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Determination of Platinum Compounds by LC-ICP-MS

Application Note Pharmaceutical

Abstract
Platinum compounds such as cisplatin were analyzed using the combination of a high performance liquid chromatograph and an 4500 ICP-MS. Good separation of two types of Pt compounds, cis - amminedichloro - (cyclohexylamine) platinum (II) and bis (acetato) ammine - dichloro (cyclohexylamine) platinum (IV), can be clearly demonstrated. The detection limit as Pt was 2 ug/L(ppb), corresponding to the amount of 0.1 ng Pt.

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Introduction
Cisplatin is an antineoplastic drug with proven effectiveness against numerous animal and human tumors. With its broad spectrum of antitumour activity, cisplatin is one of the most important drugs in clinical oncology developed in the second half of this century. However, cisplatin has some side effects such as nephrotoxicity and nausea, which brought on a search for second generation platinum compounds with less nephrotoxicity and greater anti-tumor activity. In order to investigate the behavior of platinum compounds, a selective and sensitive analytical method is required. Until now, high performance liquid chromatography (HPLC) using either off-line AAS detection or on-line UV detection is widely used for platinum compounds determination. However, it is time consuming and there is a lack of sensitivity or/and specificity. ICP-MS is now a mature technique to analyze trace level of elements with high throughput. This application note describes the determination of platinum compounds by the 4500 ICP-MS with liquid chromatography.

Plasma gas flow rate Aux. gas flow rate Carrier gas flow rate RF Power Nebulizer Spray chamber Spray chamber temp ICP torch injector Sampler cone Skimmer cone Sampling depth Points/mass Acquired mass Integration time/mass Total acquisition time/sample Table 1. 4500 ICP-MS Operating Parameters

16.0 L/min 1.0 L/min 0.84 L/min 1250 W Concentric - type Glass, double pass 1C Quartz, 2.5 mm Nickel Nickel 7 mm 1 5 mass: 194Pt, 195Pt, 196Pt, 115In and 205Tl 0.2 sec 2500 sec

(80 cm x 0.3 mm I.D.). Parameters for 4500 ICP-MS are given in Table 1.

Results
The chromatogram for 3.73 mg/L of JM118, which corresponds to 1.95 mg/L Pt is shown in Fig. 1. The sharp peak at around 500 seconds is the signal due to cis - aminedichloro (cyclohexylamine) platinum (II). The ratios of signals on m/z=194, 195 and 196 fit the Pt isotope ratio, which identifies the peaks. Some other peaks are also shown, which might be derivative of Pt compounds. JM216 was also analyzed to check the level of cis - aminedichloro (cyclohexylamine) platinum (II). The chromatogram for 2.29 mg/L of JM216 is shown in Fig. 2. The peaks for bis (acetato) amine - dichloro (cyclohexylamine) platinum (IV) is shown at about 2300 sec. Among the several impurity peaks, the peak for cis - aminedichloro (cyclohexylamine) platinum (II) can be detected, and its concentration was about 165 ug/L, which corresponds to 72 mg/g in solid.

Sample Preparation
Eluent used was the mixture of 1 mM heptanesulphonic acid, 0.1% acetic acid, and 20% methanol. Platinum compounds analyzed were cis - amminedichloro (cyclohexylamine) platinum (II) (JM118) and bis (acetato) ammine - dichloro (cyclohexylamine) platinum (IV) (JM216) purchased from Johnson Matthey Electronics (Ward Hill, MA, USA). Each compound was diluted by 0.1% Triton X - 100 and 0.1% HNO3. In and Tl were used as the internal standard elements. A 500 ug/L of mixed internal standard solution was added on-line at the time of analysis using a peristaltic pump. The flow rate of internal standard solution was 20 uL/min, which makes approximately 10 ug/L of In and Tl in a final solution. This solution was also used for tuning of the 4500 ICP-MS.

Experimental
Instrumentation The high performance liquid chromatography used in this work consisted of Agilent 1050 LC system with 50 uL loop and a ODS C18 column (150 mm x 4.6 mm I.D., 5 um particle size). The column temperature was set at 30 C, and the sample flow rate was 1 mL/min. The eluent from the HPLC column was directly connected to the nebulizer of 4500 ICP-MS using ETFE tubing

Fig. 1 Chromatogram of platinum in JM118

Fig.2 Chromatogram of platinum in JM216

The detection limit defined as the amount producing a signal three times the standard deviation of the noise was 2 ug/L of Pt, corresponding to the amount of 0.1 ng Pt. The detection limit could be increased if only one mass was analyzed or a larger sample loop used. Although the internal standard elements were not used this time, it would be useful for the correction of signal drift when analyzing samples for a long time.

Conclusion
Platinum compounds can be detected with simple treatment (just dissolution) by 4500 ICP-MS with HPLC. The combination of HPLC separation capabilities and the 4500 ICP-MS high sensitivity and selectivity enable the determination of platinum compounds down to ng levels.

Agilent Technologies shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing, performance or use of this material. Information, descriptions and specifications in this publication are subject to change without notice. Visit our website at http:/www.agilent.com/chem/icpms Copyright 2000 Agilent Technologies, Inc. Printed 4/2000 Publication number 5968-8185E

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