Urinary cyclophosphamide assay as a method for biological monitoring of occupational exposure to cyclophosphamide

Transcription

1 Internatlol Archivm of Int Arch Occup Environ Health ( 1986) 58: O {ltilail} 11 s Springer-Verlag 1986 Urinary cyclophosphamide assay as a method for biological monitoring of occupational exposure to cyclophosphamide C T A Evelo, R P Bos, J G P Peters, and P Th Henderson Department of Toxicology, Faculty of Medicine, University of Nijmegen, P O Box 9101, NL-6500 HB Nijmegen, The Netherlands Summary Urine of twenty hospital workers was monitored for the excretion of the cytostatic drug cyclophosphamide using GC-MSD The drug was found to be present above the detection limit of 0 5 g g/24 h urine in five cases A clear relationship between cyclophosphamide handling and the detectability of excretion existed This method developed can be of use for biological monitoring studies directed toward the finding of exposure hazards. Key words: Cyclophosphamide Urine Hospital workers Biological monitoring Introduction Handling of cytostatic agents by medical and pharmaceutical personnel is considered to be an occupational health hazard Increases in urinary thioether excretion (Jagun et al 1982) and in urinary mutagenicity (Falck et al 1979 ; Bos et al 1982) have been demonstrated after occupational exposure to cytostatic agents Chromosome damage and increases in SC Es (Waksvik et al 1981) have indicated a possible genetic risk in such cases. One of the most widely used cytostatics is cyclophosphamide Since cyclophosphamide is known to be a human carcinogen (IARC 1981), exposure of hospital workers to this agent may give rise to carcinogenic risks. Specific monitoring of cyclophosphamide uptake has mainly been directed at determinations in patients having received therapeutic doses Pantarotto et al ( 1974) developed a GC method for the determination of cyclophosphamide in body fluids This method was modified by several workers in the field, but the limited sensitivity did not allow biomonitoring of hospital personnel. Offprint requests to: C T A Evelo at the above address

2 152 C T A Evelo et al. Recently, Hirst and coworkers ( 1984) determined cyclophosphamide in urine of nurses, using an extraction procedure on total 24-h urine samples We modified the clean-up and concentration procedure, and used it to determine cyclophosphamide excretion in urine of personnel in four hospitals in the Netherlands. Materials and methods Chemicals Cyclophosphamide and iphosphamide were kindly supplied by Asta (Bielefeld, FRG). Amberlite, type XAD-2, was from Serva (Heidelberg, FRG) Isooctane was of Uvasol quality from E Merck (Darmstadt, FRG) All other chemicals were of analytical purity. Sampling Cyclophosphamide workers and control persons started the collection of 24-h urine samples early Thursday morning All samples were collected between April and June 1985 Handling of cyclophosphamide was registered from Monday morning until the end of the sampling period Preparation and administration were registered as separate handlings The amount of cyclophosphamide handled each time ranged from 100 to 1000 mg (mean ± 350 mg). To check recovery and sensitivity each day of analysis, three control urine samples (from a female laboratory worker) spiked with 2 gg cyclophosphamide were analysed as well. Concentration on XAD g Ig Iphosphamide was added to 10 % of a 24-h urine sample as an internal standard The urine was eluted (flow rate 3-5 ml/min) through an amberlite XAD-2 column with a 6-cm 3 bed volume The column was washed three times with 5 ml of aqua dest to elute the more hydrophilic components The adsorbed material was eluted with 10 ml of ethylacetate Each elution step was completed by air-pressure to elute the residue of the solvents mentioned. Further purification To the eluted ethylacetate, 5 5 ml of a 10 % saturated Na HCO 3 solution was added After mixing and separation, the aqueous layer was extracted once more with 10 ml ethylacetate Subsequently 5 ml 0 05 N H Cl was added to the combined ethylacetate layers And again the resulting aqueous layer was reextracted with 20 ml ethylacetate The combined ethylacetate layers were evaporated at 40 0 C under nitrogen and the residue was dissolved in 0 5 ml methanol/ water ( 90:10 v/v) The methanol/water phase was washed with 1 ml hexane (v d Bosch and de Vos 1980) and the solvent was removed by evaporation under nitrogen. The recovery of cyclophosphamide throughout the whole concentration and purification procedure was % in comparison with directly analysed solution of cyclophosphamide in ethylacetate. GC analysis The residue was dissolved in 50 Il ethylacetate, and 50 gl Trifluor-Acetic-Anhydride (TFAA) was added The mixture was allowed to react at 70 C for 20 min The ethylacetate and excess TFAA were removed by evaporation under nitrogen at 40 C The residue was redissolved in 50 1 l iso-octane One gl of this sample was injected into a Hewlett-Packard 5790 gaschromatograph A capillary column packed with cross-linked dimethyl silicone (film thickness 0 3 jim, length 16 m, internal diameter 0 2 mm) was used, with a helium flow of 0 75 ml/min at a pres-

3 Biological monitoring of urinary cyclophosphamide 153 sure of 10 psi The injector temperature was 250 C The splitless injection method was used. The initial temperature of the column was 80 C After a valve time of 0 8 min this temperature was increased by 75 C/min to C After each run the temperature was raised to C in order to clean the column. The GC was interfaced with a Hewlett-Packard 5970 mass selective detector (a quadrupole with an electron energy of 70 e V) The interface temperature was 270 C and the vacuum was 6 x 10-5 torr. The retention times of the TFA derivates from cyclophosphamide and the internal standard iphosphamide were 4 3 and 5 1 min, respectively. Two characteristic ions, m/z = 307 and m/z = 309, were detected by selected monitoring (dwell times of 200 ms for each ion) as described by Lartigue-Mattei et al ( 1984). Results Cyclophosphamide handling during the week in which samples were collected was relatively low In total only twenty persons handled cyclophosphamide, most of them fewer than five times Cyclophosphamide excretion above the detection limit of 0 5 lig/24-h urine was found in urine of five of these workers. Table 1 Urinary cyclophosphamide excretion by Dutch hospital workers, related to frequency of cyclophosphamide handling Person Sex Age Smoking Frequency of CP handling CP excretion habits D-1 D-2 D-3 D-4 Total tg/24 h 1 m 30 N f 41 N f 22 N f 22 S f 21 S f 27 N f 30 S f 28 S f 27 N f 29 N f 26 N f 55 S f 29 S f 24 S f 54 N f 43 N f 23 S f 31 S f 31 S f 39 S D-1 = Monday, D-2 = Tuesday, D-3 = Wednesday, D-4 = Thursday s = smoker; N = nonsmoker

4 154 C T A Evelo et al. The frequency of cyclophosphamide handling during the week, the amount of cyclophosphamide excreted, and some individual data on the subjects under investigation are listed in Table 1. In order to test the analytical method for false positives, 12 urine samples from a non-exposed female laboratory worker and 21 control urines of hospital workers who had not handled cyclophosphamide were analysed as well The persons carrying out the analyses were unacquainted with the origin (i e cyclophosphamide worker or control person) of the samples In only one case (a 54- year-old, female hospital administration worker) was an unidentified substance detected as " 1 1 tig cyclophosphamide/24-h urine". Discussion None of the workers involved in this study handled cyclophosphamide more than incidentally on the collection day and the two days before; furthermore all of them claimed to have taken regular safety precautions (i e at least wearing gloves during the handling of cyclophosphamide) Nevertheless cyclophosphamide excretion was detected in five cases There is an obvious relationship between frequency of cyclophosphamide handling and urinary excretion In four out of five cases the persons with detectable urinary cyclophosphamide concentration had handled cyclophosphamide ten times or more during the week. All of the five persons with detectable cyclophosphamide excretion were smokers This may or may not be a coincidence, since there were no non-smokers with a high frequency of cyclophosphamide handling If excretion for smokers is really higher than for non-smokers, this may be due to a higher uptake (finger shunt) This is also one of the explanations Bos et al ( 1982) gave for their finding of an increased urinary mutagenicity of smoking nurses handling cytostatic drugs in comparison with smoking controls, while urinary mutagenicity of non-smoking nurses was not increased in comparison with non-smoking controls. Another striking feature is that nurses with detectable cyclophosphamide excretion handled most, and in two cases all, cyclophosphamide on Monday and Tuesday Therefore, urinary excretion may have been even higher earlier in the week On the other hand there may also be a lag phase in the excretion, especially when uptake of cyclophosphamide was through the skin In order to elucidate this question, studies on the time course of cyclophosphamide excretion are in progress. Hirst et al ( 1984) found no cyclophosphamide excretion after topical application of 1 mg cyclophosphamide by a male volunteer, whereas they did find excretion after treatment of four female volunteers in the same way It is not known whether this is due to an influence of sex on cyclophosphamide uptake, metabolism and excretion Therefore, the applicability of the method to biological monitoring of male personnel is uncertain Since only one male worker was involved in this study, this uncertainty still remains.

5 Biological monitoring of urinary cyclophosphamide 155 It was not possible to establish whether the one positive value in the control group is due to a methodological failure or to an unexpected non-occupational exposure to cyclophosphamide. Since exposures are expected to be higher in situations where cyclophosphamide is handled routinely (e g in oncologic clinics), it is to be expected that the method described is well applicable for biological monitoring of cyclophosphamide excretion after occupational exposures Routine analyses are possible, although the method depends on the availability of GCMS facilities The substitution of the extraction of cyclophosphamide from complete 24-h urine, as used by Hirst et al ( 1984), by concentration on XAD-2 of only 10 % of the urine, makes the method far more suitable as an exposure test for routine purposes. The primary value of studies on exposures of workers to genotoxic cytostatics may be to discover working areas or manipulations with relatively high exposures and to evaluate the effectiveness of safety precautions, the use of protective materials etc In this way they may be useful to reduce the exposures to levels "as low as possible". Evaluations of the demonstrated cyclophosphamide excretion in terms of health risk are not yet possible, since the effects at low exposure levels are still unknown. Acknowledgements This project was part of a biomonitoring study on hospital workers handling cytostatic drugs which was carried out in collaboration with the Medical Department of the Dutch Governmental Medical Services Financial support was given by the General Directorate of Labour, Dutch Ministry of Social Affairs. References Bos RP, Leenaars AO, Theuws JLG, Henderson P Th ( 1982) Mutagenicity of urine from nurses handling cytostatic drugs; influence of smoking Int Arch Occup Environ Health 50: vd Bosch N, de Vos D ( 1980) Some aspects of the gas-liquid chromatographic analysis of cyclophosphamide in plasma J Chromatogr 183:49-56 Falck K, Sorsa M, Vainio H ( 1979) Use of the bacterial fluctuation test to detect mutagenicity in urine of nurses handling cytostatic drugs Mutat Res 85: Hirst M, Tse S, Mills DG, Levin L, White DF ( 1984) Occupational exposure to cyclophosphamide Lancet I: IARC Monographs ( 1981) Some antineoplastic and immunosuppressive agents IARC 26: , Lyon Jagun O, Ryan M, Waldron HA ( 1982) Urinary thioether excretion in nurses handling cytostatic drugs Lancet II: Lartigue-Mattei C, Chabard JL, Touzet C, Bargneux H, Petit J, Berger JA ( 1984) Plasma cyclophosphamide assay by selective ion monitoring J Chromatogr 310: Pantarotto C, Bossi A, Belvedere G, Martini A, Donelli MG, Frigerio G ( 1974) Quantitative GLC determination of cyclophosphamide and isophosphamide in biological specimens. J Pharmacol Sci 63: Waksvik H, Klepp O, Br O ger A ( 1981) Chromosome analyses of nurses handling cytostatic agents Cancer Treat Rep 65: Received October 21, 1985 / Accepted March 6, 1986