lorqnn! of Chromzro~hy, 147 (1978) 507-508 @Eke* scimsc publishing campany, Am&darn-PEnted
~TIxeNethedands
CHROM. 10,375
lmjwoved procedure-for the determination of niridazole in biological fluids
by-high-performance liquid chromatography
J. J. MILE& B. M. JONES.P. R MASSEYandJ. R SALAMTAN K.R.U.F.In.sti&ute of Red Disease, CardiflRoyal Ii$rnwys Car&jf(Great Britmiz~ (Recekd
June 26th,l977)
Niridazole, l-(5-nitro-2-thiazoiyl~2-yl)-2-imidazolidinone, is marketed by Ciba under tie trade-nameAmbii. Although this drug has been used for many years for the treatmentof schistosomiasisfit is only recentlythat niridazolehas heenfound to be immunosuppressive2d.A previous gas-liquid chromatographicmethod’ for the determinationof niridazoiein urineand serumwas sensitiveonly to 250 &ml of sampleand had a retentiontime for niridazoleof 27.75 min. The proceduredescribed here allows for the determinationdown to 50 ng/ml of sampleand a much improved time period for assay (7.5 min). MATERlAI_S
use.
AND
METHODS
An&r-grade reagentswere used throughout. Methanol was redistilledbefore
High-performance liquid chrotnaiography High-performanceliquid chromatography(F%?LC) was carriedout using an Anachem 4153 chromatograph(Anachem, Luton, Great Britain)fittedwith an Altex 153 .analyticalUV detector with an 8-,~l flow cell. The detector was fitted with a 365-runHter. The column was 250 mm x 4.6 mm I-D. packedwith Spherisorb ODS, mesh size 5 ,UII, and injectionof sampleson to this column was achievedvia a loop
injector-The isobaric flow-rate was 0.5 ml/min using methanol-water (4:1) as Solvent. Under these conditionsit was found that niridazolehad a retentiontime of 7.5 min.
Extrmtion procedure A l-ml sample of eitherurine or serum was acidifkd with 1 ml of 1 M HCl
and extracted by shaking with 5 ml of 1,2dichloroethane. The whole was allowed td
stand for 5-10 min -beforethe lower organic phase was removed and concentrated by rotary evaporation. The sample was redissolvedin the methanol-water eluent (1 ml) and aliquots weresubsequentlyinjectedinto the high-petiormanceliquidchromatograph.
508
NOTES
.
DISCUSSION
There was found to be a good linearresponsefor HPLC of a known concentrationof niridazole(100 pg-50 ng) whichweredissolvediu the elueut.Further,under the conditions employed all urine and serum samples tested to date by HPLC have been free from interferingpeaks. The use of a wavelengthtiter at 365 mu to detectthe 5-n&0 group presentin niridazole (absorption maximum 380 nm) results in the greatestsensitivityfor the detection of the parent compound. However, it has been found possible to use a 2%nm filter for the detection of niridazole in biological fluids, with an obvious resultantfall in the sensitivityof the assay. Using this filtersystemit was only possible to detectlevels of 100 ng/ml of sample. RESULTS
As has previouslybeen reportedniridazolehas been used for many years for the treatmentof schistosomiasisbut not as an immunosuppressant1~3. We arecurrently involvedin a clinicaltrial comparingniridazole+ azathioprine+ prednisolonewith the more standard immunosuppressiveregime of azathioprine + prednisolonein kidney transplant recipients,a stucl~ which requiresan improved, more sensitive assay for niridazole.These requirementshave now been attainedby the use of HPLC. ACKNOWLEDGEMENTS
We are grateN to the Medical ResearchCouncil and the K.R.U.F. Institute of Renal Disease for finaucialsupport. RITERENCES 1 M. A. Mandel, 2 3. R. Saknan, 9 (1977) 989. 3 J. R Salaman, 23 (1977) 29. 4 J. R Sahman,
A. A. R. Mahmoud and K. S. Warren, Plart. Zeconwr. Surg, 35 (1975) 76. M. Bird, A. M. Godfrey, B. M. Jones, D. Milk and J. J. Milk, Trampbmt. Pro=.,
M. Bird, A. M. Godfrey, M. Bird, A. M. Godfrey,
B. M. Jones, D. MiIlar and J. J. Miller, Trampfantation,
B. M. Jones, D. Milk
and J. J. Milk,
Micrumrgerry,
(1977) in press. 5 B. M. Jones, M. Bird, M. Howells, D. MUar, J. J. Miller, S. Reeves 2nd J. R. Saknan, Tronsplantatkm, 24 (1977) 134. 6 S. V. Lucas, J. C. DanieLs, R. D. Schubert, J. M. Simpson, A. A. R. Mahmoud, K. S. Warren, j_ R David and L. T. Webster, J. ImnuuwZ_, 118 (1977) 418. 7 J. J. Miller and It J. Oake, J. Chrumatogr., 131 (1977) 442.
~TIxeNethedands
CHROM. 10,375
lmjwoved procedure-for the determination of niridazole in biological fluids
by-high-performance liquid chromatography
J. J. MILE& B. M. JONES.P. R MASSEYandJ. R SALAMTAN K.R.U.F.In.sti&ute of Red Disease, CardiflRoyal Ii$rnwys Car&jf(Great Britmiz~ (Recekd
June 26th,l977)
Niridazole, l-(5-nitro-2-thiazoiyl~2-yl)-2-imidazolidinone, is marketed by Ciba under tie trade-nameAmbii. Although this drug has been used for many years for the treatmentof schistosomiasisfit is only recentlythat niridazolehas heenfound to be immunosuppressive2d.A previous gas-liquid chromatographicmethod’ for the determinationof niridazoiein urineand serumwas sensitiveonly to 250 &ml of sampleand had a retentiontime for niridazoleof 27.75 min. The proceduredescribed here allows for the determinationdown to 50 ng/ml of sampleand a much improved time period for assay (7.5 min). MATERlAI_S
use.
AND
METHODS
An&r-grade reagentswere used throughout. Methanol was redistilledbefore
High-performance liquid chrotnaiography High-performanceliquid chromatography(F%?LC) was carriedout using an Anachem 4153 chromatograph(Anachem, Luton, Great Britain)fittedwith an Altex 153 .analyticalUV detector with an 8-,~l flow cell. The detector was fitted with a 365-runHter. The column was 250 mm x 4.6 mm I-D. packedwith Spherisorb ODS, mesh size 5 ,UII, and injectionof sampleson to this column was achievedvia a loop
injector-The isobaric flow-rate was 0.5 ml/min using methanol-water (4:1) as Solvent. Under these conditionsit was found that niridazolehad a retentiontime of 7.5 min.
Extrmtion procedure A l-ml sample of eitherurine or serum was acidifkd with 1 ml of 1 M HCl
and extracted by shaking with 5 ml of 1,2dichloroethane. The whole was allowed td
stand for 5-10 min -beforethe lower organic phase was removed and concentrated by rotary evaporation. The sample was redissolvedin the methanol-water eluent (1 ml) and aliquots weresubsequentlyinjectedinto the high-petiormanceliquidchromatograph.
508
NOTES
.
DISCUSSION
There was found to be a good linearresponsefor HPLC of a known concentrationof niridazole(100 pg-50 ng) whichweredissolvediu the elueut.Further,under the conditions employed all urine and serum samples tested to date by HPLC have been free from interferingpeaks. The use of a wavelengthtiter at 365 mu to detectthe 5-n&0 group presentin niridazole (absorption maximum 380 nm) results in the greatestsensitivityfor the detection of the parent compound. However, it has been found possible to use a 2%nm filter for the detection of niridazole in biological fluids, with an obvious resultantfall in the sensitivityof the assay. Using this filtersystemit was only possible to detectlevels of 100 ng/ml of sample. RESULTS
As has previouslybeen reportedniridazolehas been used for many years for the treatmentof schistosomiasisbut not as an immunosuppressant1~3. We arecurrently involvedin a clinicaltrial comparingniridazole+ azathioprine+ prednisolonewith the more standard immunosuppressiveregime of azathioprine + prednisolonein kidney transplant recipients,a stucl~ which requiresan improved, more sensitive assay for niridazole.These requirementshave now been attainedby the use of HPLC. ACKNOWLEDGEMENTS
We are grateN to the Medical ResearchCouncil and the K.R.U.F. Institute of Renal Disease for finaucialsupport. RITERENCES 1 M. A. Mandel, 2 3. R. Saknan, 9 (1977) 989. 3 J. R Salaman, 23 (1977) 29. 4 J. R Sahman,
A. A. R. Mahmoud and K. S. Warren, Plart. Zeconwr. Surg, 35 (1975) 76. M. Bird, A. M. Godfrey, B. M. Jones, D. Milk and J. J. Milk, Trampbmt. Pro=.,
M. Bird, A. M. Godfrey, M. Bird, A. M. Godfrey,
B. M. Jones, D. MiIlar and J. J. Miller, Trampfantation,
B. M. Jones, D. Milk
and J. J. Milk,
Micrumrgerry,
(1977) in press. 5 B. M. Jones, M. Bird, M. Howells, D. MUar, J. J. Miller, S. Reeves 2nd J. R. Saknan, Tronsplantatkm, 24 (1977) 134. 6 S. V. Lucas, J. C. DanieLs, R. D. Schubert, J. M. Simpson, A. A. R. Mahmoud, K. S. Warren, j_ R David and L. T. Webster, J. ImnuuwZ_, 118 (1977) 418. 7 J. J. Miller and It J. Oake, J. Chrumatogr., 131 (1977) 442.
