL final concentration) was added to each tube. Next, 500 μL of methyl-tert-butyl ether was added followed by vortexing for 2 min on a Vortex Genie-2 set at 10 (Model G-560 Scientific Industries, Bohemia, NY, USA). Samples were centrifuged at 12,000 × g at room temperature for 5 min. Samples were placed in a −80 °C freezer for 25 min. Supernatants were transferred to 12 mm × 75 mm borosilicate glass tubes and evaporated to dryness under a stream of nitrogen at 37 °C. Dried residues were redissolved in 100 μL of acetonitrile: water: formic acid (50:50:0.1, v/v/v). After a brief vortexing, the supernatants were transferred to autosampler vials, followed by injection of 5 μL into the LC- MS/MS system.
2.6. Validation procedures
2.6.1. Calibration curve and lower limit of quantitation (LLOQ)
Calibration standards and blanks were prepared (see paragraph 2.4 and 2.5) and analyzed in triplicate (and in 3 independent runs) to establish the calibration range with acceptable accuracy and precision, as previously described [7]. Two Independent batches of PTX, 6-α-OH-PTX, and 3-p-OH-PTX stocks were compared to ensure accurate stock preparation.
2.6.2. Accuracy and precision
The accuracy and precision of the assay were determined by analyzing samples at the LLOQ, QCL, QCM, and QCH concentrations (N=5 per run, 3 independent runs), as previously described [7, 8].
2.6.3. Selectivity and specificity
To investigate whether endogenous matrix constituents interfered with the assay, six individual batches of control, drug-free human plasma were processed and analyzed according to the described procedure (sections 2.2–2.5). Responses of analytes at the LLOQ concentrations were compared with the response of the blank samples. Although PTX, 6-α-OH-PTX, and 3-p-OH-PTX did not co-elute, relevant cross-talk of each individual analyte was characterized by detection in other MRM channels at 10 times the upper limit of quantitation (ULOQ) for each analyte.
2.6.4. Extraction recovery and matrix effect
We determined the extraction recoveries and matrix effects of PTX and its metabolites from plasma, as previously described [7].
2.6.5. Stability
Long term stability experiments were performed for PTX and metabolites, by comparing with fresh stock solution made from powder. In addition, the stabilities of PTX and metabolites in stock solution at room temperature for 6 h were determined in replicates of four. All stability testing in plasma was performed in replicates of four or more at the QCL, QCM and QCH concentrations. The stability of PTX and its metabolites in plasma at–80 °C were determined by assaying samples before and after storage. The effect of 3 freeze/thaw cycles analyte concentrations on plasma was evaluated by assaying samples after they had been frozen (−80 °C) and thawed on 3 separate days and comparing the results with those of freshly prepared samples. The stabilities of PTX and metabolites in plasma during sample preparation were evaluated by assaying samples before and after 6 h of storage at room temperature. To evaluate the autosampler stability of PTX and metabolites in reconstituted samples in the autosampler, we re-injected QC samples and calibration curves approximately 72 h after the first injection and compared the concentrations derived from the second injection with those derived from the first injection. The results of the second runs were expressed as a percentage of their respective values in the first runs.
2.7. Application of the assay
To document the potential applicability of the assay, we determined the pharmacokinetics of PTX and its respective metabolites up to 48 h after administration of a 175 mg/m2 IV dose of PTX to a patient enrolled on the ongoing Phase 1 combination study (NCI #8808, ClinicalTrials.gov Identifier: NCT01366144). The pharmacokinetic parameters from this patient (half-life (t1/2), maximum plasma concentration (Cmax), time of Cmax (Tmax), area under the plasma concentration time profile (AUC0-inf), volume of distribution at steady state (Vss), and clearance (Cl)) were determined non-compartmentally using PK Solutions 2.0 (Summit Research Services, Montrose, CO).
2.8. Incurred sample reanalysis and cross-validation
Upon completion of the validation, incurred sample reanalysis utilizing the identical assay was performed on samples from an ongoing clinical trial being supported with this assay (ClinicalTrials.gov Identifier: NCT01366144). ISR was done on samples that had values above LLOQ and the time frame between assay dates varied from 217 to 364 days.
Available study samples had previously been analysed for only PTX concentrations using a previously reported assay, which was based on solid phase extraction and different chromatography and mass spectrometric detection [9]. We re-analyzed these samples as a means of cross-validation for PTX values only.
3. Results and Discussion
3.1. Development
We explored methanol protein precipitation, and ethyl acetate and MTBE liquid–liquid extractions. Ethyl acetate and MTBE extraction resulted in larger peak areas than methanol protein precipitation. Carry-over was assessed by injecting a prepared plasma sample with 100,000 ng/mL PTX, 10,000 ng/mL 6-α-OH-PTX and 5,000 ng/mL 3-p-OH-PTX, followed by serial plasma blank injections. Carry-over in the final assay was 0.013%, 0.0038%, and 0.0025%, respectively. Linearity for PTX was achieved from 10–10000 ng/mL while 6-α-OH-PTX and 3-p-OH-PTX were linear from 1–1000 ng/ml, which suited our target clinical concentration range.
3.2. Validation of the assay
3.2.1. Chromatography
The approximate retention times of each compound were as follows: PTX at 3.3 min, 3-p-OH-PTX at 2.3 min, 6-α-OH-PTX at 2.5 min, [13C6]-PTX at 3.3 min, and [D5]-6-α-OH-PTX at 2.5 min. They had a corresponding capacity factory of 5.2, 3.3, 3.7, 5.2, and 3.7, respectively, with a void time of 0.53 min. Representative chromatograms of each compound (blank and LLOQ), and internal standards in plasma are displayed in Fig. 2.
Fig. 2.
Representative chromatograms of: A) PTX (m/z 854.5>286.0; 3.3 min) added to control plasma at the LLOQ concentration of 10 ng/mL (top trace with an offset of 200 counts) and control human plasma (bottom trace with an offset of 100 counts); B) 6-α-OH-PTX (m/z 870.5>286.0; 2.5 min) added to control plasma at the LLOQ concentration of 1 ng/mL (top trace with an offset of 40 counts) and control human plasma (bottom trace); C) 3-p-OH-PTX (m/z 870.5>569.5; 2.3 min) added to control plasma at the LLOQ concentration of 1 ng/mL (top trace with an offset of 40 counts) and control human plasma (bottom trace); D) [13C6]-PTX internal standard (m/z 860.5>292.0; 3.3 min) added to control plasma at a concentration of 500 ng/mL (top trace with an offset of 2000 counts) and control human plasma (bottom trace with an offset of 1000 counts); E) [D5]-6-α-OH-PTX internal standard (m/z 875.5>291.0; 2.5 min) added to control plasma at a concentration of 500 ng/mL (top trace with an offset of 2000 counts) and control human plasma (bottom trace with an offset of 1000 counts).
3.2.2. Calibration curve and LLOQ
The selected assay range of 10–10,000 ng/mL for PTX, and 1–1,000 ng/mL for 6-α-OH-PTX, and 3-p-OH-PTX fulfilled the FDA criteria [10] for the LLOQ concentration and the calibration curve. Accuracies and precisions at the different concentrations from triplicate calibration curves on 3 separate days are reported in Suppl.Table 1. Representative calibration curves and corresponding correlation and regression coefficient are shown in Suppl.Fig. 1.
3.2.3. Accuracy and precision
The range of QC based accuracies was 94.3 to 110.4% for PTX, 95.1 to 104.7% for 6-α-OH-PTX, and 100.5 to 112.8% for 3-p-OH-PTX. The intra- and inter-assay precisions for the tested concentrations (LLOQ, QCL, QCM, QCH) were all within the defined acceptance criteria (Table 1) [10].
Table 1.
Assay performance data for the quantitation of LLOQ, QCL, QCM and QCH of PTX and metabolite concentrations in human plasma.
| Concentration (ng/mL) | Accuracy (%) | Intra-assay precision (%) | Inter-assay precision (%) |
|---|---|---|---|
| 10 (LLOQ) | 109.9 | 3.5 | 3.0 |
| 20 (QCL) | 110.4 | 2.0 | 1.2 |
| 500 (QCM) | 102.3 | 4.0 | 6.4 |
| 8000 (QCH) | 94.3 | 4.9 | 7.6 |
| 1 (LLOQ) | 101.2 | 9.2 | NA* |
| 2 (QCL) | 104.7 | 4.0 | 3.2 |
| 50 (QCM) | 100.1 | 2.6 | 11.3 |
| 800 (QCH) | 95.1 | 3.1 | 7.0 |
| 1 (LLOQ) | 112.8 | 7.3 | 1.9 |
| 2 (QCL) | 109.1 | 3.9 | 1.1 |
| 50 (QCM) | 100.5 | 1.7 | 5.8 |
| 800 (QCH) | 100.7 | 3.2 | 5.0 |
N=15; 5-fold results, each in 3 separate runs, for each concentration.
The mean square of the within runs was greater than the mean square of the between runs, indicating that there was no significant additional variation due to the performance of the assay in different runs [6].
3.2.4. Selectivity and specificity
Chromatograms of six individual control plasma samples contained no co-eluting peaks >20% of the analyte areas at the LLOQ concentration (interference <4.8% for PTX, <8.1% for 3-p-OH-PTX, and <5.8% for 6-α-OH-PTX).
Cross talk calculations were performed and tabulated in Suppl.Table 2. Maximum cross-talk at ULOQ (analytes) or applied concentrations (IS) would result in contributions to other analytes less than 20% of respective LLOQs: PTX, 10.000*0.0013%=0.13 ng/mL 6-α-OH-PTX; 6-α-OH- PTX, 1.000*0.033%=0.33 ng/mL PTX; 3-p-OH-PTX, 1.000*0.063%=0.63 ng/mL PTX; 13C6-PTX, 50*0.24%=0.12 ng/mL PTX; 6-α-OH-PTX IS, 50*0.21%=0.11 ng/mL PTX. Cross talk does not appear to be a major issue with any of the analytes.
3.2.5. Extraction recovery and matrix effect
The recoveries of PTX ranged from 85.0% to 91.7% (CV 10.4% to 12.0%), 3-p-OH-PTX from 74.2 to 86.7% (CV 10.4% to 19.0%), 6-α-OH-PTX from 75.8.3 to 83.4% (CV 11.0% to 15.1%). PTX matrix effect ranged from −2.2 to 5.1%, 3-p-OH-PTX from −2.3 to 6.2%, 6-α-OH- PTX from −3.5 to 2.3%, with CVs less than 11% (Suppl.Table 3). The minor apparent concentration dependent nature of the recovery (earlier experiments showed recovery dropping from 90 to 60% at increasing concentration) was adequately corrected for by the internal standards used, as is evident from the appropriate accuracies across the concentration ranges (Table 1 and Suppl.Table 1).
3.2.6. Stability
The stabilities of PTX, 3-p-OH-PTX, and 6-α-OH-PTX, stock solutions at room temperature for 6 h were 100.5%, 103.7%, and 100.2%, respectively (Table 2). Stabilities in stock solutions for 13.5 months at −80 °C were 99.7%, 101.7%, and 107.9%, respectively. The stability of the analytes after 3 freeze thaw cycles (−80 °C to RT) were between 90.2 and 107.0%. Long-term stabilities of the analytes in plasma at −80 °C for 37 months were adequate with recoveries between 89.4 and 112.6%. The concentrations derived for plasma extracts of PTX and metabolite at the quality control concentrations, when reconstituted and kept in the autosampler for 72 h, were 93.4 to 103.5% of the initial concentrations (CV 1.7–4.6%). Importantly, the reinjection run passed the requirements of any run set by the FDA [10].
Table 2.
Stability of PTX and metabolites stock and plasma under varying conditions.
| Storage Condition | Concentration (ng/mL) | Stability (%) | CV (%) | Replicates | |
| PTX | |||||
| Stock Solution | 6 h ambient temp | 1,000,000 | 100.5 | 6.8 | 4 |
| 13.5 months −80 °C | 1,000,000 | 99.7 | 12.2 | 3 | |
| Plasma | 6 h ambient temp | 10 (LLOQ) | 104.4 | 6.8 | 6 |
| 20 (QCL) | 101.4 | 5.6 | 6 | ||
| 500 (QCM) | 96.0 | 4.3 | 6 | ||
| 8000 (QCH) | 105.0 | 4.9 | 6 | ||
| 3 freeze-thaw cycles −80 °C | 10 (LLOQ) | 90.2 | 7.0 | 5 | |
| 20 (QCL) | 101.0 | 5.4 | 5 | ||
| 500 (QCM) | 97.9 | 9.6 | 5 | ||
| 8000 (QCH) | 98.4 | 10.5 | 5 | ||
| 37 months at −80 °C | 20 (QCL) | 89.4 | 8.6 | 4 | |
| 500 (QCM) | 91.7 | 4.9 | 4 | ||
| 8000 (QCH) | 100.5 | 8.5 | 4 | ||
| Autosampler | 72 h at 4 °C | 20 (QCL) | 103.4 | 1.9 | 4 |
| 500 (QCM) | 98.2 | 2.2 | 5 | ||
| 8000 (QCH) | 96.6 | 4.6 | 5 | ||
| Storage Condition | Concentration (ng/mL) | Stability (%) | CV (%) | Replicates | |
| 3-p-OH-PTX | |||||
| Stock Solution | 6 h ambient temp | 100,000 | 103.7 | 5.1 | 4 |
| 13.5 months −80 °C | 100,000 | 101.7 | 11.4 | 3 | |
| Plasma | 6 h ambient temp | 1 (LLOQ) | 104.7 | 9.5 | 6 |
| 2 (QCL) | 108.2 | 3.9 | 6 | ||
| 50 (QCM) | 90.7 | 2.0 | 6 | ||
| 800 (QCH) | 91.4 | 3.6 | 6 | ||
| 3 freeze-thaw cycles −80 °C | 1 (LLOQ) | 107.0 | 14.0 | 5 | |
| 2 (QCL) | 104.0 | 7.6 | 5 | ||
| 50 (QCM) | 98.8 | 6.0 | 5 | ||
| 800 (QCH) | 95.9 | 8.7 | 5 | ||
| 37 months at −80 °C | 2 (QCL) | 112.6 | 15.9 | 4 | |
| 50 (QCM) | 100.9 | 9.1 | 4 | ||
| 800 (QCH) | 102.3 | 10.1 | 4 | ||
| Autosampler | 72 h at 4 °C | 2 (QCL) | 103.5 | 3.5 | 4 |
| 50 (QCM) | 93.6 | 2.0 | 5 | ||
| 800 (QCH) | 93.4 | 3.3 | 5 | ||
| Storage Condition | Concentration (ng/mL) | Stability (%) | CV (%) | Replicates | |
| 6-PTX-OH-PTX | |||||
| Stock Solution | 6 h ambient temp | 100,000 | 100.2 | 6.0 | 4 |
| 13.5 months −80 °C | 100,000 | 107.9 | 9.2 | 3 | |
| Plasma | 6 h ambient temp | 1 (LLOQ) | 93.5 | 11.4 | 6 |
| 2 (QCL) | 99.7 | 10.7 | 6 | ||
| 50 (QCM) | 96.8 | 3.7 | 6 | ||
| 800 (QCH) | 102.0 | 4.8 | 6 | ||
| 3 freeze-thaw cycles −80 °C | 1 (LLOQ) | 95.5 | 10.9 | 5 | |
| 2 (QCL) | 101.0 | 4.8 | 5 | ||
| 50 (QCM) | 96.3 | 4.3 | 5 | ||
| 800 (QCH) | 101.5 | 5.8 | 5 | ||
| 37 months at −80 °C | 2 (QCL) | 101.7 | 16.8 | 4 | |
| 50 (QCM) | 96.9 | 6.6 | 4 | ||
| 800 (QCH) | 100.0 | 3.2 | 4 | ||
| Autosampler | 72 h at 4 °C | 2 (QCL) | 102.9 | 4.7 | 4 |
| 50 (QCM) | 100.8 | 1.7 | 5 | ||
| 800 (QCH) | 102.4 | 3.7 | 5 |
3.3. Application of the assay
As seen in Fig. 3, the assay was capable of quantitating PTX, 3-p-OH-PTX, and 6-α-OH- PTX in this patient. Non-compartmental analysis yielded the following pharmacokinetic parameters: PTX t1/2 11.7 h, Cmax 5056 ng/mL, Tmax 2.95 h, AUC0-inf 19.1 μg/mL•h, Vss 72.7 L/m2, Cl 9.98 L/h/m2; 3-p-OH-PTX t1/2 11.8 h, Cmax 148 ng/mL, Tmax 3.1 h, AUC0-inf 0.832 μg/mL•h; 6- α-OH-PTX t1/2 5.5 h, Cmax 560 ng/mL, Tmax 3.11 h, AUC0-inf 1.98 μg/mL•h.
Fig. 3.
Plasma concentrations of PTX (○), 3-p-OH-PTX (□), and 6-α-OH-PTX (Δ) in a patient after a 3 h intravenous infusion of 175 mg/m2 PTX.
3.4. Incurred sample reanalysis and cross-validation
Incurred sample reanalysis of 102, 96, and 98 samples respectively yielded the following results (%samples with a difference larger than 20% / average difference / average absolute difference): PTX 8.8% / −7.1% / 11.5%; 3-p-OH-PTX 25.0%/−10.0% / 16.6.0%; and 6-α-OH-PTX 16.3.0% / −6.4% / 15.1%.
Utilizing different extraction techniques (SPE vs LLE), instruments (Quattro Micro vs AB4000Q) monitoring different ions (sodium vs proton adduct), operated by different personnel, and with a time between assays of 700–2200 days, based on 892 samples, 86.1% of the PTX re- analysis results had a difference within 20% of the mean. The median difference was −0.9%, with a median absolute difference of 10.8%. There were no trends with respect to concentration, patients, time between assays, or nominal sample time, see Fig. 4. The cross-validation effort spanned a period longer than the period covered by formal long term stability experiments. However, the apparent lack of bias when looking at samples from this longer period suggests that even longer term stability experiments could be successful.
Fig. 4.
Absence of trends with respect to concentration, patients, time between assays, or nominal sample time of re-assaying clinical samples for PTX concentrations utilizing a different sample preparation, instrument, operated by different personnel. This analysis was based on 892 samples, with 86.1% of the re-analysis results having a difference within 20% of the mean. The median difference was −0.9%, with a median absolute difference of 10.8%. For visual purposes, the y-axis was limited between −100 and 100%, which resulted in 4 points being excluded from this graphic depiction.
4. Conclusion
The objective of this study was to develop and validate an analytical method for the quantitation of PTX and its metabolites in human plasma. We accomplished this using reversed phase chromatography for separation with triple quadrupole mass spectrometric MRM detection, and followed the most recent FDA guidance [10]. Previously reported mass spectrometric assays for the quantitation of PTX and metabolites in human plasma are listed in Suppl.Table 4. As can be seen from our application data and comparing assay characteristics, our assay has relevant concentration ranges for all analytes without sample dilutions, a small sample volume, a relatively simple and easily implementable sample preparation, and a very modest run time, utilizing the best available set of stable isotope internal standards. We can focus specifically on the two assays with shorter run-times than our 8 min. The assay with a 7 min run-time[11], though similar in sample volume and chromatography, uses a related taxane as IS, reducing robustness, while the concentration range for PTX and 3-p-OH-PTX do not cover the clinically relevant range, necessitating frequent dilutions. The assay with a 5 min run-time [12], though using a stable isotope PTX IS for all analytes, requires 4 times the plasma volume compared to our assay, and the dynamic range of each of the analytes is inadequate to capture the clinically relevant range, again necessitating frequent dilutions. Our analytical method will be a valuable tool in characterizing the pharmacology of PTX and its main metabolites in the setting of liver dysfunction where we are currently applying this assay (ClinicalTrials.gov Identifier: NCT01366144). While paclitaxel has been studied in patients with liver dysfunction, the data for the 3 h infusion are scarce (approximately 90–12 patients), and PK sampling was limited. In addition, the differential impact of liver dysfunction on CYP2C8 and CYP3A4 activity phenotype may be assessed with our assay. Lastly, our assay may be utilized in the setting of therapeutic drug monitoring of paclitaxel. Recently, the time above a plasma concentration of 0.05 μM (Tc>0.05), as determined from a single blood sample on day 2, has been shown to correlate with toxicity. [13–16].
Supplementary Material
Highlights.
Assay for paclitaxel (10–10,000 ng/mL), 3-para-OH, and 6-alpha-OH metabolite (1–1,000 ng/mL)
Phenotypic readout of CYP2C8 and CYP3A4 metabolic pathways
Plasma stability documented for 37 months
Acknowledgments
Funding
Support: Grant UM1-CA186690 (NCI-CTEP) and R50CA211241. This project used the UPCI Cancer Pharmacokinetics and Pharmacodynamics Facility (CPPF) and was supported in part by award P30-CA47904. Declarations of interest: none.
Footnotes
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