A ovel Approach to Quantify Unbound Cisplatin, Carboplatin, and xaliplatin in Human Plasma Ultrafiltrate by Measuring Platinum-DDTC Complex Using LC/M/M Min Meng, Ryan Kuntz, Al Fontanet, and Patrick K. Bennett; Tandem Labs, alt Lake City, Utah Presented at the 2006 AM Conference, eattle, WA, May 2006 verview Purpose To develop and validate a LC/M/M method for the determination of unbound cisplatin, carboplatin, and oxaliplatin anti-cancer drugs in human plasma ultrafiltrate. Methods Carboplatin-, cisplatin-, or oxaliplatin-fortified human plasma ultrafiltrate was mixed with palladium acetate (ITD) and 5% diethyldithiocarbamate (DDTC). The mixture was incubated first and then extracted by methyl-t-butyl ether (MtBE). The LC/M/M system was either a ciex API4000 or API3000 with an Ionics HID + upgrade and coupled with a himadzu LC-10AD pump HPLC system. The instrument was operated in the positive-ionization mode using the TurboIonpray source. The MRM transitions were monitored for the compounds of interest. The liquid chromatography was optimized on a Phenomenex Gemini C 18 2 x 50 mm HPLC column with isocratic conditions using 0.1% formic acid in water and acetonitrile. A dual valve switching system was utilized to divert earlier eluting peaks to waste and to flush the analytical column between injections. Results Direct infusion of the three platinum complexes showed that only carboplatin and oxaliplatin can be ionized directly. There are no detectable ions found for cisplatin. The infusion of cisplatin, carboplatin, and oxaliplatin DDTC derivatives generate identical Q1 full scan spectra with a predominant ion
at m/z 492, which correlates with Pt-(DDTC) 2 (M.W. 491). This suggests that after DDTC derivatization, carboplatin, cisplatin, and oxaliplatin were converted to Pt-(DDTC) 2. Palladium-(DDTC) 2 was selected as ITD to quantify platinum-(ddtc) 2. The optimized sample preparation procedure was achieved using a MtBE liquid-liquid extraction. The incorporation of dual switching valves proved to be essential for improving the overall performance of the assays. Three sets of standard curves in plasma were prepared by spiking either carboplatin, cisplatin, or oxaliplatin at 1.00-1,000 ng/ml. The correlation coefficients are 0.9967, 0.9947, and 0.9958, respectively. The precision and accuracy of the low, medium, and high QC are 103.8±2.73, 96.1±4.44, and 95.5±5.79. This method has been utilized to support sample quantitation for discovery studies. Introduction Platinum complexes, cisplatin, carboplatin, and oxaliplatin, are widely used in cancer treatment. ormally, platinum complexes are heavily bound to protein. Approximately 90% of bound platinum has no cytotoxicity. nly approximately 10% of free platinum was present in plasma ultrafiltrate as an intact drug. The quantitation of platinum in plasma ultrafiltrate, especially for cisplatin, is very challenging. Various conventional analytical techniques, such as HPLC-UV, FAA, or HPLC-IPA-M, were reported for the quantitation of platinum complexes. However, the detection limits and selectivity were less satisfactory. Compared to carboplatin and oxaliplatin, the detection of cisplatin is more challenging because it has neither chromophore for spectrophotometric detection nor detectable ions for mass spectrometric detection. We previously reported a simple dilute-and-shoot LC/M/M method to measure carboplatin in human plasma ultrafiltrate at LLQ of 50.0 ng/ml. In this presentation, a novel LC/M/M methodology was developed to quantify unbound platinum from cisplatin, carboplatin, and oxaliplatin at a much lower LLQ in plasma ultrafiltrate using a derivatization reagent followed by a liquid-liquid extraction. 2 Tandem Labs
Figure 1: Chemical tructures of Cisplatin, Carboplatin, xaliplatin, Palladium (II) Acetate, and Diethyldithiocarbamate (DDTC) Cl Cl Pt H 3 H 3 + Pt Cisplatin DDTC Pt(DDTC) 2 M.W. 491.1 Pt H 3 H 3 + Pt Carboplatin DDTC Pt(DDTC) 2 M.W. 491.1 Pt H H + Pt xaliplatin DDTC Pt(DDTC) 2 M.W. 491.1 Pd + Pd Palladium Acetate DDTC Pd(DDTC) 2 M.W. 401.5 Tandem Labs
METHD ample Preparation 1. 2. 3. 4. 5. 6. 7. 8. 9. Add 100 µl of internal standard Pd-acetate (1000 ng/ml in water) into 100 µl platinumfortified human plasma ultrafiltrate. Add freshly prepared 5% diethyldithiocarbamate (DDTC) in 0.2 ah into all samples. Incubate all samples at 45ºC for 30 minutes. Add 2 ml of methyl-t-butyl ether (MtBE). hake samples for 15 minutes. Freeze aqueous lay in acetone-dry ice bath. Transfer top organic layer to clean tubes. Evaporate samples completely in turbovap at 45 o C for approximately 20 minutes. Reconstitute sample with 200 µl MeC. Chromatographic Conditions Column: Phenomenex Luna Gemini C 18, 50 x 2 mm, 5 µ Mobile Phases: LC Program: A: 0.1% formic acid in water B: MeC C: 90/10 acetone/water 500 µl/min Isocratic 0.3 ml/min @ 80% B Injection Volume: 5 25 µl Column Temperature: A Temperature: Diverting Time: 30 o C Room temperature 0.8 2.2 minutes into M Tandem Labs
Mass pectrometer Conditions Instrument: Ionization ource: Ionization Mode: ource Temperature: ciex API4000 or 3000 w/ Ionics HID + upgrade TurboIonpray Positive ion mode 400 o C RM Transition: Platinum-(DDTC) 2 492 426 Palladium-(DDTC) 2 403 254 Tandem Labs
Figure 2 Figure 3 Figure 4
Figure 5: Representative Product Ion can Figure 6: Representative RM Chromatogram of Control Blank
Figure 7: Representative RM Chromatogram of LLQ Figure 8: Representative xaliplatin std Curve [1.00-1,000 ng/ml, platinum-(ddtc) 2 ]
Figure 9: Representative Cisplatin std Curve [1.00-1,000 ng/ml, platinum-(ddtc) 2 ] Figure 10: Representative Carboplatin std Curve [1.00-1,000 ng/ml, platinum-(ddtc) 2 ]
Cisplatin Calibration tandards 28-Dec-2005 Mean.D. %CV %Bias n 5.00 4.52, 5.34 4.93 0.579 11.8-0.07 2 10.0 9.67, 10.9 10.3 0.869 8.5 0.285 2 50.0 51.6, 49.3 50.4 1.55 3.1 0.4 2 250 246.5, 242.9 245 2.61 1.1-0.25 2 500 519.8, 480.9 500.4 27.5 5.5 0.35 2 750 738.7, 702.5 721 25.5 3.6-29.4 2 1,000 1109.5, 977.9 1040 93.1 8.9 43.7 2 This table presents the back-calculated concentrations of calibration standards for cisplatin in human plasma ultrafiltrate. Run number 1; all concentrations are expressed as ng/ml. Cisplatin Quality Control 28-Dec-2005 Mean.D. %CV %Bias %Theoretical n 30.0 31.9, 30.2, 30.6, 31.9 31.1 0.870 2.8 3.67 103.7 4 800 817.3, 740.2, 752.1, 766.9 769.1 33.9 4.4-3.88 96.1 4 1,000 1005.4, 955.7, 878.1, 981.9 955.1 55.1 5.8-4.49 95.5 4 This table presents the back-calculated concentrations of quality control for cisplatin in human plasma ultrafiltrate. Run number 1; all concentrations are expressed as ng/ml. 10 Tandem Labs
results M Tuning Direct infusion of three platinum complexes showed that only carboplatin and oxaliplatin can be ionized directly (m/z 372 for carboplatin and m/z 398 for oxaliplatin). There are no detectable ions found for cisplatin. Various solvents and acid modifiers were tested but failed to produce ions for cisplatin. The infusion of carboplatin, cisplatin, and oxaliplatin DDTC derivatives generate identical Q1 full scan spectra with a predominant ion at m/z 492. This correlates with Pt-(DDTC) 2 (M.W. 491), suggesting that after DDTC derivatization, carboplatin, cisplatin, and oxaliplatin were converted to Pt-(DDTC) 2. The product ion scan of Pt-(DDTC) 2 showed a major ion at m/z 462 (loss of two methyl groups). Two metallic elements, zinc and palladium, were evaluated for internal standard. Palladium- DDTC derivatives Q1 full scan spectra showed a predominate ion at m/z 403, which correlates with Pd-(DDTC) 2 (M.W. 403), suggesting that after DDTC derivatization, palladium was converted to Pd-(DDTC) 2. The product ion scan of Pd-(DDTC) 2 showed a major ion at m/z 254 (loss of one DDTC). Finally, palladium-(ddtc) 2 (RM 403 254) was selected as ITD to quantify Pt-(DDTC) 2 (RM 492 462), which is derived from carboplatin, cisplatin, or oxaliplatin. ample Extraction To optimize the sample preparation procedure, DDTC amount, incubation time, and temperature were evaluated. The best condition was established using freshly prepared 5% diethyldithiocarbamate (DDTC) in 0.2 ah for derivatization. Various organic solvents for liquid-liquid extraction and various PE columns under different solvent, ph, column types, and washing-elution conditions were also investigated. The best condition was obtained using methyl-t-butyl ether (MtBE) liquid-liquid extraction. Tandem Labs 11
HPLC Development everal types of HPLC reverse d-phase columns were screened for the best retention and peak shape. The optimized condition was determined using Gemini C 18 under high organic composition condition. During HPLC chromatography development, five common phospholipid ions, 496 184 (lysophospholipid), 524 184 (lysophospholipid), 704 184 (glycerophospholipid), 758 184 (glycerophospholipid ), and 806 184 (glycerophospholipid ), were monitored so to ensure that the analytes were separated from endogenous phospholipids. The incorporation of dual switching valves was essential to improve the overall performance of the assays. The diversion of the earlier elutors kept the TurboIonpray interface clean on the API4000 and reduced the tendency for divergent calibration curves. The backflush of the analytical column with strong solvent can efficiently wash out retaining endogenous phospholipids. Detection Limit and Linearity Using 100 µl aliquot size, the LLQ of Pt-(DDTC) 2 on API4000 is 1.00 ng/ml with / ratio of 100/1. The sensitivity for carboplatin was improved dramatically, compared to the original LLQ for carboplatin method, which is 50.0 ng/ml on an API4000. Three sets of standard curves in plasma ultrafiltrate were prepared by spiking either carboplatin, cisplatin, or oxaliplatin. The final concentration range was at 1.00-1,000 ng/ml. After DDTC derivatization and liquid-liquid extraction, RM transition of Pt-(DDTC) 2 was monitored. The correlation coefficients of the three curves were > 0.99. Precision and Accuracy of Cisplatin tandard and QC Additional work was conducted for quantitation of cisplatin in order to support discovery work. This work was conducted on an API3000 with an Ionics HID + upgrade at linear range of 5.00-1,000 ng/ml. The precision and accuracy of the three levels of QC are 103.7±2.8, 96.1±4.4, and 95.5±5.8. This method has been utilized to support discovery work. CCLUI A sensitive and selective method was developed to quantify carboplatin, cisplatin, or oxaliplatin at LLQ of 1.00 ng/ml in plasma ultrafiltrate. This methodology was utilized to support discovery sample analysis for the quantitation of cisplatin in human plasma ultrafiltrate.
References Andrews PA, et al. A high performance liquid chromatographic assay with improved selectivity for cisplatin and active platinum (II) complexes in plasma ultrafiltrate. Analytical Biochemistry 1984; 143:46-56. Bennett PK, et al. Identification of the major endogenous and persistent compounds in plasma, serum, and tissue that cause matrix effects with electrospray LC/M techniques. Presented at the 2003 AAP Annual Meeting and Exposition, alt Lake City, Utah, ctober 2003. Meng M, et al. imple and rapid determination of carboplatin in human plasma ultrafiltrate using LC/M/M by direct injection coupled with post column addition. Presented at the 2003 AAP Annual Meeting and Exposition, alt Lake City, Utah, ctober 2003. Answering pharmaceutical questions with discipline and ingenuity. Tandem Labs-alt Lake City Tandem Labs-ew Jersey Tandem Labs-ew England 801/293/2400 609/434/0044 781/933/2769 x 123 tandemlabs.com