Quantification of Desferrioxamine in Blood Plasma ... - Clinical Chemistry

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few problems during this evaluation. Our find- ..... molarity. Practical sensitivity. Practically, the sensitivity of the described method exceeds. 1 mgIL. Using a 0.5- ...

In conclusion, we find the Vision procedure for theophylline procedure suitable for clinical use. It is sufficiently precise and produces results reliably and rapidly, and we encountered few problems during this evaluation. Our finding that capillary samples collected by finger stick and whole-blood samples obtained by venipuncture have similar theophylline values demonstrates that blood samples obmined via finger stick in clinics, emergency rooms, or physicians’ offices can provide valid, transferable results for theophylline. This study Laboratories.





by a grant



References 1. Hendeles L, Weinberger M. Theophylline: a ‘state of the art’ review. Pharmacotherapy 1983;3:2-44. 2. McDonald JM, Ladenson JH, Turk J, et al. Theophylline toxicity. Clin Chem 1979;24:1603-8. 3. Rangsithienchai P, Newcomb RW. Aminophylline therapy in children:


4. Schultz SG, centrifugation 1985;31:1457-63.

for dosage. J Pediatr


Holen JT, Donohue JP, et al. Two-dimensional for desk-top clinical chemistry. Clin Chem

CLIN. CHEM. 33/1,


in Blood Plasma

Sylvle Bourdon,1


Pascal Houz#{233},2 and Raymond

A sensitive method, inductively coupled plasma atomic emission spectroscopy, is used to measure desferrioxamine in blood plasma. The desferrioxamine is transformed into its iron chelate, terrioxamine, which is extracted into benzyl alcohol, then re-extracted into HCI (0.5 mol/L), which is used as the sample for the spectroscopy. For a 0.5-mL plasma sample, the detection limit (1 g/mL) suffices for following the concentration of desferrioxamine in plasma after its subcutaneous or intramuscular injection (40 mg per kg of body weight). Neither blood pigments nor trace metals interfere. Keyphrases: compared

thalassemia .

6. Doumas BT, Perry BW, Sasse EA, et al. Standardization bilirubin assays: evaluation of selected methods and stability bilirubin solutions. Clin Chern 1973;19:984-93.





encephalopathy chronic renal fail-


Chem 1985;31:1415-6. 8. Alles GA, Hawes RC. Cholinesterases in the blood of man. J Biol Chem 1940;133:375-90. 9. Elm RJ, Ruddel M. Discrepant results in determination of theophylline in serum from a patient with renal failure [Letter]. Clin Chem 1983;28:1879. 10. Patel JA, Clayton LT, LeBel CP, et al. Abnormal theophylline levels in plasma by fluorescence polarization immunoassay in patients with renal disease. Ther Drug Monit 1984;6:458-60. 11. Compton R, Lichti D, Ladenson JH. Influence o uremia on four assays for theophylline: improved results with a monoclonal antibody in the TDX procedure. Clin Chem 1985;31:152-4. 12. Breiner R, McComb R, Lewis S. Positive interference immunoassay of theophylline in serum of uremics [Letter]. Chem 1985;31:1575-6.

by Inductively

with Clin


Vol. 33, No. 1, 1987




amine into children with thalassemia major and into adults with chronic renal failure. The following technique is based on the formation and extraction offemoxamine, with subsequent measurement of the chelate by inductively coupled plasma atomic emission spectroscopy (icxs). The assay’s sensitivity permits measurement of desferrioxainine in a plasma sample as small as 200 p.L, if necessary. The same technique is easily applied to urine, but in most cases the sensitivity of the colorimetric method is sufficient for use with urine. Materials

and Methods

Instrumentation used:

#{149} an IPS neaux,


spectrometer Colombes,



68, Rue




#{149} a plasma torch argon (Philips, 105, Rue de Paris, 93002Bobigny, France); #{149} a cross-flow pneumatic nebulizer fed by a peristaltic (minipulse) pump (Gilson, 72, Rue Gambetta, 95400-Vuhers le Bel, France) at a flow rate of 600 L/min (Table 1). Reagents 40 mmol/L: To 800 mL of distilled water g of nitrilotriacetic acid and 10.80 g of ferric chloride (FeCl3 6H20); adjust the pH to 6-7 with ammonia; dilute to 1000 mL with distilled water. Perchloric acid (d = 1.67), diluted 10-fold. Sodium hydroxide solution (d = 1.33), diluted 10-fold. Ferric

Department of Analytical Chemistry, Rene Descartes University, Paris, France. 2LaIyjratoiy of Toxicology, Fernand Widal Hospital 200, Rue du Faubourg Saint Denis, 75010 Paris, France. (Address correspondence to RB., at this address.) Received June 23, 1986; accepted September 20, 1986. 1



7. Ferron LA, Weber J-P, Hebert J, et al. Theophylline concentrations in serum, plasma, and whole blood compared [Letter]. Clin


Described as a chelating agent (1-2), desferrioxamine is commonly used in the treatment of major thalassemia and aluminum encephalopathy, to increase the removal of iron and aluminum (2-9). Despite much work on optimal conditions for its use, monitoring desferrioxamine remains difficult, mainly because of the poor sensitivity (in blood plasma) of reported methods (10-11). We attempted to establish a sensitive method for use with small-volume samples obtained after injection of desferriox-




Quantification of Desferrioxamine Emission Spectrometry

Additional colorimetry

5. Chan K-M, Arriaga C, Landt M, et al. Interference by hemolysis with various methods for total calcium and its correction by trichloroacetic acid precipitation. Clin Chem 1983;29:1497-500.





Benzyl alcohol with boiling point at standard pressure = 204.7#{176}C. pH 9 buffer: Dissolve 5.35 g of ammonium chloride in 50 mL of distilled water, add 7 mL of ammonia (d = 0.91), dilute to 100 mL with distilled water. HCI (d = 1.15): “Suprapur” grade, diluted 20-fold. Anhydrous magnesium sulfate. Desferrioxamine standard solution, 5.0 gIL: This solution can be used for 15 days if stored at -20 #{176}C.


Precision and accuracy. A plasma sample containing added desferrioxamine (12.5 mg/L) and processed 20 times gave the following results (mean ± SD): 11.1 ± 0.6 mg/L (95 ± 2.4% analytical recovery). Reproducibility. Patients’ samples, measured during two different runs, gave a between-run CV of 5.3%. Correlation with colorimetric measurements. We could assess such a correlation only for desferrioxamine concentrations ‘25 mgfL because of the poor sensitivity of the colorimetric assay (see Table 2).

Procedures Deproteinization, chelation, and extraction. In a centrifuge thoroughly mix 0.5 mL of blood plasma, 1.5 mL of distilled water, and 0.8 mL of perchloric acid reagent. After 10 mm, centrifuge, remove the clear supernatant liquid, and set it aside in a second centrifuge tube (glass stoppered). Again add 0.8 mL of perchloric acid reagent to the protein precipitate, re-extract as before, and combine the second extract with the first. To the combined extract add (in the following order): 1.3 mL of diluted sodium hydroxide solution (caution: pH should be near 3 and always s5), 0.10 mL of the ferric reagent, and 0.5 mL of pH 9 buffer. Mix, and let stand for 15 mm. Then add 1.5 mL of benzyl alcohol and 5 g of anhydrous magnesium sulfate. Stir for 15 mm, then centrifuge. Remove 0.5 mL of the supernate and mix it with 4 mL of diluted HC1, again mix, and centrifuge. Standardization. Using pooled plasma as the matrix, prepare four standards by adding 0,50, 100, or 200 L of 20fold diluted desferrioxamine standard solution to 0.5 mL of plasma (equivalent to a sample concentration of 0, 25, 50, or 100 mgIL in a 0.5-mL plasma sample). Every standard is then deproteinized, chelated, and extracted as described above for a plasma sample. Quantification. When the spectrometer is ready for use (see Table 1), prepare and record the standard curve. Every measurement is defined by the difference between photons counted at the top (259.940 ±0.002 mn) and foot (260.000 ± 0.002 nm) of the peak (exclusive of the continuous emission background). With a 2.5-s integration time, the counts for the 100 mgfL standard are roughly 106 photons. As usual in IcpAxs the calibration graph is perfectly linear. Measure every sample under the same conditions as the standard; the tube,


and OperatIng

50 MHz; power: 2.2 kW Monoblock silica: solvent




2. Comparison





27 ±5 25 ±2

25 ±5 42 ±2

29 ±5 31 ±2

46 ±5 52 ±3


The chemical basis of this method was that proposed by et al. (11): chelation of desferrioxamine by iron to form ferrioxamine, extraction with benzyl alcohol, and colorimetry of the organic layer. Unfortunately, that method is neither accurate (results are pH dependent) nor sufficiently sensitive; every sample must contain at least 25 g of desferrioxamine for such colorimetric results to be satisfactory. Moreover, its specificity is questionable, being affected by pigments and drugs. Given that the concentration of desferrioxamine in plasma is frequently 15 mgfL, at least 2 mL of plasma is needed, an unacceptably large volume for pediatric studies. By ICPAES, the detection limit for iron is roughly 2 g/L; thus measurements of iron at 10 pgIL are excellent. When one takes into account the concentration of desferrioxamine in plasma and the molar ratio ironldesferrioxamine in ferrioxamine (1/2), a 1 mg/L concentration of desferrioxamine theoretically can be detected in a 0.2-mL plasma sample, corresponding to 4 mL of re-extraction aqueous phase, the volume required for ICPAES measurement. When possible, however, we routinely use a 0.5mL plasma sample. Deproteinization. To obviate adsorption of the chelate, the proteins must be eliminated. The cation, desferrioxamine, is soluble in water in acidic medium. Thus use of 0.3 mol/L HC1O4 simultaneously deproteinizes the sample and extracts the desferrioxamine. To get an extraction yield 96%, however, a second extraction is necessary. Chelate formation and interferences. Besides Fe3, many ions are chelated by desferrioxamine, although their stability constants are generally lower than for ferrioxamine (12). Under the conditions we describe, Cu2, Al3, and Zn2 do not inhibit the ferrioxamine formation (Table 3). Consefor 10 Plasma or Serum Samples as DetermIned by


(11) and by Desferrioxamine






of desferrioxamine


Observation height: 13 mm Plasma flow rate: 19 Limin Auxiliary flow rate: 0.2 L/min Carrier flow rate: 0.9 Llmin Holographic grating: 2400 grooves/mm Resolution: 0.2 nm/mm


to Patients

treatment of thalassemia major-by subcutaneous injection of 40 mg per kilogram of body weight-showed, for 100 samples, an average concentration of roughly 13 mg/L at pseudo steady state (12). The measured concentrations of desferrioxamine in patients with chronic renal failure were, quite often, much higher (‘150 mgIL) and correlated poorly with the excretion of aluminum in the fluids analyzed.

computer calculates the sample concentration (plus the blank value). The result for the 0 mg/L standard (i.e., the reagent blank) should be subtracted from the result for each plasma sample.

Generator: Table 1. ICPAES Facilities


54 ±6 56 ±3


concn, mg/L 103 ±10 114 ±5

97 ±9 95 ±5


63 ±6

37 ±5

54 ±6

68 ±3

44 ±2

60 ±3


Vol. 33, No. 1, 1987



3. Effect

of Adding Blood Interfsrent

Potential Samples and concn,


to Four


AI3, i Cu2, 2 m2’, 2 Desferrioxamine concn, mg/L, mean of n = 2 determinations

26 31





17 48

16.5 48.5


25.5 30.5 17.5



quently, desferrioxamine can be quantified in aluminumrich plasma samples, such as from patients with aluminum encephalopathy who are being treated with desferrioxamine; aluminum concentrations ‘20 pxnol/L do not interfere. We use the reagent proposed by Summers et al. (11), nitrilotriacetic acid-ferric complex, but animonium ferric citrate also can be used, in the same molarity. Practical sensitivity. Practically, the sensitivity of the described method exceeds 1 mgIL. Using a 0.5-mL sample of plasma, one counts 106 photons for 100 mg/L or iO photons for 1 mgfL. The standard deviation being ‘5#{149} 102 photons for every measurement (top and foot of the line for the sample and for the standard), the total error of counting is i03 photons, equivalent to 0.2 mgfL for a 0.5-mL sample or 0.5 mg/L for a 0.2-mL sample. Usefulness of desferrioxamine quantification. The biological half life of desferrioxamine is very short (11), but the equilibrium between the free drug and its complexes is much slower. Consequently, it is very useful to ascertain the best sequence of times for subcutaneous injections, that associated with the highest rate of excretion of iron or aluminum (in urine or dialysis fluids); for details see 12 and 13.

References 1. Schwarzenbach G, Schwarzenbach K. Hydroxamate complexes. I. The stability of the iron(ffl) complexes of simple hydroxamic acids and desferrioxamin B. Helv Chixn Acts 1963;46:1390-400.




Vol. 33, No. 1, 1987

2. Schwarzenbach G, Schwarzenbach K. Iron exchange between sideramines and complexones. A discussion of the formation constants of the hydroxamate complexes. Ibid., 1409-22. 3. Westlin WF. 1971;4:597-602.

as a chelating



Clin Toxicol

4. Baker LR, Barnett MD, Brozovic B, et al. Haemosiderosis in a patient on regular haemodialysis: treatment by desferrioxamine. Clin Nephrol 1976;6:326-8. MJ, Cailender ST, Letsky in transfusion-dependent 1978;i:1178-80.

5. Pippard

EA, et al. Prevention



6. Pogglitsch H, Peter W, Wawschinek 0, et al. Treatment stages of dialysis encephalopathy by aluminum depletion Lancet 1981;ii:1344-5.

7. Ackrill P, Ralston AJ, Day JP, et al. Successful aluminum from patient with dialysis encephalopathy Lancet

of iron

Lancet of







8. Adhemar JP, Laederich J, Jaudon aluminum from patients with dialysis Lancet 1980;ii:1311.


et al. Removal




9. Aria RS, Parkinson IS, Cartlidge NE, et al. Reversal of aluminum dialysis encephalopathy after desferrioxainine treatment [Letter]. Lancet;ii:1116.

10. Fielding J Clin


J, Brunstram



of ferrioxamine


Pathol 1964;17:395-8.

11. Summers MB, Jacobs A, Tudway D, Perera P, Ricketts C. Studies in desferrioxamine and ferrioxanilne metabolism in normal and iron-loaded subjects. Br J Haematol 1979;42:547-55. 12. Llados EA, Girot R, House P, Bourdon B, Lenoir G. Cinetique de la desferrioxamine perfusee par voie sous-cutan#{233}e et mouvemerits du fer chez les enfants thalass#{233}miques[Oral cominunication]. 2#{232}me J Pharmacol Clin. Paris. Facult#{233} de Lariboisi#{232}re, June 1986.

13. Llados EA, Girot R, House P, Bourdon R, Lenoir G. Cinetique de Ia desferrioxamine perfus#{233}epar voie sous-cutan#{233}e et mouvements du fer chez les enfants thalass#{233}miques [Oral communication and abstract]. Reunion de Groupe Latin de P#{233}diatrie, Pavie, May 1986.

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