Separation and identification of desferrioxamine and its iron chelating metabolites by...

ANALYTICAL BIOCHEMISTRY 187,212-219 (1990)

Separation and Identification of Desferrioxamine and Its Iron Chelating Metabolites by High-Performance Liquid Chromatography and Fast Atom Bombardment Mass Spectrometry: Choice of Complexing Agent and Application to Biological Fluids

Surinder Singh,* Robert C. Hider,* and John B. Porter? *Department of Pharmacy, King’s College, University of London, Manresa Road, London S W3 6LX, United Kingdom, and TDepartment of Haematology, University College Hospital and Middlesex School of Medicine, 98 Chenies Mews, London WClE 6HX, United Kingdom

Received November 17.1989

An HPLC-based method capable of separating desferrioxamine (DFO) and its iron chelating metabolites from uv-absorbing species present in biological fluids has been developed. This method relies on the use of nitrilotriacetic acid (NTA) as the complexing agent in the mobile phase, instead of EDTA, previously used in HPLC methods. The use of NTA ensures that iron contamination present in buffers and bound to the column does not interfere with analysis. The disadvantages of using EDTA are discussed. The identity of the iron chelating metabolites of DFO present in the urine of patients with &thalassemia major has been established using FAB mass spectrometry. The metabolism of DFO, reported in this study, takes place almost exclusively at the N-terminal region of the molecule and is in many respects similar to the degradation of the amino acid lysine. In addition, a metabolite which corresponds to N- hydroxylation of the terminal amino group has been identified. 0 1990 Academic Press, Inc.

Desferrioxamine B (DFO)’ is a naturally occurring siderophore produced on an industrial scale by fermen- tation of the fungi, Streptomycespilous. It contains three hydroxamic acid groups and reacts stoichiometrically with tripositive metal ions such as iron and aluminium to form octahedral complexes with very high binding

’ Abbreviations used: DFO, desferrioxamine; DFO-met, desferrioxamine metabolite; FO, ferrioxamine; MeCN, acetonitrile; MeOH, methanol; NTA, nitrilotriacetic acid, NTBI, non-transferrin-bound iron; RT, retention time; FO-met, ferrioxamine metabolite.

212

affinities. It is widely used to treat iron overload disor- ders such as thalassemia major which result from fre- quent blood transfusions and diseases related to a chronic increase in dietary iron absorption such as idio- pathic hemochromatosis (l-5). The excess iron (particu- larly in the case of thalassemia patients where venesec- tion is not possible) must be removed by chelation therapy in order to prevent death due to failure of organs such as the heart, liver, and endocrine tissue. DFO is also used to remove aluminium from patients suffering from impaired renal function undergoing hemodialysis treatment (6,7).

One of the major drawbacks of DFO is its lack of oral activity. When administered as a single intramuscular injection, it is only capable of removing a relatively small amount of iron and is unable to keep pace with iron accumulated from the diet and transfusion regimen in thalassemia major patients. In order to achieve negative iron balance, DFO must be infused subcutaneously over a period of 8-10 h for 5-7 days a week. This is partly due to its short plasma half-life (8) and the limited amount of chelatable iron present at any one time.

Serious side effects have been observed when doses of 40 mg/kg are administered to hemodialysis patients (9). These toxic effects are similar to those encountered with thalassemic patients taking higher doses of DFO (5). The cause of this toxicity is presently unknown, but it has been suggested that the removal of other vital metal ions such as zinc and copper may be responsible (10). However, certain metabolites of DFO may also be responsible (11).

Despite having been introduced into clinical use in the early sixties (12), very little is known about the metabo-

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SEPARATION OF IRON CHELATING METABOLITES OF DESFERRIOXAMINE 213

lism of DFO. Neither the metabolic profile nor a complete pharamacokinetic study of the drug and its iron complex has been reported to date. This is absolutely critical, both in terms of determining the possible toxicity of the metabolites and in terms of providing information on the dose required for maximal therapeutic bene- fit. The above information has not been forthcoming largely due to the lack of a sensitive and specific chromatographic method that is capable of separating both free and iron-bound forms of the drug and their respec- tive metabolites.

Early analytical techniques employed to study the metabolism (13) and pharmacokinetics (8) of DFO both in the free and in the iron-bound forms relied on the spectroscopic properties of ferrioxamine (FO). These methods are influenced by other species present in serum and urine which are capable of complexing with iron and forming interfering chromophores. However, the major limitation of such techniques is their inability to distin- guish between DFO and its iron chelating metabolites, as they both have identical spectroscopic properties. More recently, methods based on inductively coupled plasma atomic emission spectrometry (14) and atomic absorption spectrometry (15) were used to quantify DFO in plasma for pharmacokinetic analysis. These methods rely on the addition of iron to biological samples in which DFO is presumed to be converted to FO and subsequently extracted into benzyl alcohol. The above assays are indirect in that they depend on the quantifica- tion of extracted iron bound to the drug rather than measurement of DFO or its metabolites. Such analytical procedures are prone to errors because it is possible that added iron, bound to other ligands present in serum or urine, could be extracted into benzyl alcohol. The binding region in nearly all the metabolites of DFO remains unaffected and hence they retain similar metal chelating abilities. The major weakness of these methods, like those of the calorimetric assays, is their inability to dis- tinguish between DFO and its metabolites. In principle, an HPLC-based method could provide a more complete picture for the formation and clearance of the two forms of the drug and their metabolites. A number of HPLC- based methods have been developed (11,16-21). How- ever, some are not readily applicable to biological fluids (16,19) and others are influenced by iron contamination (17). Some methods are unable to obtain sufficient reso- lution to separate the iron chelating metabolites and the free drug from other uv-absorbing species present in biological fluids (18). A method specifically developed to overcome all the problems mentioned above is presented in this paper.

MATERIALS AND METHODS

Chemicals

DFO was obtained from Ciba-Geigy (Horsham, UK); NTA (disodium salt), EDTA (disodium salt), ammo-

nium formate, potassium dihydrogen phosphate, dipo- tassium hydrogen phosphate, and trifluoroacetic acid were obtained from Aldrich. HPLC grade methanol (MeOH) and acetonitrile (MeCN) were purchased from BDH (Poole, UK) Mini-Q water was used throughout the study.

Competition Study

Competition studies were performed using a 1:l ratio of 25 yM iron:DFO against varying concentrations of EDTA and NTA prepared in either Chelex loo-treated 50 mM phosphate buffer (pH 7.0) or 50 mM ammonium formate (pH 3.8). The decrease in absorbance of the ferrioxamine chromophore at 430 nm was monitored against time using a Perkin-Elmer Lambda 5 spectro- photometer.

Sample Storage and Preparation

Serum samples obtained from patients undergoing continuous DFO infusion were stored frozen at -20°C within 1 h of collection, until time of analysis. The above procedure was used to minimize the loss of metabolite D which can undergo slow oxidation. Urine samples obtained from the same patients were stored in an identical manner.

The only preparative step performed was ultrafiltration of serum using Amicon Centriflow CF-25 filter cones. The above procedure was not required for urine due the low protein content. Typically 30 ~1 of urine or serum ultrafiltrate was injected onto an HPLC column.

HPLC System

A Hewlett-Packard Model 1090M complete with an autoinjector, autosampler, and diode-array detector at- tached to a HP900-300 data station was used. An LKB-Bromma 2212 fraction collector was used to collect fractions from the LC. Waters (Milford, MA) radial compression columns (10 cm X 8 mm i.d.; 4 pm, Novapak ODS) equipped with a precolumn cartridge (Waters, 10 pm, Bondapak ODS) were used for analysis of biological samples. A steel column (Hewlett-Packard, 10 cm X 4.6 mm i.d.; 5 pm, Hypersil ODS) was used to demonstrate the presence of iron contamination in conventional HPLC systems. The chromatographic conditions used in this study are as follows: flow rate = 1.5 ml/min; pump A (20 mM phosphate, pH 7.0, containing 2 mM NTA); pump B (20 mM phosphate, pH 7.0, containing 2 mM

NTA in 50% acetonitrile). The following gradient program was used (min/%B): O/O; l/O; 20/l& 24/25; 28/35; 31/35; 32/O. A post run time of 10 min was allowed for reequilibration of the column between injections.

FAB Mass Spectrometry of Isolated Metabolites

Fractions containing the iron-complexed metabolites were desalted using Sep Pak ODS cartridges (Waters,

214 SINGH, HIDER, AND PORTER

Ferrioxamine

ksferrioxamine

J

Retenton time for Ferrioxamme

Time (min)

FIG. 1. (A) HPLC chromatogram of DFO on a conventional stainless-steel column in the absence of a complexing agent. Conditions: mobile phase, A = 20 mM phosphate buffer (pH 7); B = MeCN, 0% B + 40% B (30 min, linear gradient); column, Hypersi15 ODS (10 cm X 4.6 mm i.d.); flow rate, 1 ml/min; uv, 235 nm. (B) HPLC analysis of DFO in the presence of a complexing agent (2 mM NTA). No peak corresponding to FO is present, confirming the removal of interfering iron contamination. Column, Waters radial compression column, No- vapak 4 ODS (10 cm X 8 mm i.d.); flow rate, 1.5 ml/min; uv, 235 nm. For mobile phase composition and gradient program, refer to Materi- als and Methods.

Milford, MA). The samples were applied to the cartridges, washed with 2 ml of water, followed by 2 ml of 0.1% trifluoroacetic acid solution, and finally eluted with 3 ml of MeOH/MeCN (l/l, v/v). The solvent was then evaporated to dryness under a stream of nitrogen and redissolved in MeOH. FAB mass spectrometry was carried out using a Kratos MS-890 mass spectrometer fitted with an argon atom gun and FAB source. Samples were loaded in a glycerol/MeOH solution onto a stainless- steel target and bombarded with argon atoms having 6 keV of energy.

RESULTS AND DISCUSSION

Selection of Mobile Phase Chelating Agent

When DFO is injected onto a conventional HPLC system equipped with a stainless-steel reversed-phase Hy- persil ODS column, two peaks result (Fig. 1A). The first

peak absorbs both in the visible and in the uv region of the spectrum and corresponds to FO, and the second corresponds to that of DFO. All buffers, unless pretreated with a chelating resin, are contaminated with variable amounts of iron and this almost certainly accounts for much of the iron bound by DFO. Another important source of iron for chelation by DFO is iron bound to free silanol groups of the stationary phase. This is character- istic of the broadening seen for the FO peak (Fig. 1A). Cramer et al. (16) overcame this problem by the addition of the complexing agent EDTA in their mobile phase and were able to simultaneously monitor DFO and FO at 220 and 430 nm. Unfortunately their assay as described is not readily applicable to biological systems, An alternative method of overcoming the problem of iron contamination is to use an “iron-free” HPLC system (19,21). To be a truly iron-free system, all parts made of stainless steel such as the pumps, injection system, connecting tubing, and columns must be replaced by in- ert materials such as glass, Teflon, and titanium. In addition, the mobile phase should be treated with a chelating resin to remove iron contamination which would otherwise bind to the silanol groups of the stationary phase and build up to relatively high concentrations. This does not appear to have been carried out in the iron-free HPLC methods that have been described to date.

Although the use of an iron-free system probably pro- vides the ideal answer, it is a rather specialized and ex- pensive alternative to the simple incorporation of a complexing agent in the mobile phase. Unfortunately, the use of EDTA under many assay conditions is unsatisfac- tory, since it is capable of removing iron from FO (21). The effectiveness of iron removal depends on the concentration ratio of the ligand and the pH of the solution.

Time(minb

FIG. 2. Competition by NTA and EDTA for FO (25 PM) iron at different pH values and concentration ratios with respect to time: (0) NTA (4000:1, pH 7); (m) EDTA (200~1, pH 7); (V) EDTA (400:1, pH 7); (0) EDTA (1600~1, pH 7); (0) EDTA (200:1, pH 3.8).


300 -

200-

? -

100 -

1 10 20 30

Time (min)

FIG. 3. Separation of FO and DFO under chromatographic conditions used in this study (see Materials and Methods).

Due to its much lower pK, values, EDTA has a relative ability to bind iron greater than that of DFO at acidic pH values. A competition study was carried out in order to assess the above problem under the assay conditions (pH 7). The requirements of the ideal complexing agent are that it should remove all iron contamination present in the mobile phase, column, connecting tubing, and me- chanical parts of the system without competing for iron at high complexing agent to FO ratios, in order to opti- mize scavenging efficiencies. The concentration of the complexing agent in the mobile phase is typically milli- molar, whereas the concentration of FO or its metabolites injected onto the column is in the micromolar range. Additionally the contact volume between the complexing agent and the FO must also be considered. This value depends on the flow rate of the mobile phase and the retention time of the iron complex. Thus the concentration ratio of ligand to FO under most analytical conditions is always likely to be in excess of lOOO- fold. At pH 3.8, a 200-fold excess of EDTA results in nearly 90% of the iron being removed from FO over a 30-min period (Fig. 2). In contrast, under neutral and otherwise identical conditions, only 3.6% of the iron is removed. However with a 1600-fold excess of the ligand, greater than 50% of the iron was removed (Fig. 2). Such exchange would lead to a gross underestimation of the amounts of iron bound to the drug. Clearly an alternative ligand with a lower binding affinity for iron is required for this type of assay. This ligand should be one which will not compete for FO iron, but yet scavenge trace metals. Such a compound is unlikely to be a hexadentate ligand and would ideally contain acidic pK, values. One such compound is NTA, which does not compete for FO iron even at a 4000-fold excess at pH 7.0 (Fig. 2). Its ability to remove all iron contamination present in the system was confirmed by injecting DFO. No peak corresponding to FO was detectable (Fig. 1B). NTA was

subsequently used at a concentration of 2 mM in the mobile phase.

Development of Elution Conditions

It was decided to limit the number of sample preparation steps for the sake of accuracy and reproducibility. Since DFO and its metabolites have different net charges, preparative ion-exchange cartridges are unsuitable. Liquid-liquid extractions with adjustment of pH are also unsuitable and at extremes of pH there is the possibility of iron release by the ligands or ferric-hydrox- ide formation. In principle Sep Pak ODS cartridges could be used to remove polar compounds typically found in the biological fluids. However, this step was not introduced as such polar components elute early during HPLC analysis and do not generally interfere with the peaks of interest. Thus the only preparative step performed was deproteinization of serum by ultrafiltration.

Time (min)

mo

200

2

100

10 20 30 J

Time (mtn)

FIG. 4. HPLC chromatogram of thalassemic urine monitored simultaneously at (A) 430 nm and (B) 235 nm.


FO Metkmlite D

FIG. 5. Three-dimensional plot of iron complexes of DFO metabolites monitored at wavelengths, 230-600 nm (chromatogram corresponds to that presented in Fig. 4).

A reversed-phase ODS radial compression column was used routinely in this assay as it minimizes interparticle void volumes and reduces the “wall effect” found in stainless-steel columns. This, coupled with the use of 4- pm ODS-bound silica particles as column packing mate- rial, achieved the high efficiency (10,000 plates/l0 cm) required for successful analysis.

In order to enhance both the efficiency and the reproducibility of the analysis, a method capable of simultaneously quantifying DFO and FO, without the need for multiple wavelength detection or comparison with iron- saturated samples, was designed. Separation of FO, DFO, and its metabolites (both free and iron bound) from uv-absorbing species present in serum and urine was achieved. This elution system resulted in good reso- lution and peak shape (Fig. 3).

Standard curves of both peak area and height against varying concentrations of DFO and FO resulted in linear behavior with correlation coefficient values 2 0.99. The on-column detection limits (30 ~1 injection volume) of this assay are 1.5 and 1.0 pg/ml for DFO and FO, respectively. An increase in injection volume from 30 to 150 ~1 reduces the absolute limit of detection of DFO and FO to 0.3 and 0.2 Kg/ml, respectively.

Metabolism of Desferal

Early studies on DFO suggested that the major site of metabolism (as determined by the iron binding ability of

DFO) occurs in blood serum (13). The enzyme responsible was reported to be a serum a2-globular protein. The decrease of DFO concentration in serum was reported to be most rapid in rats, cats, pigs, rabbits, bovines, and guinea pigs (in decreasing order). A striking feature however was the low activity of human or dog serum. More recently it was suggested that the destruction of DFO could be due to hydrolysis of the drug by various hydro- lytic enzymes (11). However, in the present work, DFO and FO, when incubated with fresh human serum at 37°C remained unchanged for periods up to 5 h as esti- mated by HPLC. That DFO is unaffected when incubated in fresh human blood serum for prolonged periods indicated that, if hydrolysis is occurring, it is a slow process.

Analysis of urine samples, obtained from a patient with thalassemia major undergoing continuous infusion of DFO at a dose of 100 mg/kg/24 h during the time of sampling, demonstrated the presence of five peaks when monitored at 430 nm (Fig. 4A). The metabolites so detected are named according to their order of elution. This column eluant was also monitored in the uv region (235 nm; Fig. 4B). A near baseline separation of both free and iron-bound metabolites of the drug from other uv-absorbing species was achieved. These metabolites have identical spectroscopic properties (Fig. 5); i.e., they contain identical chromophores. Thus the environment around the metal ion is largely unaffected for all the me-


I 6

0 00 450 500 550 600

i)

(M+l

-

00 450 500 550 600 Ii) 453 676 600

628

h I 1

1

400 450 500 550 600 650

iv) 423

(M+H)+

417 466 692 614

435

IIll Iltl llhil II 1111 I I I I III

400 450 500 550 600 6si

FIG. 6. FAB mass spectra (iron complexes) of(i) DFO, (ii) metabolite A, (iii) metabolite B, (iv) metabolite C, and (v) metabolite D.

tabolites and they are therefore likely to retain similar iron binding abilities.

In contrast to the urine samples of the above patient, serum samples were only found to contain DFO metabolites bound to iron. Iron-free DFO metabolites, if present, were at levels below the detection limit. Identical results were found with either serum or plasma samples

from a number of thalassemia major patients undergoing similar treatment regimes. The concentration of DFO metabolite iron complexes in serum samples was always found to be much lower than the corresponding urine levels. Thus a typical concentration of iron complex of metabolite B in serum is 4-8 PM, whereas in urine the levels approach 150-300 PM. Varying amounts of un- complexed DFO and DFO metabolites (dependent on the iron status of the patient) are also present in the urine.

The two main peaks separated by HPLC (Fig. 4) correspond to FO (RT = 20.1 min) and the iron complex of metabolite B (RT = 18.4 min), the major metabolite. The identity of FO was confirmed by its protonated molecular ion m/z = 614 (Fig. 6i). The iron complex of DFO-met B has a protonated molecular ion (M + H)+ of 629 (Fig. Giii). This species corresponds to the same oxidative deamination product of the parent drug first characterized by Keberle (22) and more recently by Leh- man and Heinrich (21).

Metabolite A (iron complex) possesses a protonated molecular ion (M + H)+ of 601 (Fig. 6ii), which can be formed from metabolite B via P-oxidation where two methylene groups are removed (Scheme I). The reduc- tion in the chain length of metabolite A is consistent with the observed decrease in the retention time of its iron complex relative to that metabolite B (Fig. 4). The two additional metabolites which correspond to the iron complexes of metabolite C (RT = 21.8 min) and metabolite D (RT = 25.1 min) have positive FAB molecular ions of 585 (Fig. 6iv) and 630 (Fig. 6v), respectively. Metabo- lite C is likely to be formed via decarboxylation of metabolite B (Scheme I). Formation of metabolite C, like most decarboxylation reactions of amino acids, is probably catalyzed by a pyridoxal phosphate-dependent de- carboxylase. Despite the chain length of metabolite C being considerably reduced compared to that of the parent drug, its corresponding iron complex elutes after FO. This can be explained by the net neutral charge of the iron complex of metabolite C under chromatographic conditions, in contrast to FO, which is positively charged. DFO is also metabolized via the classic route for aliphatic primary amines, where N-hydroxylation takes place to form metabolite D, the hydroxylamine deriva- tive of the drug. This compound, typical of most hydrox- ylamines, has limited stability and can undergo slow oxidation to form the nitroso and nitro derivatives. Metabolites C and D have not been previously reported.

The metabolism of DFO, which can be considered as a small peptide, parallels that of the basic amino acid, lysine (Scheme I). This is reflected by metabolites A, B, and C which result from oxidative deamination, P-oxidation, and decarboxylation of DFO. The above enzymatic reactions are not known to take place extracellularly. By analogy with lysine metabolism (23), the predominant site of metabolism is probably liver mitochondria. An


14-c -(cH,),-N-C- -

a 8

Monooxygenase j MW. 629 Mytabolite D

J- HO-C+H&-N-C---

8 Hb !

MW 628 Metabolite 6

oxidation

O=N--(CH.&-N-C-

Hb ! I !

Further

oxidation

* 0,N-(CH2)5-N-C--

Hd a

SCHEME I. An overview of the metabolic profile of DFO. All the metabolites were isolated as iron complexes.

overview of the metabolic pathway of DFO is depicted in Scheme I.

Implications of DFO Metabolism

From the scheme of metabolism for DFO (Scheme I) a number of inferences can be made regarding the likely distribution of DFO and its metabolites between extracellular and intracellular compartments. The uptake and efIlux of chelators into cells will be influenced by several factors including the size, shape, lipid solubility, and net charge of the free chelate and iron complex. It is likely that the net charge of DFO and its metabolites will have an important bearing on the relative distribution of these species intracellularly or extracellularly. Due the Nernst potential, charged species distribute across membranes until an electrochemical equilibrium is at- tained. For a membrane potential of 70 mV, an equilibrium distribution ratio of 15 will result for singularly charged species. Thus positively charged species are accumulated by cells due to the negative interior of cells, whereas negatively charged molecules accumulate extracellularly. Thus it is likely that DFO which is positively charged will be accumulated by cells.

When the hexadentate DFO molecule coordinates iron(III), three protons are displaced, thus FO, like DFO, possesses a net positive charge. As a consequence of this charge both will tend to be accumulated by cells. FO, unlike DFO, possesses a bulky spherical shape and once formed is unlikely to be metabolized or permeate biological membranes rapidly. In contrast the linear DFO molecule is rapidly metabolized and both the par-

ent compound and its metabolites can penetrate cells, possibly via facilitatory transport systems. Intracellular metabolism of DFO to negatively charged metabolites thus leads to a relatively fast efflux of the molecules from the cells. Metabolites which fail to coordinate iron intracellularly will tend to efhux, whereas the fraction which coordinates intracellular iron will efflux much more slowly, due to their bulky shape. It is likely therefore that most of FO and its iron containing metabolites detected in the serum of iron-overloaded patients have resulted from complexation of extracellular iron. A possible ex- ception to this scheme may be biliary excretion of DFO, FO, and metabolites which almost certainly occurs by an active process (24).

This extracellular origin of the iron coordinated by FO and its metabolites (FO-met) is supported by prelimi- nary clinical observations measuring non-transferrin- bound iron (NTBI) in iron-overloaded patients treated with DFO. At high serum NTBI levels, a lower proportion of metabolites has been observed, presumably because less DFO is available to influx into cells following the formation of FO extracellularly. Conversely in patients with low NTBI levels, a higher proportion of metabolites has been seen. Thus a low [FO-met]/[FO] ratio in serum may be indicative of a high serum NTBI concentration which might suggest that the dose of DFO could be increased. This relationship requires further systematic investigation because it may be possible to adjust the treatment of iron-overloaded patients with DFO by reference to the [FO-met]/[FO] ratio in serum so as to minimize the potentially harmful NTBI.


ACKNOWLEDGMENTS 10.

We thank Drs. L. A. Damani and H. H. Peter for the helpful discus- 11. sion and comments made, Mrs. M. Bird for preparation of the artwork, Mr. A. Cakebread and R. Tye for performing the mass spectrometric analysis, and Ms. Elena Marchesi for preparation of the manuscript.

12.

13.

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