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A Novel Approach to Quantify Unbound

Cisplatin, Carboplatin, and Oxaliplatin in

Human Plasma Ultrafiltrate by Measuring

Platinum-DDTC Complex Using LC/MS/MS

Overview

Purpose

To develop and validate a LC/MS/MS 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 (ISTD) and 5% diethyldithiocarbamate (DDTC). The mixture was incubated first and then extracted by methyl-t-butyl ether (MtBE). The LC/MS/MS system was either a Sciex API4000 or API3000 with an Ionics HSID+ upgrade and coupled with a Shimadzu LC-10AD pump

HPLC system. The instrument was operated in the positive-ionization mode using the TurboIonSpray source. The MRM transitions were monitored for the compounds of interest. The liquid chromatography was optimized on a Phenomenex Gemini C18 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

Min Meng, Ryan Kuntz, Al Fontanet, and Patrick K. Bennett; Tandem Labs, Salt Lake City, Utah Presented at the 2006 ASMS Conference, Seattle, WA, May 2006

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2 Tandem Labs

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 ISTD 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.

Normally, platinum complexes are heavily bound to protein. Approximately 90% of bound platinum has no cytotoxicity. Only 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-MS, 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/MS/MS method to measure

carboplatin in human plasma ultrafiltrate at LLOQ of 50.0 ng/mL. In this presentation, a novel LC/MS/MS methodology was developed to quantify unbound platinum from cisplatin, carboplatin, and oxaliplatin at a much lower LLOQ in plasma ultrafiltrate using a derivatization reagent followed by a liquid-liquid extraction.

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Tandem Labs 

Figure 1: Chemical Structures of Cisplatin, Carboplatin, Oxaliplatin, Palladium (II) Acetate, and Diethyldithiocarbamate (DDTC)

Pt Cl Cl NH3 NH3 N S S + Pt O O O O NH3 NH3 H N Pt N H O O O O S S Pt S S N N N S S + S S Pt S S N N N S S + S S Pt S S N N Cisplatin DDTC Pt(DDTC)2 M.W. 491.1 Carboplatin DDTC Oxaliplatin DDTC Pd O N S S + S S Pd S S N N Palladium Acetate DDTC O O O Pt(DDTC)2 M.W. 491.1 Pt(DDTC)2 M.W. 491.1 Pd(DDTC)2 M.W. 401.5

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 Tandem Labs

MetHOd

Sample Preparation

Add 100 µL of internal standard Pd-acetate (1000 ng/mL in water) into 100 µL platinum-fortified human plasma ultrafiltrate.

Add freshly prepared 5% diethyldithiocarbamate (DDTC) in 0.2N NaOH into all samples. Incubate all samples at 45ºC for 30 minutes.

Add 2 mL of methyl-t-butyl ether (MtBE). Shake 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 45oC for approximately 20 minutes.

Reconstitute sample with 200 µL MeCN.

Chromatographic Conditions

column: Phenomenex Luna Gemini c18, 50 x 2 mm, 5 µ

Mobile Phases:

A: 0.1% formic acid in water B: Mecn

c: 90/10 acetone/water 500 µL/min Lc Program: isocratic 0.3 mL/min @ 80% B injection volume: 5 – 25 µL

column temperature: 30oc

AS temperature: room temperature diverting time: 0.8 – 2.2 minutes into MS 1. 2. 3. 4. 5. 6. 7. 8. 9.

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Tandem Labs 

Mass Spectrometer Conditions

Instrument: Sciex API4000 or 3000 w/ Ionics HSID+ upgrade

Ionization Source: TurboIonSpray Ionization Mode: Positive ion mode Source Temperature: 400oC

SRM Transition: Platinum-(DDTC)2 492 ’ 426

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Figure 2

Figure 3

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Figure 5: Representative Product Ion Scan

Figure 6: Representative

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Figure 7: Representative SRM Chromatogram of LLOQ

Figure 8: Representative Oxaliplatin std Curve [1.00-1,000 ng/mL, platinum-(DDTC)2]

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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]

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10 Tandem Labs

Cisplatin Calibration Standards

28-Dec-2005 Mean S.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 S.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.

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Tandem Labs 11

reSuLtS

MS 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 (SRM 403 ’ 254) was selected as ISTD to quantify Pt-(DDTC)2 (SRM 492 ’ 462), which is derived from carboplatin, cisplatin, or oxaliplatin.

Sample 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 0.2N NaOH for derivatization.

Various organic solvents for liquid-liquid extraction and various SPE 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.

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HPLC Development

Several types of HPLC reverse d-phase columns were screened for the best retention and peak shape. The optimized condition was determined using Gemini C18 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 TurboIonSpray 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 LLOQ of Pt-(DDTC)2 on API4000 is 1.00 ng/mL with S/N ratio of 100/1. The sensitivity for carboplatin was improved dramatically, compared to the original LLOQ 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, SRM transition of Pt-(DDTC)2 was monitored. The correlation coefficients of the three curves were > 0.99.

Precision and Accuracy of Cisplatin Standard 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 HSID+ 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.

cOncLuSiOnS

A sensitive and selective method was developed to quantify carboplatin, cisplatin, or oxaliplatin at LLOQ 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.

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Answering pharmaceutical questions with discipline and ingenuity.

tandem Labs-Salt Lake city tandem Labs-new Jersey tandem Labs-new england

801/293/2400 609/434/0044 781/933/2769 x 123

tandemlabs.com

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/MS techniques. Presented at the 2003 AAPS Annual Meeting and Exposition, Salt Lake City, Utah, October 2003.

Meng M, et al. Simple and rapid determination of carboplatin in human plasma ultrafiltrate using LC/MS/MS by direct injection coupled with post column addition. Presented at the 2003 AAPS Annual Meeting and Exposition, Salt Lake City, Utah, October 2003.

References

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