Measurement of Platinum in Plasma and Urine bv ...

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Jan 1, 1988 - This paper is dedicated to the memory of Professor John M. i Department of Otorhinolaryngology. i To whom correspondence should be ...

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Measurement of Platinum in Plasma and Urine bv Direct Current Plasma Atomic Emission Spectrometry*

Published on 01 January 1988. Downloaded by University of Regina on 26/10/2014 14:54:53.

S. Mctoughlin, D. Bowdlert and N. B. Roberts* Department of Chemical Pathology, Royal Liverpool Hospital, Prescot Street, Liverpool L7 SXW, UK

The measurement of Pt in biological fluids has been developed using a d.c. plasma emission spectrometer. The analyses were validated by standard additions and spectral overlay procedures using a dynamic wavelength scanner and showed no background or spectral interferences. The detection limits were 0.2,0.04 and 0.02 pg ml-1 for plasma, urine and ultrafiltrate, respectively, and the assay was linear up to 20 pg mi-1. The recovery of Pt added to plasma and urine averaged 100% ( n = 66) and the precision over the range 1-5 pg ml-1 was 4.6-7.1% within batch and 2.&6.6% between batch. Comparison with an electrothermal atomisation atomic absorption procedure (x) gave highly significant correlations ( P < 0.001) for urine ( y = 0 . 9 6 7 ~- 0.01 1, r = +0.98, n = 23) and plasma ( y = 1 . 0 8 8 ~ 0.03, r = +0.971, n = 68). Free Pt levels in vitro declined logarithmically with a t+ of 165 min. The initial ultrafilterable (free) Pt accounted for 93-97% of the total plasma Pt levels. Recovery of cisplatin from ultrafiltered aqueous solutions was 95 & 8% at a Pt concentration of 1.3 yg ml-1, Cisplatin infusion over 8 h was carried out in nine patients and u p to 30% of the dose was excreted in urine within 3 d. Total Pt levels in plasma were maximal a t the end of infusion with mean values & 1 SD of 2.53 k 0.19 ( n = 4) and 1.58 k 0.38 pg ml-1 ( n = 5) with doses of 100 and 50 m g m-2, respectively. Nine hours after completion of the infusion, the total Pt was 75 k 11% of the peak level and thereafter declined with a ti of 81 k 11 h.


Keywords: cis- Diamminedichloroplatinum( 11) : plasma and urinary platinum; direct current plasma atomic emission spectrometry; electrothermal atomisation atomic absorption spectrometry; dynamic wavelength scanning

cis-Diamminedichloroplatinum(I1) (cisplatin) is the most widely used of a series of &configuration Pt” complexes having anti-neoplastic properties. Its effectiveness in the treatment of malignancies, especially tumours of the testis, ovary, head and neck has been well established.1-4 However, the drug has several toxic side-effects including a dose limiting cumulative nephrotoxicity , myelosuppression, neurotoxicity and ototoxicity.s.6 Administration of the drug has been carried out intravenously, intraperitoneally,7 orally8 and at the tumour site by single, monthly and by consecutive daily dosage.6.9 Prior intravenous hydration and concomitant administration of thiosulphate have been employed in an attempt to reduce the side-effects at higher doses.7J)Jl The pharmacokinetics of cisplatin are frequently studied in attempts to establish suitable dosage regimes whereby maximum tumour “kill” is obtained with minimum toxicity. The methods used include monitoring of a radioactively labelled dose. 12 X-ray fluorescencel3.14 and flame and electrothermal atomisation atomic absorption techniques. 1.1-17 All these methods have their shortcomings with undue patient stress in the first, poor sensitivity with the fluorescence procedures and in the last, restricted linearity within the desired range, the necessity for standard additions, matrix problems and the need for wet ashing. In this paper we describe an analytical technique for the determination of platinum in plasma, plasma ultrafiltrate and urine using direct current plasma atomic emission spectrometry (DCP-AES). The method requires only a simple dilution of the sample, is linear for more than three orders of magnitude above the detection limit and enables the element to be measured quickly and the pharmacokinetics to be studied effectively. The emission spectra obtained from aqueous standard solutions, plasma and urine were studied using a dynamic wavelength scanner to check for any spectral or background interference. * This paper is dedicated to the memory of Professor John M. Ottaway . i Department of Otorhinolaryngology. i To whom correspondence should be addressed.

The method was finally validated by standard additions procedures and by comparing the results with those obtained by an electrothermal atomisation atomic absorption (ETAAAS) method.

Experimental Reagents

Nitric acid and Triton X-100 were of analytical-reagent grade and were obtained from BDH (Poole, Dorset , UK). Solhtions were prepared in doubly de-ionised water (Spectrum System, Elga Products, High Wycombe, Buckinghamshire, UK). The platinum primary standard (1 mg 1-1 of Pt), as the chloride, was Spectrosol atomic absorption grade (BDH). Cisplatin, Platinex (0.5 mg ml-1) was obtained from Bristol Myers Pharmaceuticals, (Langley, Slough, Berkshire, UK). For centrifugal ultrafiltration, Centriflo CF-50 conical filters, with a molecular cut-off of 50 000 u (Amicon, Danvers, MA, USA) were used. All working dilutions of standards and tests were prepared in the assay diluent, 1% V/V nitric acid containing 0.01% V/V Triton X-100. Urine and plasma specimens not containing Pt, a plasma spiked with 2 pg ml-1 of Pt and a plasma spiked with 1.67 pg ml-1 of cisplatin (1.08 pg ml-1 of Pt) were assayed throughout the experiments as controls. Atomic Absorption Procedure

Samples were analysed for Pt using a Perkin-Elmer 2280 atomic absorption spectrometer with an HGA-300 graphite furnace and an AS 40 autosampler (Perkin-Elmer, Beaconsfield, UK). Pyrolytic graphite coated tubes fitted with L’vov platforms were used to increase the sensitivity. The heating programme used was as recommended by Perkin-Elmer.

Sample preparation All plasma samples were diluted before assay with an equal volume of 0.5% V/V Triton X-100 (to assist in the dispersion and solubilisation of the sample material) and urine with 0.2% VIV H N 0 3 . For the Pt assay the following standard additions

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procedure was used: plasma or urine was mixed with an equal volume of 0.2% V/VHN03to give an initial absorbance, and a second absorbance measured after dilution with 0.2% V/V H N 0 3 containing 1 pg ml-1 of Pt. De-ionised water was analysed as a blank following each standard addition.

Published on 01 January 1988. Downloaded by University of Regina on 26/10/2014 14:54:53.

Plasma Emission Procedures

Instrument conditions The Spectraspan IIIa single-channel d.c. plasma emission spectrometer (Applied Research Laboratories, Luton, Bedfordshire, UK) was set up using an argon (high-purity grade, BOC Prescot, Merseyside, UK) flow of 2 1 min-1 across the tungsten and carbon electrodes. The samples were nebulised through a ceramic cross-flow nebuliser with argon carrier gas at 7 1 min-1. The sample was aspirated at 1ml min-1 (Minipuls peristaltic pump, Anachem, Luton, Bedfordshire, UK) and analysed over 2 x 5-s or 3 x 3-s integration periods. The exit slits of the spectrometer were set at 200 pm vertically and 500 pm horizontally and the entrance slits were set at 300 pm horizontally and 100 pm vertically. Platinum atomic emissions were measured at 265.95 nm, using the 85th order, to give maximum sensitivity and signal to noise ratio. Dynamic wavelength scanner (background corrector) The Spectraspan IIIa high-resolution kchelle monochromator was modified by fitting a quartz refractor plate behind the entrance slit. Movement of the refractor plate to give limited wavelength displacement at the exit slit was controlled by an Apple IIe microcomputer via a scanner controller and limited rotation motor. Wavelength positioning was achieved through an 8-bit digital to analogue converter (DAC) and data acquisition through a 12-bit analogue to digital converter (ADC). The 8-bit DAC permits 256 possible intensity measurement points within a wavelength scan of 0.015 nm. This modification was carried out by the Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow in conjunction with Professor J. M. Ottaway. Platinum assay procedures Plasma and urine. Aliquots (0.2 ml) of standards, plasma and urine were diluted with 1.8 ml of assay diluent. Several of the plasma samples from patients were cloudy, i.e., they contained protein precipitates caused by storage at 4°C and -20 "C. These were mixed thoroughly before dilution. The instrument was calibrated between 0 (diluent only) and 10 pg ml-1 of Pt and the unknown concentrations determined directly as pg ml-1 of Pt. Standard additions procedure. For the standard additions procedure tests were diluted five-fold (1 ml of test solution + 4 ml of assay diluent) and to 1 ml of this was added 1 ml of standard Pt solution to give 5.0,2.5,1.25,0.625 and 0 pg ml-1 of Pt. The solutions were then analysed using the calibrated instrument as above. Determination of non-protein bound platinum. In vitro experiments were used to measure the rate of binding of cisplatin to plasma proteins. Normal human plasma (25 ml) was pre-incubated at 37 "C for 20 min in the dark (to simulate in vivo conditions) and 200 pl of cisplatin were added (to give a final concentration of 2.6 pg ml-1 of Pt). The solution was mixed continuously and kept in the dark; 2-ml portions were removed at timed intervals (up to 9 h) and the free fraction separated by ultrafiltration. Plasma samples (2 ml) were centrifuged in GF-50 conical filters, pre-soaked in de-ionised water for 30 min, using the Amicon ultrafiltration system at 1000 g for 15 min. An aqueous Pt standard (1 pg ml-1) and a solution of cisplatin (1.3 pg ml-1 of Pt) were put through with each analysis to correct for losses of Pt on the ultrafiltration membrane. The ultrafiltrates were then diluted two-fold in the assay diluent and

assayed for free Pt, i.e. , the non-protein bound fraction. The half-life (t4) decay constant is then defined as the time (min) in which 50% of the cisplatin remains unbound. Clinical Studies

Nine patients with advanced head and neck malignancies, eight with advanced squamous cell carcinoma and one with an advanced salivary tumour, were treated with cisplatin and their Pt levels studied. The dosage of cisplatin was determined by reference to the endogenous creatinine clearance; a full dose of 100 mg m-2 (body surface area) was given when the clearance exceeded 60 ml min-1 and a half-dose of 50 mg m-2 for values between 50 and 60 ml min-1. Cisplatin was not administered to patients with clearances less than 50 ml min-1. The creatinine clearances ranged from 51 to 133 ml min-1 and the cisplatin dosage from 60 to 180 mg. Four patients were given a full dose and five patients a half-dose. Cisplatin was administered by 8-h intravenous infusion in 11 of isotonic saline containing 12.5 g of mannitol (to aid diuresis) in combination, via a Y-connector, with 500 ml of isotonic saline containing 5 mg kg-1 of Maxolon (an anti-emetic). Prior to treatment each patient was pre-hydrated with 2 1 of normal saline over 16 h. Following completion of the cisplatin infusion a further 1 1 of isotonic saline was infused. Blood samples were taken from patients at timed intervals during and after infusion into lithium heparin polypropylene tubes (Sarstedt, Leicester, UK). Urine was collected into 2.5-1 polypropylene bottles containing 10 g of boric acid as preservative, for up to 5 d. Aliquots were then stored at -20 "C before the determination of Pt.

Results Interference and Specificity

A comparative spectral analysis using the dynamic wavelength scanner of water, urine and human plasma (diluted as for the Pt assay) showed symmetrical spectra for Pt at 265.95 nm with no background or spectral interference (Fig. 1). Those elements present in relatively high concentrations in the biological samples, e.g., Na, K, Ca and Mg did not interfere with the platinum assay.




Fig. 1. Wavelength scans across the 265.95-nm platinum peak showing Pt emissions in ( a ) aqueous, ( b )plasma and (c) urine matrices at a concentration of 10 pg rnl-l of Pt

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Published on 01 January 1988. Downloaded by University of Regina on 26/10/2014 14:54:53.

Table 1. Accuracy and precision data, within and between batch, for the determination of platinum in plasma and urine by DCP-AES

Sample Within batch: Plasma .. Plasma .. Urine.. . . Urine. . . .


Between batch. Plasma .. Urine.. . . Urine.. . .

. .

Pt added*/ pgml- 1


Pt recovered/ pgml-1

+1 SD

Mean (range) recovered, '/o




12 10 9 9

1.08* 2.0 2.5 5.0

1.10 1.98 2.51 5.03

0.07 0.14 0.12 0.23

102 (92-1 12) 99 (92-1 13) 100 (94-108) 100 (95-105)

7.1 4.8 4.6

. . ..

10 8 8

1.08* 2.5 5.0

1.09 2.47 4.91

0.07 0.11 0.14

101 (93-112) 99 (92-105) 98 (94-102)

4.5 2.8

.. ..


* As cisplatin.





Platinum -6






Platinum addedipg ml-l Fig. 2. Standard additions experiments on urines (A)(1-4) and plasmas (V) (5-7) from patients treated with cisplatin. Values obtained by direct assay and by extrapolation: 1,

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