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Ofloxacin

Molecular formula: C18H20FN3O4


Molecular weight: 361.4
CAS Registry No.: 82419-36-1

SAMPLE
Matrix: blood
Sample preparation: 250 JJLL Serum + 50 |xL pipemidic acid solution + 200 JJLL chloroform,
extract. Extract the organic phase with 200 |xL 100 mM NaOH, inject a 20 |xL aliquot.
HPLCVARIABLES
Column: C18
Mobile phase: MeCN-.buffer 13:87 (Buffer was 10 mM NaH2PO4 and 5 mM tetrabutylammonium hydrogen sulfate, pH 2.7.)
Flow rate: 1.4
Injection volume: 20
Detector: F ex 282
CHROMATOGRAM
Internal standard: pipemidic acid
Limit of quantitation: 50 ng/mL
KEY WORDS
serum; pharmacokinetics
REFERENCE
Klepser, M.E.; Patel, K.B.; Nicolau, D.R; Quintiliani, R.; Nightingale, CH. Comparison of the bactericidal activities of ofloxacin and ciprofioxacin alone and in combination with ceftazidime and piperacillin against clinical strains of Pseudomonas aeruginosa. Antimicrob.Agents Chemother., 1995, 39,

2503-2510
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 50 |xL 180 jxg/mL enoxacin in MeOH, shake briefly,
add 3 mL MeCN, shake at 1000 rpm for 30 s, centrifuge at 2600 g for 10 min. Remove 3
mL of the supernatant and evaporate it to dryness under a stream of nitrogen at 60,
reconstitute the residue in 100 |xL 60 mM KH2PO4 adjusted to pH 2.6 with orthophosphoric acid, inject a 30 |xL aliquot.
HPLCVARIABLES
Column: 150 X 3 7 |xm Separon SGX C18 (Tessek, Prague)
Mobile phase: THF: buffer 5.5:94.5 (Buffer was 60 mM KH2PO4 adjusted to pH 2.6 with
orthophosphoric acid: triethylamine 97:3 containing 50 fxg/mL sodium azide. Caution! Sodium azide is carcinogenic and toxic and must not be discharged to the plumbing system!)
Flow rate: 0.7
Injection volume: 30
Detector: F ex 282 em 450
CHROMATOGRAM
Retention time: 6.7
Internal standard: enoxacin (5.0)
Limit of detection: 8 ng/mL
Limit of quantitation: 20 ng/mL

KEYWORDS
plasma
REFERENCE
Macek, J.; Ptacek, P. Determination of ofloxacin in human plasma using high-performance liquid chromatography and fluorescence detection. J.Chromatogr.B, 1995, 673, 316-319

SAMPLE
Matrix: blood
Sample preparation: 500 |xL Serum + 100 |xL water + 1 mL MeCN, shake mechanically
for 30 s, centrifuge at 10000 g for 5 min. Remove 1.5 mL of the supernatant and evaporate
it to dryness under reduced pressure at 40, reconstitute the residue in 300 JJIL mobile
phase, inject a 25 (xL aliquot.
HPLCVARIABLES
Guard column: 30 X 4 30-40 jim Perisorb RP-18 (Merck)
Column: 125 X 4 5 |xm Nucleosil 100 SA
Mobile phase: MeCN: buffer 75:25, adjusted to pH 3.82 with concentrated phosphoric acid,
final sodium concentration 23 mM (Buffer was 6.74 mL concentrated phosphoric acid and
40 mL 2 M NaOH made up to 990 mL with water, adjust pH to 2.92 with concentrated
phosphoric acid, make up to 1 L with water.)
Flow rate: 1.5
Injection volume: 25
Detector: F ex 295 em 525
CHROMATOGRAM

Retention time: 8.0


Internal standard: ofloxacin
OTHER SUBSTANCES
Extracted: sparfloxacin
KEYWORDS
ofloxacin is IS; serum; protect from light
REFERENCE
Borner, K.; Borner, E.; Lode, H. Determination of sparfloxacin in serum and urine by high-performance
liquid chromatography. J.Chromatogr., 1992, 579, 285-289

SAMPLE
Matrix: blood
Sample preparation: 500 |xL Serum + 500 |xL 7% perchloric acid, vortex for 10 s, centrifuge at >700 g for 10 min, inject a 20 JJLL aliquot of the supernatant.
HPLCVARIABLES
Column: 100 X 8 jxBondapak C18 Radial-PAK
Mobile phase: MeOH: 18 mM KH2PO4 containing 0.13 mM heptanesulfonic acid !concentrated phosphoric acid 30:70:0.1
Injection volume: 20
Detector: F ex 294 em 475
CHROMATOGRAM

Retention time: 5.6


KEYWORDS
serum

REFERENCE
Griggs, D.J.; Wise, R. A simple isocratic high-pressure liquid chromatographic assay of quinolones in
serum. J.Antimicrob.Chemother., 1989, 24, 437-445

SAMPLE

Matrix: blood
Sample preparation: 50 |xL Plasma + I m L 100 mM pH 7.0 K2HPO4 adjusted to pH 7.0
with 85% orthophosphoric acid + 100 |xL 300 |xg/mL nalidixic acid in water + 3 mL
dichloromethane: isoamyl alcohol 9:1, shake vigorously for 10 min, centrifuge at 2270 g
for 10 min. Remove 2 mL of the organic phase and evaporate it to dryness under a stream
of nitrogen at 40. Reconstitute residue in 100 |xL MeOH: 50 mM NaOH 2:1, vortex, inject
a 10 |xL aliquot.
HPLCVARIABLES

Column: 150 X 4.6 5 |xm Chemcosorb 5-ODS-H


Mobile phase: MeOH: 5 mM sodium lauryl sulfate 2:1, adjusted to pH 2.5 with 85% phosphoric acid (Better separation obtained at pH 2.35, J.Chromatogr. 1990, 530, 186.)
Column temperature: 40
Flow rate: 0.6
Injection volume: 10
Detector: UV 300
CHROMATOGRAM

Retention time: 5.9


Internal standard: nalidixic acid (5.0)
Limit of detection: 150 ng/mL
OTHER SUBSTANCES

Extracted: felbinac, fenbufen, norfloxacin


Interfering: enoxacin
KEYWORDS

plasma; rat
REFERENCE
Katagiri, Y.; Naora, K.; Ichikawa, N.; Hayashibara, M.; Iwamoto, K. Simultaneous determination of
ofloxacin, fenbufen and felbinac in rat plasma by high-performance liquid chromatography.
J.Chromatogr., 1988, 431, 135-142

SAMPLE

Matrix: blood
Sample preparation: 1 mL Plasma + 2 mL 100 mM pH 7.0 phosphate buffer + 100 |xL
1 mg/mL flufenamic acid in phosphate buffer + 6 mL chloroform, agitate on a rotary
mixer for 15 min, centrifuge at 4000 g at 4 for 5 min. Remove the organic phase and
evaporate it to dryness under nitrogen at 37. Suspend the residue in 100 uX 100 mM
pH 7.0 phosphate buffer, vortex for 45 s, centrifuge at 9000 g for 2 min, inject a 20 |xL
aliquot of the supernatant.
HPLCVARIABLES

Column: 150 X 4.5 5 |xm Hypersil ODS


Mobile phase: MeCN: 40 mM phosphoric acid 45:55, final pH 2.56-2.59
Column temperature: 17
Flow rate: 2
Injection volume: 20
Detector: F ex 370 em 400-700

CHROMATOGRAM
Retention time: 3.83
Internal standard: flufenamic acid (2.67)
Limit of quantitation: 2.5 ng/mL
KEYWORDS
plasma
REFERENCE
Notarianni, L.J.; Jones, R.W. Method for the determination of ofloxacin, a quinolone carboxylic acid
antimicrobial, by high-performance liquid chromatography. J.Chromatogr., 1988, 431, 461-464

SAMPLE
Matrix: blood, CSF
Sample preparation: Serum. 1 mL Serum + 500 |xL 500 mM pH 7 phosphate buffer + 8
mL dichloromethane, rotate for 10 min at 20 rpm, centrifuge at 4000 rpm for 15 min.
Remove the organic layer and evaporate it to dryness at 40 in a vortex evaporator. Dissolve in 1 mL mobile phase, inject a 100 JULL aliquot. CSF. Inject a 20 |xL aliquot directly.
HPLCVARIABLES
Column: 250 X 4 5 |jim Nucleosil C18
Mobile phase: MeCN: 40 mM pH 2.2 phosphate buffer containing tetrabutylammonium
chloride as the ion-pairing reagent 15:85
Flow rate: 1
Injection volume: 20 (CSF), 100 (serum)
Detector: F ex 295 em 475
CHROMATOGRAM
Limit of detection: 90 ng/mL (serum); 70 ng/mL (CSF)
KEYWORDS
serum
REFERENCE
Bitar, N.; Claes, R.; Van der Auwera, P. Concentrations of ofloxacin in serum and cerebrospinal fluid of
patients without meningitis receiving the drug intravenously and orally. Antimicrob.Agents Chemother., 1989, 33, 1686-1690

SAMPLE
Matrix: blood, intestinal efflux
Sample preparation: Intestinal efflux. Freeze intestinal efflux at -80, lyophilize, reconstitute with 1 mL MeOH: 100 mM phosphoric acid 50:50, centrifuge at 3000 rpm for 10
min, inject a 20 (xL aliquot. Serum. Deproteinize serum with MeOH.
HPLCVARIABLES
Column: 150 X 3.9 Novapack C18
Mobile phase: MeOH:buffer 25:75 (Buffer was 10 mM pH 3.0 potassium phosphate buffer
containing 25 mM sodium heptanesulfonate (PIC B7) and 20 mM triethylamine.)
Flow rate: 1.5
Injection volume: 20
Detector: F ex 330 em 440
CHROMATOGRAM
Internal standard: ofloxacin

OTHER SUBSTANCES
Extracted: ciprofloxacin
KEYWORDS
serum; rat; ofloxacin is IS
REFERENCE
Rubinstein, E.; Dautrey, S.; Farinoti, R.; St.Julien, L.; Ramon, J.; Carbon, C. Intestinal elimination of
sparfloxacin, fleroxacin, and ciprofloxacin in rats. Antimicrob.Agents Chemother., 1995, 39, 99-102

SAMPLE
Matrix: blood, saliva, sputum
Sample preparation: 200 |xL Serum, sputum, or saliva + 200 jxL 1.5 |xg/mL pipemidic
acid in 100 raM pH 7.0 phosphate buffer H- 5 mL dichloromethane, shake for 15 min,
centrifuge at 1600 g for 10 min. Remove 4 mL of organic phase and evaporate to dryness
under a stream of nitrogen. Dissolve the residue in 200 |xL mobile phase, sonicate for 10
min, inject.
HPLCVARIABLES
Column: 150 X 6 5 jim YMC-Pack ODS-AM
Mobile phase: MeCNrIO mM phosphoric acid (pH 7.0) containing 20 mM tetrabutylammonium hydrogen sulfate 1:10
Flow rate: 1.3
Injection volume: 200
Detector: F ex 285 em 460
CHROMATOGRAM
Retention time: 8
Internal standard: pipemidic acid (5)
Limit of quantitation: 10 ng/mL
KEYWORDS
serum
REFERENCE
Koizumi, R; Ohnishi, A.; Takemura, H.; Okubo, S.; Kagami, T.; Tanaka, T. Effective monitoring of concentrations of ofloxacin in saliva of patients with chronic respiratory tract infections. Antimicrob. Agents Chemother., 1994, 38, 1140-1143

SAMPLE
Matrix: blood, tissue, urine
Sample preparation: Serum, plasma. Dilute serum or plasma 1:2 to 1:10 with 30 mM
phosphoric acid, centrifuge, inject a 20 (JiL aliquot of supernatant. Urine. Dilute urine 1:
10 to 1:100 with 30 mM phosphoric acid, centrifuge, inject a 20 |xL aliquot of supernatant.
Tissue (lung, gut). Cut tissue with a scalpel, homogenize with 1-3 mL buffer, centrifuge
at 9600 g for 5 min three times, inject a 20 (JLL aliquot. Tissue (chondral). Cut tissue with
a scalpel, homogenize with 3-6 mL buffer in an ice bath for 2-3 min, centrifuge at 9600
g for 5 min four or five times, inject a 100 |xL aliquot. Pleural. Dilute human pleural
samples with buffer, centrifuge, inject a 20 |xL aliquot. (Buffer was 66.6 mM K2HPO4
adjusted to pH 7.40 with KH2PO4.)
HPLCVARIABLES
Column: 200 X 4 5 [xm Nucleosil C18
Mobile phase: MeOH: MeCN: buffer 13:7:80, adjusted to pH 3.0 with phosphoric acid (Buffer was 15 mM phosphoric acid adjusted to pH 3.0 with tetrabutylammonium hydroxide.)

Flow rate: 1
Injection volume: 20-100
Detector: F ex 278 em 446
CHROMATOGRAM
Limit of detection: 10 ng/mL
OTHER SUBSTANCES
Simultaneous: ciprofloxacin, norfloxacin
KEYWORDS
serum; plasma; lung; gut; pleural; chondral
REFERENCE
Knoller, J.; Konig, W.; Schonfeld, W.; Bremm, K.D.; Roller, M. Application of high-performance liquid
chromatography of some antibiotics in clinical microbiology. J .Chromatogr., 1988, 427, 257-267

SAMPLE
Matrix: blood, urine
Sample preparation: 1 mL Plasma or serum or 200 |JLL urine + 1 mL pH 7 buffer + 5
mL dichloromethane, shake, centrifuge at 2500 g for 5 min. Remove 4 mL organic layer
and evaporate it under nitrogen, dissolve the residue in 200 |JLL mobile phase.
HPLCVARIABLES
Guard column: CT 30/6/4 Resolvosil-BSA-7 (Macherey-Nagel)
Column: 150 X 4 ET 150/8/4 Resolvosil-BSA-7 (Macherey-Nagel)
Mobile phase: Isopropanol: 200 mM pH 8.0 phosphate buffer 3:97
Flow rate: 1
Injection volume: 100
Detector: F ex 298 em 458
CHROMATOGRAM
Retention time: 5.2 (-), 7.5 (+)
Limit of detection: 3 ng/mL (plasma); 80 ng/mL (urine)
KEYWORDS
plasma; serum; chiral
REFERENCE
Lehr, K.-H.; Damm, P. Quantification of the enantiomers of ofloxacin in biological fluids by high-performance liquid chromatography. J.Chromatogr., 1988, 425, 153-161

SAMPLE
Matrix: blood, urine
Sample preparation: 500 \xL Plasma or serum or 200 |xL urine + 1 mL pH 7 buffer + 2
mL dichloromethane, shake, centrifuge at 2500 g for 5 min. Remove 1 mL organic layer
and add it to 1 mL dichloromethane containing 20 jxL diphenylphosphinyl chloride and
20 fxL triethylamine, vortex for 10 s, add 500 |xL L-leucinamide solution, shake for 10
min, extract with 200 [xL 1 M HCl (500 JJLL 0.5 M HCl for urine samples), inject 100 JJLL
of aqueous supernatant. (Prepare L-leucinamide solution by adding 10 mL dichloromethane and 1 mL 5 M NaOH to 500 mg L-leucinamide hydrochloride, shake, discard upper
aqueous layer, store dichloromethane layer over anhydrous sodium sulfate.)
HPLCVARIABLES
Column: 125 X 4.6 5 jim Nucleosil 120-5C18

Mobile phase: MeCN: 200 mM phosphoric acid adjusted to pH 1.85 with tetraethylammonium hydroxide 20:80
Column temperature: 40
Flow rate: 1.5
Injection volume: 100
Detector: F ex 298 em 458
CHROMATOGRAM
Retention time: 2.6 (-), 3.8 (+)
Limit of detection: 3 ng/mL (plasma); 80 ng/mL (urine)
KEYWORDS
plasma; serum; derivatization; chiral
REFERENCE
Lehr, K.-H.; Damm, P. Quantification of the enantiomers of ofloxacin in biological fluids by high-performance liquid chromatography. J.Chromatogr., 1988, 425, 153-161

SAMPLE
Matrix: blood, urine
Sample preparation: Plasma. 500 jxL Plasma + 50 |xL 200 jxg/mL desmethylpefloxacine
in 10 mM HCl + 1.5 mL 50 mM pH 9 borate buffer containing 50 mM KCl and 21 mM
NaOH, vortex for 10 s, add 8 mL dichloromethane, shake for 5 min, centrifuge at 1500 g
for 5 min. Remove 7 mL of the organic phase and add it to 200 |xL 100 mM HCl, shake
for 5 min, centrifuge at 1500 g, inject a 5 |xL aliquot of the aqueous layer. Urine. Dilute
with water 1:40, add 100 |xg/mL desmethylpefloxacine, inject a 5 \xL aliquot.
HPLCVARIABLES
Column: 300 X 3.9 10 jxm jxBondapak C18
Mobile phase: MeCN: water 10:90 containing 1.36 g/L KH2PO4 and 40 mL/L 5 mM aqueous
tetrabutylammonium phosphate, pH adjusted to 2.9 with formic acid
Flow rate: 0.7
Injection volume: 5
Detector: F ex 290 em 500
CHROMATOGRAM
Retention time: 8.7
Internal standard: desmethylpefloxacine (10.2)
Limit of detection: 20 ng/mL
KEYWORDS
plasma
REFERENCE
Mignot, A.; Lefebvre, M.A.; Fourtillan, J.B. High-performance liquid chromatographic determination of
ofloxacin in plasma and urine. J.Chromatogr., 1988, 430, 192-197

SAMPLE
Matrix: blood, vitreous humor
Sample preparation: Serum. 20 [xL Serum + 130 fxL mobile phase, mix, filter, inject a
100 JJLL aliquot of the filtrate. Vitreous humor. 15 |xL Vitreous humor + 135 |xL mobile
phase, mix, filter, inject a 100 |JLL aliquot of the filtrate.
HPLCVARIABLES
Column: 220 X 2.1 5 |im Nucleosil C18
Mobile phase: MeCN: 100 mM pH 3.82 phosphoric acid 75:25

Flow rate: 0.2


Injection volume: 100
Detector: UV 240; UV 280; F ex 280 em 445
CHROMATOGRAM
Limit of detection: 10 ng/mL
KEYWORDS
rabbit; serum; pharmacokinetics
REFERENCE
Perkins, R.J.; Liu, W.; Drusano, G.; Madu, A.; Mayers, M.; Madu, C; Miller, M.H. Pharmacokinetics of
ofloxacin in serum and vitreous humor of albino and pigmented rabbits. Antimicrob.Agents Chemotker., 1995, 39, 1493-1498

SAMPLE
Matrix: bronchoalveolar lavage fluid
Sample preparation: 1 mL Bronchoalveolar lavage fluid + 20 |JLL 30 |xg/mL IS + 1 mL
100 mM pH 7.0 phosphate buffer, vortex, add 5 mL chloroform, shake horizontally at 2
strokes/s for 10 min, centrifuge at 1500 g for 10 min. Remove the organic layer and
evaporate it to dryness under a stream of nitrogen at 40. Reconstitute residue in 400 |xL
mobile phase, inject a 20 |JLL aliquot.
HPLCVARIABLES
Column: 150 X 4.6 5 |xm TSK ODS-80TM (Tosoh)
Mobile phase: THF: 0.5% KH2PO4 adjusted to pH 2.9 with orthophosphoric acid 1:16
Flow rate: 1
Injection volume: 20
Detector: F ex 290 em 460
CHROMATOGRAM
Retention time: 12
Internal standard: 9-fluoro-2,3-dihydro-3-methyl-10-(l-imidazoyl)-7-oxo-7H-pyrido[l,2,3de][l,4]benzoxazine-6-carboxylic acid (8)
Limit of quantitation: 0.5 ng/mL
REFERENCE
Matsubayashi, K.; Une, T.; Osada, Y. Determination of ofloxacin in bronchoalveolar lavage fluid by highperformance liquid chromatography and fluorimetric detection. J.Chromatogr., 1989, 495, 354-357

SAMPLE
Matrix: cell lysate
Sample preparation: Sonicate lysed cells for 5 min, filter (0.2 |xm PTFE), add timolol,
inject an aliquot.
HPLCVARIABLES
Guard column: 4 X 4 5 ^m LiChrospher 100 RP-18
Column: 250 X 4 5 |xm LiChrospher 100 RP-18
Mobile phase: MeCN: buffer 18:82 (Buffer was 0.5% triethylamine adjusted to pH 2.5 with
78% phosphoric acid.)
Flow rate: 1
Injection volume: 100
Detector: UV 293
CHROMATOGRAM
Retention time: 5.40

Internal standard: timolol (7.70)


Limit of detection: 18 ng/mL
REFERENCE
Fresta, M.; Spadaro, A.; Cerniglia, G.; Ropero, LM.; Puglisi, G.; Furneri, P.M. Intracellular accumulation
of ofloxacin-loaded liposomes in human synovial fibroblasts. Antimicrob.Agents Chemother., 1995,39,
1372-1375

SAMPLE
Matrix: cells
Sample preparation: 100 |xL Cell suspension + 100 |xL cefoperazone solution + 100 (xL
Hanks balanced salt solution, sonicate 30 min, add 800 JxL MeCN, centrifuge at 13000 g
for 5 min, remove supernatant. Dry supernatant under air, dissolve in 100 |xL mobile
phase, inject 75 JULL.

HPLCVARIABLES
Column: jxBondapak C18
Mobile phase: MeCN: 5 mM pH 2.0 tetrabutylammonium hydrogen sulfate 10:90
Flow rate: 1
Injection volume: 75
Detector: UV 280
CHROMATOGRAM
Retention time: 10.6
Internal standard: ciprofloxacin
Limit of detection: 100-1000 ng/mL
REFERENCE
Darouiche, R.O.; Hamill, R.J. Antibiotic penetration of and bactericidal activity within endothelial cells.
Antimicrob.Agents Chemother., 1994, 38, 1059-1064

SAMPLE
Matrix: cells
Sample preparation: Incubate cells in 2 mL 100 mM pH 3.0 glycine-HCl buffer for 2 h at
room temperature, centrifuge at 5600 g for 5 min, inject an aliquot.
HPLCVARIABLES
Column: Bondapak C18
Mobile phase: MeCN: 25 mM phosphoric acid adjusted to pH 3.0 with tetrabutylammonium hydroxide 25:75
Flow rate: 1.5
Detector: F ex 340 em 425
OTHER SUBSTANCES
Also analyzed: ciprofloxacin, fleroxacin, lomefloxacin, norfloxacin, temafloxacin
REFERENCE
Pascual, A.; Garcia, L; Conejo, M.C; Perea, E.J. Fluorometric and high-performance liquid chromatographic measurement of quinolone uptake by human neutrophils. Eur.J.Clin.Microbiol.Infect.Dis.,
1991, 10, 969-971

SAMPLE
Matrix: formulations
Sample preparation: Dissolve 40 mg freeze-dried nanoparticles in 25 mL acetone:MeOH
90:10 containing a few drops of 100 mM HCl, filter (0.45 |xm), inject an aliquot.

HPLCVARIABLES

Column: 250 X 4.6 5 |jim Partisil 10-ODS-l C18


Mobile phase: MeCN: 10 mM KH2PO4: triethylamine 14:86:0.2
Flow rate: 1.5
Injection volume: 20
Detector: UV 292
OTHER SUBSTANCES

Simultaneous: pefloxacin (UV 275)


KEYWORDS

nanoparticles
REFERENCE
Fresta, M.; Puglisi, G.; Giammona, G.; Cavallaro, G.; Micali, N.; Furneri, P.M. Pefloxacine mesHate and
ofloxacin-loaded polyethylcyanoacrylate nanoparticles: Characterization of the colloidal drug carrier
formulation. J.Pharm.ScL, 1995, 84, 895-902

SAMPLE

Matrix: hair
Sample preparation: Wash hair successively with 0.1% sodium dodecyl sulfate and water
for 30 min, repeat twice, blot between 2 sheets of paper towel, allow to dry at room
temperature. Take a 1 cm fragment of hair, add 500 |xL 1 M NaOH, heat at 80 for 30
min, cool, add 500 |xL 1 M HCl, add 1 mL 100 mM pH 4.6 potassium hydrogen citrate
buffer, add 50 uX 1 |xg/mL IS in water. Add the mixture to a Bond-Elut C8 SPE cartridge,
elute with 2 mL THF: 25 mM orthophosphoric acid 20:80, evaporate eluate to dryness in
vacuum, dissolve residue in 150 jxL mobile phase, vortex, inject a 60 |xL aliquot.
HPLCVARIABLES

Column: 150 X 4.6 Tosoh 5 ^m TSKgel ODS-80Ts


Mobile phase: MeCN: 25 mM orthophosphoric acid adjusted to pH 3.0 with 0.5 M tetra-nbutylamine hydroxide 5:95
Column temperature: 40
Flow rate: 1
Injection volume: 60
Detector: F ex 295 em 490
CHROMATOGRAM

Retention time: 8.1


Internal standard: (R)-9-fluoro-2,3-dihydro-3-methyl-10-(4-ethyl-l-piperazinyl)-7-oxo-7Hpyrido[l,2,3-de][l,4]benzoxazine-6-carboxylic acid (DS-4632) (10.2) (determine at F ex 280
em 445)
Limit of detection: 0.2 ng/mL
OTHER SUBSTANCES

Extracted: ciprofloxacin, norfloxacin (determine at F ex 280 em 445)


KEYWORDS

SPE
REFERENCE
Mizimo, A.; Uematsu, T.; Nakashima, M. Simultaneous determination of ofloxacin, norfloxacin and ciprofloxacin in human hair by high-performance liquid chromatography and fluorescence detection.
J.Chromatogr.B, 1994, 653, 187-193

SAMPLE
Matrix: hair
Sample preparation: Wash hair several times with 0.1% sodium dodecyl sulfate and with
water. Add 500 |xL 1 M NaOH to 5-10 cm hair, heat at 80 for 30 min, cool, add 500 JJIL
1 M HCl, add 1 mL 500 mM pH 7.0 phosphate buffer, add 100 JULL 2.5 |xg/mL IS, add 5
mL chloroform, shake mechanically for 20 min, centrifuge at 1670 g for 10 min. Remove
the organic layer and evaporate it to dryness under a stream of nitrogen at 50, reconstitute the residue in mobile phase, inject a 100 ^xL aliquot.
HPLCVARIABLES
Guard column: 15 X 4.6 TSKguardgel ODS-120T (Tosoh)
Column: 250 X 4.6 5 |xm TSKgel ODS-120T (Tosoh)
Mobile phase: MeCN: buffer 18:82 (Buffer was 6.8 g/L KH2PO4 adjusted to pH 2.6 with
phosphoric acid.)
Flow rate: 0.8
Injection volume: 100
Detector: F ex 290 em 460
CHROMATOGRAM
Retention time: 10
Internal standard: 9-fluoro-2,3-dihydro-3-methyl-10-(l-imidazolyl)-7-oxo-7H-pyrido[l,2,3de][l,4]benzoxazine-6-carboxylic acid (DL-8357) (8)
KEYWORDS
pharmacokinetics (Ther.Drug Monit. 1995, 17, 101)
REFERENCE
Miyazawa, N.; Uematsu, T.; Mizuno, A.; Nagashima, S.; Nakashima, M. Ofloxacin in human hair determined by high performance liquid chromatography. Forensic Sci.Int., 1991, 51, 65-77

SAMPLE
Matrix: solutions
Sample preparation: Filter (0.45 |xm) a solution in MeCN: water 10:90, inject an aliquot
of the filtrate.
HPLCVARIABLES
Column: 250 X 4 5 |xm LiChrospher 100 RP-18
Mobile phase: MeCN: buffer 7:93 (Buffer was 25 mM phosphoric acid adjusted to pH 3.89
with 100 mM tetrabutylammonium hydroxide.)
Flow rate: 1
Injection volume: 10
Detector: UV 295
CHROMATOGRAM
Retention time: 8.8
OTHER SUBSTANCES
Simultaneous: ciprofloxacin (UV 280), enoxacin (UV 280), fleroxacin (UV 280), norfloxacin
(UV 280), pipemidic acid (UV 280)
REFERENCE
Barbosa, J.; Berges, R.; Sanz-Nebot, V. Solvatochromic parameter values and pH in aqueous-organic
mixtures used in liquid chromatography. Prediction of retention of a series of quinolones.
J.ChromatogrA, 1996, 719, 27-36

SAMPLE

Matrix: solutions
HPLC VARIABLES

Guard column: 20 mm long Supelguard LC-18S (Supelco)


Column: 250 X 4.6 Suplecosil LC-18S
Mobile phase: MeCN: buffer: water 10:3.5:86.5 (Buffer was 400 mM tetrabutylammonium
hydroxide adjusted to pH 2.85.)
Flow rate: 1.8
Detector: UV 280
REFERENCE
Sinko, RJ.; Hu, P. Determining intestinal metabolism and permeability for several compounds in rats.
Implications on regional bioavailability in humans. Pharm.Res., 1996, 13, 108113

SAMPLE

Matrix: solutions
Sample preparation: Prepare a 20 fxg/mL solution in MeCN: water 10:90, filter (0.45 |xm),
inject an aliquot.
HPLCVARIABLES

Column: 250 X 4 5 jxm LiChrospher 100 RP-18


Mobile phase: MeCN: 25 mM phosphoric acid 7:93, adjusted to pH 3.09 with 100 mM
tetrabutylammonium hydroxide
Flow rate: 1
Injection volume: 10
Detector: UV 295
CHROMATOGRAM

Retention time: 6.2


OTHER SUBSTANCES
Simultaneous: ciprofloxacin (UV 280), norfloxacin (UV 280), pipemidic acid (UV 280)
REFERENCE
Barbosa, J.; Berges, R.; Sanz-Nebot, V. Linear solvation energy relationships in reversed-phase liquid
chromatography. Prediction of retention of several quinolones. J.Liq.Chromatogr., 1995, 18, 34453463

SAMPLE

Matrix: solutions
HPLCVARIABLES

Column: 125 X 4.6 3 |jim ODS-Hypersil C18


Mobile phase: MeOH: THF: 670 mM pH 3.0 phosphate buffer 20:0.8:79.2 plus 2 g/L tetrabutylammonium hydrogen sulfate and 2 mL/L 85% phosphoric acid
Flow rate: 1
Injection volume: 20
Detector: UV 286
CHROMATOGRAM

Retention time: 6.38

OTHER SUBSTANCES
Simultaneous: photodegradation products, fieroxacin
Interfering: ciprofloxacin
REFERENCE
Tiefenbacher, E.-M.; Haen, E.; Przybilla, B.; Kurz, H. Photodegradation of some quinolones used as
antimicrobial therapeutics. J.Pharm.ScL, 1994, 83, 463-467

SAMPLE
Matrix: tears
Sample preparation: Dry tear sample under nitrogen.
HPLCVARIABLES
Column: 250 X 4.6 5 p,m Ultrasphere ODS
Mobile phase: MeCN:phosphoric acid:sodium lauryl sulfate 40:1:0.2:58.8
Flow rate: 1.2
Detector: F ex 358 em 495
CHROMATOGRAM
Retention time: 6.7
Internal standard: triamterene (8.1)
Limit of detection: 10 ng
REFERENCE
Richman, J.; Zolezio, H.; Tang-Liu, D. Comparison of ofloxacin, gentamicin, and tobramycin concentrations in tears and in vitro MICs for 90% of test organisms. Antimicrob.Agents Chemother., 1990, 34,
1602-1604

SAMPLE
Matrix: tissue
Sample preparation: Condition a 500 mg Bond Elut C18 SPE cartridge with 5 mL MeOH
and 10 mL water. Homogenize a 5 g tissue sample with 100 mL MeCN: 0.2% metaphosphoric acid 30:70 at high spped, filter through ca. 2 mm Hyflo Super-Cel coated on a
suction funnel. Evaporate nitrate under reduced pressure at 50 to about 30 mL. Apply
remaining solution to the SPE cartridge, wash with 20 mL water, elute with 10 mL
MeOH. Evaporate eluate to dryness under reduced pressure, dissolve in 1 mL mobile
phase, inject a 10 |xL aliquot.
HPLCVARIABLES
Column: 150 X 4.6 Wakosil II 5C18 HG (Wako)
Mobile phase: MeCN: 50 mM pH 2.4 phosphate buffer 20:80 containing 2.5 mM 1-heptanesulfonic acid
Flow rate: 0.6
Injection volume: 10
Detector: F ex 295 em 455
CHROMATOGRAM
Retention time: 10
Limit of detection: 10 ng/g
OTHER SUBSTANCES
Extracted: benofloxacin, danofloxacin, enrofloxacin
KEYWORDS
chicken; SPE

REFERENCE
Horie, M.; Saito, K.; Nose, N.; Nakazawa, H. Simultaneous determination of benofloxacin, danofloxacin,
enrofloxacin and ofloxacin in chicken tissues by high-performance liquid chromatography.
J.Chromatogr.B, 1994, 653, 69-76

SAMPLE
Matrix: urine
Sample preparation: 1 mL Urine + 1 mL MeCN, centrifuge at 4000 g for 15 min, inject
a 10 |xL aliquot of the supernatant.
HPLCVARIABLES
Column: 150 X 4.5 5 \xm Hypersil ODS
Mobile phase: MeCN: 40 mM phosphoric acid 45:55, final pH 2.56-2.59
Column temperature: 23
Flow rate: 2
Injection volume: 10
Detector: UV 254
CHROMATOGRAM
Retention time: 4.63
Limit of detection: 1 u,g/mL
REFERENCE
Notarianni, L.J.; Jones, R.W. Method for the determination of ofloxacin, a quinolone carboxylic acid
antimicrobial, by high-performance liquid chromatography. J.Chromatogr., 1988, 431, 461-464

ANNOTATED BIBLIOGRAPHY
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Fabre, D.; Bressolle, R; Kinowski, J.M.; Bouvet, O.; Paganin, R; Galtier, M. A reproducible, simple and
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Lehto, P.; Kivisto, K.T. Effect of sucralfate on absorption of norfloxacin and ofloxacin. Antimicrob.Agents
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Lyon, D.J.; Cheung, S.W.; Chan, C.Y.; Cheng, A.R Rapid HPLC assay of clinafloxacin, fleroxacin, levofloxacin, sparfloxacin and tosufloxacin. J.Antimicrob.Chemother., 1994, 34, 446-448
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serum, urine, and bile by high-performance liquid chromatography. J.Liq.Chromatogr., 1986, 9,
2539-2547 [norfloxacin (IS)]
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