Development of an ultraperformance liquid chromatography/mass spectrometry method to quantify cisplatin 1,2 intrastrand guanine-guanine adducts

Abstract

Platinum chemotherapeutic agents have been widely used in the treatment of cancer. Cisplatin was the first of the platinum-based chemotherapeutic agents and therefore has been extensively studied as an antitumor agent since the late 1960s. Because this agent forms several DNA adducts, a highly sensitive and specific quantitative assay is needed to correlate the molecular dose of individual adducts with the effects of treatment. An ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay for quantification of 1,2 guanine-guanine intrastrand cisplatin adducts [CP-d(GpG)], using (15)N(10) CP-d(GpG) as an internal standard, was developed. The internal standard was characterized by MS/MS, and its concentration was validated by inductively coupled plasma mass spectrometry. Samples containing CP-d(GpG) in DNA were purified by enzyme hydrolysis, centrifugal filtration, and HPLC with fraction collection prior to quantification by UPLC-MS/MS in the selective reaction monitoring mode [m/z 412.5-->248.1 for CP-d(GpG); m/z 417.5-->253.1 for [(15)N(10)] CP-d(GpG)]. The recovery of standards was >90%, and quantification was unaffected by increasing concentrations of calf thymus DNA. This method utilizes 25 mug of DNA per injection. The limit of quantification was 3 fmol or 3.7 adducts per 10(8) nucleotides, which approaches the sensitivity of the (32)P postlabeling method for this adduct. These data suggested that this method is suitable for in vitro and in vivo assessment of CP-d(GpG) adducts formed by cisplatin and carboplatin. Subsequently, the method was applied to studies using ovarian carcinoma cell lines and C57/BL6 mice to illustrate that this method is capable of quantifying CP-d(GpG) adducts using biologically relevant systems and doses. The development of biomarkers to determine tissue-specific molecular dosimetry during treatment will lead to a more complete understanding of both therapeutic and adverse effects of cisplatin and carboplatin. This will support the refinement of therapeutic regimes and appropriate individualized treatment protocols.

Figure 1

MS isotope simulation of CP-d(GpG). There are three major masses for this adduct due to isotopes.

Figure 2

Representative SRM ion chromatograms comparing the use of singly (m/1) vs. doubly (m/2) charged state during MS/MS analysis of CP-d(GpG) analyte (A and B) and internal standards (C and D). A and C show MS/MS fragmentation in the singly charged state, while B and D show MS/MS fragmentation in the doubly charged state. The chromatogram has been cropped as no other quantifiable peaks are observed.

Figure 3

Full scan positive ion MS spectrum m/s 450 - 850 showing the in source fragmentation of CP-d(GpG). The main ions observed were [M-NH₂]⁺ m/z 807.1, [M- d(GpG)-NH₂]⁺ 497.2, [M- d(GpG)]⁺m/z 513.1, and [M-deoxyribose]⁺ m/z 708.1. All fragment ions contained the Pt characteristic isotopic profile.

Figure 4

Quantification of CP-d(GpG) in platinated ctDNA. Platinated calf thymusDNA was diluted with blank calf thymus DNA. Aliquots of 0, 25, 50, and 100 μg platinated calf thymus DNA were placed in eppendorf tubes with 500 fmol internal standard andthe corresponding amount of blank calf thymus DNA was added to bring the total amount per tube to 100 μg. Samples were processed through the hydrolysis method using HPLC clean-up and total adducts were quantified by UPLC-MS/MS.

Figure 5

Quantification of CP-d(GpG) in human ovarian carcinoma cells. Two isogenic cell lines, one sensitive (A2780) and one resistant (CP70) to cisplatin were treated with increasing (12.5-250 μM) doses of cisplatin. Where no error bars are visible, they are smaller than the symbol.

Scheme 1

Preparation and quantification of CP-d(GpG) adducts by UPLC-MS/MS. Cisplatin treated DNA is isolated. Next, internal standard is added and enzyme hydrolysis is performed. Following hydrolysis, solid phase extraction or HPLC enrichment separates the platinated adduct from nucleosides, enzymes and sodium salts. Finally, the CP-d(GpG) adduct is quantified by UPLC-MS/MS.