Nitrofurantoin

Molecular formula: C8H6N4O5

Molecular weight: 238.2
CAS Registry No.: 67-20-9, 54-87-5 (sodium salt), 17140-81-7
(monohydrate)

SAMPLE

Matrix: blood, milk

Sample preparation: 1 mL Plasma or milk + furazolidone, extract with 5 mL dichloromethane in acidic medium (pH 3), mix, centrifuge. Remove organic phase and evaporate
it to dryness at 50 under a stream of nitrogen. Take up residue in MeCN and inject an
aliquot. (Protect from light during extraction procedure.)
HPLCVARIABLES

Column: 75 X 4.6 Beckman XL 3 jxm ODS

Mobile phase: MeCN: water 35:65
Flow rate: 1
Detector: UV 364
CHROMATOGRAM

Internal standard: furazolidone

Limit of detection: 10 ng/mL
KEYWORDS

plasma
REFERENCE
Pons, G.; Rey, E.; Richard, M.O.; Vauzelle, R; Francoual, C; Moran, C; d'Athis, P.; Badoual, J.; Olive,
G. Nitrofurantoin excretion in human milk. Dev.Pharmacol.Ther., 1990, 14, 148152

SAMPLE

Matrix: cell suspensions

Sample preparation: 200 JJLL Cell suspension + 200 jxL 10% trichloroacetic acid + 100 |xL
10 |xg/mL furazolidone, centrifuge, inject a 75 JULL aliquot of the supernatant.
HPLCVARIABLES

Column: C18 (Waters)

Mobile phase: MeOH: water: glacial acetic acid 20:80:0.09, adjusted to pH 5 with NaOH
Flow rate: 1.5
Injection volume: 75
Detector: UV 370
CHROMATOGRAM

Retention time: 6
Internal standard: furazolidone (8)
REFERENCE
Minchin, R.F.; Ho, RC; Boyd, M.R. Reductive metabolism of nitrofurantoin by rat lung and liver in
vitro. Biochem.PharmacoL, 1986, 35, 575-580

SAMPLE

Matrix: eggs, tissue

Sample preparation: Blend (Stomacher) 10 g homogenized tissue with 30 mL saline for 3

min, centrifuge at 2000 g, mix 20 mL of the supernatant with 2 mL 1% sodium azide.
Dilute 10 mL homogenized egg with 10 mL saline, add 3 mL 10% sodium azide solution.
Dialyze sample using a Cuprophan membrane (10000-15000 dalton cut-off) against water
pumped at 0.36-1.44 mL/min for 3-9 min, pass the water through column A, flush the
column with pure water for 8 min, backflush the contents of column A onto column B
with mobile phase, after 5 min remove column A from the circuit, elute column B with
mobile phase, monitor the effluent from column B. To increase sensitivity a number of
sample batches can be dialyzed before the contents of column A are analyzed. (Caution!
Sodium azide is carcinogenic, mutagenic, and highly toxic! Do not discharge to the sink!)
HPLCVARIABLES
Column: A 60 X 4.6 37-50 ^m Bondapak C18/Corasil; B 250 X 4.6 5 jxm Hypersil ODS
Mobile phase: MeCN: 100 mM pH 5 acetate buffer 20:80
FIo1W rate: 1
Detector: UV 365
CHROMATOGRAM

Retention time: 8
Limit of detection: 1 ng/g (eggs); 2 ng/g (tissue)
OTHER SUBSTANCES
Extracted: furaltadone, furazolidone, nitrofurazone
KEY WORDS
protect from light; cow; muscle; dialysis
REFERENCE
Aerts, M.M.; Beek, W.M.; Brinkman, U.A. On-line combination of dialysis and column-switching liquid
chromatography as a fully automated sample preparation technique for biological samples. Determination of nitrofuran residues in edible products. J.Chromatogr., 1990, 500, 453-468

SAMPLE
Matrix: feed, formulations, milk
Sample preparation: Formulations. Dissolve formulation in DMF, filter, inject a 10 |JLL
aliquot. Feeds. Stir 1Og finely ground feeds with 40 mL DMF for 30 min, centrifuge,
filter, wash residues with DMF, dilute to 50 mL with DMF, inject a 10 jxL aliquot. Milk.
Lyophilize 200 mL milk, wash with 75 mL MeCN during 15 min. Extract residue with
15 mL DMF with stirring for 30 min, wash residue with a mixture of 25 mL MeCN+ 5
mL DMF. Combine all organic solutions and evaporate to dryness in vacuum. Treat residue with DMF, filter, dilute to 25 mL with DMF, filter before analysis, inject a 10 |JLL
aliquot.
HPLCVARIABLES
Column: 33 X 4.6 Perkin-Elmer Pecosphere 3x3 CR C18
Mobile phase: MeCNrIOO mM pH 3.2 sodium acetate/acetic acid 10:90
Flow rate: 2
Injection volume: 10
Detector: UV 360
CHROMATOGRAM

Retention time: 1.36

Limit of detection: 4.7 ng
OTHER SUBSTANCES
Simultaneous: furaltadone, furazolidone

REFERENCE
Galeano Diaz, T.; Lopez Martinez, L.; Martinez Galera, M.; Salinas, F. Rapid determination of nitrofurantoin, furazolidone and furaltadone in formulations, feed and milk by high performance liquid chromatography. J.Liq.Chromatogr., 1994, 17, 457-475

SAMPLE

Matrix: formulations
Sample preparation: Dissolve tablet in 10 mM HCl containing 90 mM KCl (pH 2.0), inject
an aliquot.
HPLCVARIABLES

Column: 50 mm long ODS Hypersil C18

Mobile phase: MeCN: 50 mM pH 7.0 phosphate buffer 30:70
Flow rate: 1
Detector: UV 257
CHROMATOGRAM

Retention time: 5.3

OTHER SUBSTANCES

Simultaneous: hydrocortisone
KEY WORDS

tablets
REFERENCE
Neervannan, S.; Dias, L.S.; Southard, M.Z.; Stella, V.J. A convective-diffusion model for dissolution of
two non-interacting drug mixtures from co-compressed slabs under laminar hydrodynamic conditions. Pharm.Res., 1994, 11, 1288-1295

SAMPLE

Matrix: formulations
Sample preparation: 5 mL Suspension + 20 mL water + 50 mL DMF, shake for 10 min,
cool to room temperature, dilute to 100 mL with DMF, centrifuge an aliquot. 4 mL Supernatant + 15 mL 65 [LgImL acetanilide in mobile phase, filter (5 \xm), discard first 2
mL, inject a 15 |xL aliquot.
HPLCVARIABLES

Column: 300 X 3.9 10 jxm jxBondapak C18

Mobile phase: MeCN: buffer 12:88 (Buffer was 6.8 g KH2PO4 in 500 mL water, add 30 mL
1 M NaOH, dilute to 1 L with water, pH was 7.0.)
Flow rate: 1.5
Injection volume: 15
Detector: UV 254
CHROMATOGRAM

Retention time: 8
Internal standard: acetanilide
KEYWORDS

suspensions
REFERENCE
Juenge, E.C; Kreienbaum, M.A.; Gurka, D.F. Assay of nitrofurantoin oral suspensions contaminated
with 3-(5-nitrofurfurylideneamino)hydantoicacid. J.Pharm.ScL, 1985, 74, 100-102

SAMPLE
Matrix: solutions
Sample preparation: Dissolve in chloroform at a concentration of 1 |xg/mL, inject an
aliquot.
HPLCVARIABLES
Column: 250 X 4 5 jim Lichrospher RP-18
Mobile phase: MeCNrIO mM sodium acetate 20:80, pH 5
Column temperature: 30
Flow rate: 1.6
Injection volume: 20
Detector: UV 365
CHROMATOGRAM
Retention time: 9.8
Limit of detection: 22 ng/mL
OTHER SUBSTANCES
Simultaneous: degradation products, carbadox, furaltadone, furazolidone, nitrofurazone
REFERENCE
Kaniou, L; Zachariadis, G.; Kalligas, G.; Tsoukali, H.; Stratis, J. Separation and determination of carbadox, nitrofurazone, nitrofurantoin, furazolidone, and furaltadone in their mixtures by thin layer
and high performance liquid chromatography J.Liq.Chromatogr., 1994, 17, 1385 1398

SAMPLE
Matrix: tissue
Sample preparation: Homogenize (Tekmar Tissuemizer SDT 1810 with SDT 182 EN
shaft/blades) 5 g finely chopped muscle tissue and 20 mL MeCN at medium speed for 45
s, centrifuge at 1500 g for 5 min, add 15 mL MeCN-saturated hexane, shake for 30 s,
discard the hexane layer, repeat the wash. Add 9 mL EtOH to the MeCN layer and
evaporate under reduced pressure to 2-5 mL at 45, add 2 mL EtOH, evaporate to 2 mL,
add 2 mL EtOH, evaporate to dryness, add 1 mL mobile phase, sonicate for 5 min, centrifuge at 15400 g for 10 min, filter (0.45 fxm) the supernatant, inject a 50 |xL aliquot of
the supernatant.
HPLCVARIABLES
Column: 200 X 4.6 5 |xm ODS Hypersil C18
Mobile phase: MeCN: 1% acetic acid 25:75
Column temperature: 40
Flow rate: 1
Injection volume: 50
Detector: UV 375
CHROIUIATOGRAM
Retention time: 3.5
Limit of detection: 1 ng/g
Limit of quantitation: 5 ng/g
OTHER SUBSTANCES
Extracted: metabolites, furazolidone, nitrofurazone
KEYWORDS
fish; muscle

REFERENCE
Rupp, H.S.; Munns, R.K.; Long, A.R.; Plakas, S.M. Simultaneous determination of nitrofurazone, nitrofurantoin, and furazolidone in channel catfish (Ictalurus punctatus) muscle tissue by liquid chromatography. J.AOAC Int., 1994, 77, 344-350

ANNOTATED BIBLIOGRAPHY
Ebel, S.; Liedtke, R.; Missler, B. [Quantitative determination of nitrofurantoin in body fluids by direct
injection HPLC]. Arch.Pharm.(Weinheim)., 1980, 313, 95-96
Jonen, H.G.; Oesch, R; Platt, K.L. 4-Hydroxylation of nitrofurantoin in the rat. A 3-methylcholanthreneinducible pathway of a relatively nontoxic compound. Drug Metab.Dispos., 1980, 8, 446-451