Quantitative determination of platinum complexes in human plasma generated from the oral antitumour drug JM216 using directly coupled high-performance liquid chromatography-inductively coupled plasma mass spectrometry without desolvation Peter Galettis, Jocelyn L. Carr, James W. Paxton and Mark J. McKeage* Department of Pharmacology and Clinical Pharmacology, Faculty of Medicine and Health Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand. E-mail: m.mckeage@auckland.ac.nz Received 6th January 1999, Accepted 19th April 1999 A new method was developed and validated for measuring platinum species generated from the clinical antitumour agent JM216 in methanol extracts of human plasma using high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS).Good separation of JM216 and three of its biotransformation products (JM118, JM518 and JM383) was achieved with a run time of 20 min using a C8 column (4.6×150 mm) and a gradient methanol–water mobile phase (pH 2.5) at a flow rate of 1 ml min-1.The presence of methanol in the mobile phase and in the sample matrix suppressed the platinum counts and the gradient step was associated with some base-line drift. However, the quantitation of JM216 and its biotransformation products (JM118, JM518 and JM383) was achieved with good intra-assay precision (range 1–12% RSD), inter-assay precision (range 2.3–11% RSD), accuracy (range 89–103%) and limits of quantitation (range 1–2 ng ml-1) without having to use a desolvation device.This new HPLC-ICP-MS technique has the advantages of greater sensitivity and eYciency compared to existing methods that use HPLC, fraction collection and the oV-line detection of platinum by AAS. biotransformation products by using ICP-MS detection Introduction systems directly coupled to a HPLC. Platinum compounds have been used in cancer chemotherapy Cairns et al.6 directly coupled an HPLC and ICP-MS using since the 1970s.Up to now, the concentrations of platinum a desolvation device in order to allow the use of HPLC containing species in blood and urine have been measured by solvents that would otherwise destabilise the argon plasma high-performance liquid chromatography with on-line ultra- and to permit the use of a HPLC solvent gradient that could violet or electrochemical detection or the oV-line analysis of be associated with baseline drift.Using this technique they HPLC fractions by graphite furnace atomic absorption spec- achieved good separation and limits of detection for JM216. trometry.1 Studies employing these techniques have demon- Before the method can be used for the quantitative analysis strated that the concentrations of low molecular weight of clinical samples, however, it must be validated and proven platinum species determine the likelihood of tumour response to be precise, accurate and sensitive in the determination of and toxicity during platinum-based cancer therapy.1 JM216 in the matrix of interest.Moreover, information on JM216 [Pt(NH3) (NHC6H11)(OCOCH3)2Cl2] is a new the plasma concentrations of the biotransformation products platinum complex currently in phase III clinical trials and is generated from JM216 is also of interest. The platinum species showing promise in the treatment of prostate cancer.2 JM216 were expected to form adducts with plasma proteins with low biological activity, and free platinum species were extracted diVers from the existing clinical platinum agents in that it is from plasma using methanol.administered by mouth rather than by intravenous injection, In an extension of the studies of Cairns et al.,6 we attempted with the potential advantages of increased patient convenience, to develop and validate a quantitative assay for JM216 and better quality of life and lower cost of delivering the treatment. three of its biotransformation products (JM118, JM518 and To date, studies of the clinical pharmacokinetics on JM216 JM383), using directly-coupled HPLC-ICP-MS with diVerent have been diYcult because of the low concentrations of chromatography conditions that made it unnecessary to platinum present in plasma ultrafiltrate (<100 ng ml-1)3 and desolvate the mobile phase before it entered the ICP-MS.the extensive biotransformation of this drug in patients.4 Although the presence of methanol in the mobile phase and In order to obtain information on the pharmacokinetic in the sample matrix suppressed the platinum counts, the assay behaviour of JM216 in cancer patients, a new analytical achieved adequate precision, accuracy, sensitivity and sample technique is required that is capable of separating the various throughput for quantitative pharmacokinetic studies.7 platinum species generated from JM216 and detecting these individual species at concentrations down to 1 ng ml-1.A HPLC-AAS technique has been developed for this purpose4 Description of experimental procedures but it has a sensitivity limit of 10–50 ng ml-1 and poor sample Instrumentation throughput due to the oV-line analysis of HPLC fractions.5 We and others6 have attempted to develop more sensitive and Samples were introduced into a HP4500 inductively coupled plasma mass spectrometer (Hewlett-Packard, Yokowaga, eYcient methods for analysing clinical samples for JM216 J.Anal. At. Spectrom., 1999, 14, 953–956 953Table 1 HPLC-ICP-MS conditions for quantifying platinum species Japan) using a Babington-type (V-groove) nebulizer and a in human plasma Scott double-pass spray chamber. The HPLC consisted of a HP1100 binary pump, a rheodyne injector and a 20 ml sample Chromatography— loop (Hewlett-Packard, Wilmington, DE, USA) and a Prodigy Stationary phase Phenomenex Prodigy C8 C8 column (4.6×150 mm) (Phenomenex, Auckland, New (4.6×150 mm) Mobile phase A 25% methanol–0.01% Zealand).The HPLC was connected to the ICP-MS nebulizer orthophosphoric acid pH 2.5 by a 10 cm piece of 0.25 mm diameter PEEK tubing. B 100% Methanol Chromatograms were processed using HP chromatographic Gradient 0–10 min 100% A analysis software. 10–20 min 80% A 20% B Flow rate 1.0 ml min-1 Injection volume 20 ml Reagents ICP-MS— HPLC grade methanol (Labscan, Dublin, Ireland), ortho- Forward power 1350 W phosphoric acid (Riedel-de Ha�en, Seelze, Germany), 0.9% Reflected power <5 W sodium chloride (v/v) (Baxter Healthcare, Old Toongabbie, Gas flow rates: Australia), Milli-Q water (Millipore, Bedford, MA, USA) and Plasma 15 l min-1 Auxiliary 1 l min-1 fresh frozen human plasma (New Zealand Blood Service, Nebuliser 1 l min-1 Auckland, New Zealand) were used for preparing standards, Sampling depth 8 mm samples and the mobile phase.JM216 [Pt(NH3)- Sample uptake rate 1 ml min-1 from HPLC (NHC6H11)(OCOCH3)2Cl2] and its biotransformation prod- Sampler (nickel ) orifice 1 mm ucts JM383 [Pt(NH3) (NHC6H11)(OCOCH3)2(OH)2], JM118 Skimmer (nickel ) orifice 0.4 mm [Pt(NH3) (NHC6H11)Cl2] and JM518 [Pt(NH3)(NHC6H11)- Spray chamber temperature 1 °C Mass range 194–195 (OCOCH3)2(OH)Cl ] were generously supplied by the Johnson Acquisition time 1 s Matthey Technology Centre (Sonning, Oxfordshire, UK).Sample preparation Results and discussion Methanolic extracts of plasma were prepared by adding 100 ml The chromatographic conditions shown in Table 1 achieved of ice-cold methanol to 100 ml of plasma and mixing the good separation of JM216 and its biotransformation products sample before leaving it to sit at -20 °C.After 18 h, the (JM118, JM518 and JM383) with a run time of about 20 min sample was centrifuged at 20 000g for 20 min and the super- (Fig. 1). A change in the mobile phase composition at 10 min natant was removed for analysis. JM216 and its biotransformto an increased methanol content was associated with suppres- ation products JM118, JM518 and JM383 were stable in sion of platinum counts by approximately 70% and smaller methanol extracts of plasma at -20 °C for at least 28 d.peaks for compounds JM216 and JM518 compared with compounds JM383 and JM118. Platinum-194 and -195 were Assay validation used in the determination of the platinum species and there was excellent agreement between the isotopes, consistent Stock solutions of JM216, JM118, JM518 and JM383 were with a lack of mass interference.made up in methanol at 1, 10 and 100 mg ml-1. Standard Calibration standards were made up in human plasma for solutions of JM216, JM118, JM518 and JM383 in plasma were made by adding the stock solutions to plasma at 8 diVerent concentrations ranging from 1 to 120 ng ml-1. Quality control samples were made up from separate stock solutions at 7.5 and 75 ng ml-1 in plasma. The standards and quality control samples were prepared for analysis by methanol extraction.Precision was defined as the relative standard deviation (RSD) on the same day (intra-assay precision) and diVerent days (inter-assay precision) of repeated measurements of JM216, JM118, JM518 and JM383 in plasma at 7.5 and 75 ng ml-1.7 Accuracy was calculated by comparing the measured and expected concentrations of JM216, JM118, JM518 and JM383 at 7.5 and 75 ng ml-1 in plasma.7 The limit of quantitation was defined as the lowest concentration that could be measured with precision and accuracy within the limits (20% RSD) that are acceptable for analytical methods validation.7 Selection of chromatography conditions The starting point was an isocratic methanol–water (10% v/v) mobile phase (pH 2.6) because these conditions have previously been used to separate platinum species generated from cisplatin.8 Methanol was chosen as an HPLC solvent because of its lower vapour pressure and lower carbon loading compared with acetonitrile since these factors could influence the stability of the argon plasma.The HPLC conditions produced Fig. 1 Chromatogram of a methanol extract of human plasma contain- a run time of over 2 h. Changing to a C8 column, increased ing 75 ng ml-1 of JM383 [Peak A, Pt(NH3) (NHC6H11)(OCOCH3)2- methanol content and a step gradient at 10 min reduced the (OH)2], JM118 [Peak B, Pt(NH3) (NHC6H11)Cl2], JM518 [Peak C, run time and achieved separation of the platinum compounds Pt(NH3) (NHC6H11)(OCOCH3)2(OH)Cl ] and JM216 [Peak D, Pt(NH3) (NHC6H11)(OCOCH3)2Cl2].at a flow rate of 1 ml min-1. 954 J. Anal. At. Spectrom., 1999, 14, 953–956each of the platinum compounds at 8 concentrations ranging from 1 to 120 ng ml-1. The standards were processed before analysis by carrying out a methanolic extraction. The calibration curves were linear over the concentration range with correlation coeYcients of greater than 0.99. Standards made up in human plasma and then processed by methanolic extraction had platinum counts that were 10–20% lower than the counts of standard solutions, which were made up in 0.9% sodium chloride (v/v) and analysed without further processing, consistent with a significant matrix eVect.The accuracy and precision of the assay were determined by making up quality control solutions of the four platinum compounds in human plasma at 7.5 and 75 ng ml-1 and analysing methanolic extracts of these solutions. The methanolic extracts of the quality control solutions were stable in storage (-20 °C for 9 d).The accuracy, intra-assay precision and inter-assay precision (Table 2) were within the limits that have been stated to be acceptable for quantitative pharmacokinetic studies.7 The limit of quantitation, defined as the lowest concentration that could be measured with acceptable accuracy and precision, was 2 ng ml-1 for compounds JM216 and JM518, and 1 ng ml-1 for compounds JM118 and JM383.The platinum compounds were unstable in human plasma at room temperature with the loss of 11% and 22% of compounds JM118 and JM216, respectively, within 2 h. The compounds were more stable in methanol extracts of blood plasma and when stored at 4 or -20 °C. When methanol extracts of plasma were stored at -20 °C all four compounds (JM216, JM118, JM518 and JM383) were stable for at least 28 d. Measurement errors related to the poor stability of the compounds could be avoided by processing samples immediately after blood has been collected by the preparation of methanol extracts of the plasma and then storing the methanol extracts at -20 °C until analysis.The other analytical method currently available for quantitating platinum species generated from JM216 in human plasma involves high-performance liquid chromatography, fraction collection and oV-line detection of platinum using atomic absorption spectrometry.4 The HPLC-ICP-MS method described here has improved eYciency compared with the HPLC-AAS technique because the detection system is directly coupled to an HPLC and there is no requirement for the collection of fractions or for the oV-line analysis of fractionated samples.The HPLC-ICP-MS method also has lower limits of detection for platinum compounds generated from JM216 compared with the HPLC-AAS technique.4 Despite the suppression of platinum counts by the presence of methanol in the mobile phase and in the sample matrix, the HPLC-ICP-MS Fig. 2 Chromatograms of methanol extracts of plasma taken from a method has the advantages of greater sensitivity and eYciency patient 40 min (a) and 4 h (b) after taking a 270 mg oral dose of JM216. over the HPLC-AAS technique. To demonstrate the feasibility of detecting platinum species in plasma from cancer patients given JM216 by mouth, samples At 40 min, JM216 appears in plasma at 307 ng ml-1 in association with a metabolite (JM118) at 844 ng ml-1. By four taken during a clinical trial5 were prepared for analysis.Fig. 2 shows chromatograms of methanol extracts of plasma taken hours the concentrations of JM216 and JM118 have fallen to 9.3 ng ml-1 and and 38 ng ml-1, respectively. At this time a from a patient 40 min and 4 h after an oral dose of JM216. Table 2 Performance of the assay Platinum compounds JM118 JM383 JM518 JM216 Plasma concentration 7.5 ng ml-1 Intra-assay precision (RSD) (n=6) 6.2 6.9 6.7 11.9 Inter-assay precision (RSD) (n=3) 4.8 11.0 2.3 4.4 Accuracy (%) 95 99 102 94 75 ng ml-1 Intra-assay precision (RSD) (n=6) 5 1 5 2 Inter-assay precision (RSD) (n=3) 9 3 7 10 Accuracy (%) 103 99 89 102 Limit of quantitation/ng ml-1 1 1 2 2 J.Anal. At. Spectrom., 1999, 14, 953–956 955D. Vaughn, C. Brassard, D. Lebwohl and R. Bukowski, Proc. Am. new platinum containing species has appeared, eluting with a Soc. Clin. Oncol., 1998 17, 314. retention time of 1.2 min.The chemical structures of this and 3 M. J. McKeage, F. Raynaud, J. Ward, C. Berry, D. O’Dell, several other uncharacterised platinum containing species L. R. Kelland, B. A. Murrer, P. Santabarbara, K. R. Harrap and found in the plasma of patients given JM216 are currently I. R. Judson, J. Clin. Oncol., 1997 15, 2691. under investigation. 4 F. I. Raynaud, P. Mistry, A. Donaghue, G. Poon, L. R. Kelland, C. F. J. Barnard, B. Murrer and K. R. Harrap, Cancer Chemother. Pharmacol., 1996, 38, 155. Acknowledgments 5 M. J. McKeage, P. Mistry, J. Ward, F. E. Boxall, S. Loh, C. O’Neill, P. Ellis, L. R. Kelland, S. E. Morgan, B. Murrer, The authors would like to thank Dr. F.I. Raynaud for P. Santabarbara, K. R. Harrap and I. R. Judson, Cancer providing the blood samples and the Johnson Matthey Chemother. Pharmacol., 1995 36, 451. 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