Cefaclor

Molecular formula: C15H14CIN3O4S Molecular weight: 367.8 CAS Registry No.: 53994-73-3, 70356-03-5 (monohydrate)

SAMPLE Matrix: blood Sample preparation: 100 |xL Serum + 100 JULL 6 |xg/mL cefotaxime + 1 mL MeCN, vortex, centrifuge at 2000 g for 10 min. Remove the aqueous phase and add it to 2.5 mL dichloromethane, vortex, centrifuge, inject a 25 |JLL aliquot of the upper aqueous layer. HPLCVARIABLES Column: 150 X 3.9 5 |xm C18 (Waters) Mobile phase: MeGN:100 mM phosphate 8:92, pH 5.6 Flow rate: 1.2 Injection volume: 25 Detector: UV 254 CHROMATOGRAM Internal standard: cefotaxime Limit of detection: 200 ng/mL Limit of quantitation: 500 ng/mL KEYWORDS serum; mouse; pharmacokinetics REFERENCE
Onyeji, CO.; Nicolau, D.R; Nightingale, CH.; Quintiliani, R. Optimal times above MICs of ceftibuten and cefaclor in experimental intra-abdominal infections. Antimicrob.Agents Chemother., 1994, 38, 1112-1117

SAMPLE Matrix: blood Sample preparation: Condition a 1 mL Bond-Elut C18 SPE cartridge with 2 mL MeOH and 2 mL 8.5% phosphoric acid. Condition an NH2 SPE cartridge with 1 mL hexane. 500 |JLL Plasma + 25 |JLL 8.5% phosphoric acid + 250 |JLL 1 mg/mL coumarin-3-carboxylic acid in water, add to the C18 SPE cartridge, wash with 500 JJLL water, wash with 1 mL 8.5% phosphoric acid, wash with 5% MeOH: 8.5% phosphoric acid 20:1, elute with 1 mL MeOH: 8.5% phosphoric acid 60:40 into the NH2 SPE cartridge. Wash the NH2 SPE cartridge with 1 mL hexane, wash with 1 mL MeCN, elute with 1 mL water: 10% ammonium sulfate 95:5, inject a 20 jxL aliquot of the eluate. HPLCVARIABLES Column: 250 X 4.6 C18 Mobile phase: Water:2 mM tetramethylammonium hydroxide in MeOH:acetic acid 60: 40:0.5 Flow rate: 0.8 Injection volume: 20 Detector: UV 262 CHROMATOGRAM Internal standard: coumarin-3-carboxylic acid (13)

OTHER SUBSTANCES Extracted: cefazolin, ceftizoxime, cephalexin KEYWORDS plasma; SPE REFERENCE
Moore, CM.; Sato, K.; Hattori, H.; Katsumata, Y. Improved HPLC method for the determination of cephalosporins in human plasma and a new solid-phase extraction procedure for cefazolin and ceftizoxime. Clin.Chim.Acta, 1990, 190, 121-123

SAMPLE Matrix: blood Sample preparation: 100 |xL Serum + 10 |xL 100 |xg/mL cephradine in water + 100 |xL MeCN, vortex, centrifuge at 9000 g for 10 min. Remove 100 |xL supernatant, evaporate to dryness at room temperature under reduced pressure, dissolve residue in 100 |xL 20 mM NaH2PO4 adjusted to pH 3.5 with phosphoric acid, centrifuge at 9000 g for 5 min, inject 20 |xL supernatant. HPLCVARIABLES Guard column: Guard Pak C18 (Waters) Column: 200 X 4.6 5 |xm Nucleosil C18 Mobile phase: MeCN: buffer 30:70, pH adjusted to 7.0 with NaOH (Buffer was 20 mM sodium phosphate and 5 mM tetrabutylammonium hydrogen sulfate.) Flow rate: 1 Injection volume: 20 Detector: UV 265 CHROMATOGRAM Retention time: 5.8 Internal standard: cephradine Limit of detection: 1 fxg/mL KEYWORDS serum REFERENCE
Lindgren, K. Determination of cefaclor and cephradine in serum by ion-pair reversed-phase chromatography. J.Chromatogr., 1987, 413, 351-354

SAMPLE Matrix: blood Sample preparation: 100 \xL Serum + 10 JJLL 5 jxg/mL cefixime in MeOH + 100 JXL MeCN, vortex for 15 s, centrifuge at 14000 g for 2 min. Remove the supernatant and evaporate it under a stream of nitrogen, reconstitute in 100 JJLL mobile phase, inject a 50-80 |xL aliquot. HPLCVARIABLES Guard column: RCSS Silica Guard Pak (Waters) Column: 150 X 4.6 5|xm Ultrasphere Octyl C8 Mpbile phase: MeOH: 12.5 mM pH 2.6 NaH2PO4 (pH adjusted with concentrated phosphoric acid) 20:80 Flow rate: 2 Injection volume: 50-80 Detector: UV 240

CHROMATOGRAM Retention time: 6

Internal standard: cefixime (11) Limit of detection: 1 fig/mL

OTHER SUBSTANCES

Extracted: cefadroxil, cephalexin, cephradine Noninterfering: acetaminophen, cimetidine, diazepam, digoxin, ibuprofen, phenytoin, propranolol, salicylic acid, warfarin
KEYWORDS

serum
REFERENCE
McAteer, J.A.; Hiltke, M.F.; Silber, B.M.; Faulkner, R.D. Liquid-chromatographic determination Qf five orally active cephalosporins-cefixime, cefaclor, cefadroxil, cephalexin, and cephradine-in human serum. Clin.Chem., 1987, 33, 1788-1790

SAMPLE

Matrix: blood Sample preparation: Filter plasma (0.22 |xm), inject a 10 [xL aliquot.
HPLCVARIABLES

Column: 250 X 4.6 5 |xm GFF-S5-80 internal-surface reversed phase "Pinkerton" (Regis) Mobile phase: 100 mM pH 4.38 sodium phosphate buffer containing 20 mM sodium dodecyl sulfate Flow rate: 0.8 Injection volume: 10 Detector: UV 254
CHROMATOGRAM Retention time: 13 KEYWORDS

plasma; direct injection

REFERENCE
Nakagawa, T.; Shibukawa, A.; Shimono, N.; Kawashima, T.; Tanaka, H.; Haginaka, J. Retention properties of internal-surface reversed-phase silica packing and recovery of drugs from human plasma. J.Chromatogr., 1987, 420, 297-311

SAMPLE

Matrix: blood Sample preparation: 300 |xL Plasma + 300 |xL IS in ice-cold MeOH: 100 mM pH 5.2 sodium acetate 70:30, vortex for 30 s, let stand at -20 for 10 min, centrifuge at 1500 g for 10 min, inject a 10 |xL aliquot.
HPLCVARIABLES

Guard column: 4 X 4 10 fim C18 Column: 300 X 4 10 ^m imBondapak C18 Mobile phase: MeCN: MeOH: 100 mM sodium acetate 8.64:0.36:91, pH 5.2 Flow rate: 2.5 Injection volume: 10 Detector: UV 254

CHROMATOGRAM

Retention time: 6 Internal standard: 8-chlorotheophylline (8.5) Limit of detection: 500 ng/mL
KEYWORDS plasma REFERENCE
Signs, S.A.; File, T.M.; Tan, J.S. High-pressure liquid chromatographic method for analysis of cephalosporins. Antimicrob.Agents Chemother., 1984, 26, 652-655

SAMPLE Matrix: blood, sinus mucosa Sample preparation: Plasma. Condition a 3 mL 500 mg Bond Elut C8 SPE cartridge with 3 mL MeOH and 2 mL 1% phosphoric acid. 500 |xL Plasma + 1 mL 1% phosphoric acid, mix, add to the SPE cartridge, wash with 3 mL 1% perchloric acid, elute with 750 fiL MeOH, inject a 50 JJLL aliquot of the eluate. Sinus mucosa. Condition a 1 mL Bond Elut C8 SPE cartridge with 1 mL MeOH and 1 mL 1% phosphoric acid. Chop sample with a scalpel, weigh out 20 mg and add it to 500 |JLL 10 mM pH 7.0 phosphate buffer, rotate at 4 for 12 h, centrifuge at 800 g for 10 min. 400 |xL Supernatant + 1 mL 1% phosphoric acid (?), mix, add to the SPE cartridge, wash with 1% perchloric acid, elute with 150 |xL MeOH, inject a 75 fxL aliquot of the eluate. HPLCVARIABLES Guard column: 20 X 4.6 5 jim C18 (Shandon) Column: 250 X 4.6 5 |xm Supelcosil LC 18 Mobile phase: MeOH:MeCN:50 mM pH 3.8 acetate buffer 10:3:87 (plasma) or 12:2:86 (sinus mucosa) Flow rate: 1 Injection volume: 50-75 Detector: UV 235
CHROMATOGRAM

Retention time: 18.2 Internal standard: cefaclor

OTHER SUBSTANCES Extracted: cefpodoxime KEYWORDS plasma; cefaclor is IS; SPE REFERENCE
Camus, R; Deslandes, A.; Harcouet, L.; Farinotti, R. High-performance liquid chromatographic method for the determination of cefpodoxime levels in plasma and sinus mucosa. J.Chromatogr.B, 1994, 656, 383-388

SAMPLE Matrix: blood, urine Sample preparation: Plasma or serum. Condition a Sep-Pak C18 SPE cartridge with 2 mL MeOH then 2 mL water, do not allow to go dry. 500 |xL Plasma or serum + 100 |xL 100 |xg/mL cephalexin in water + 50 \xh 25% acetic acid, mix, add to SPE cartridge, wash with two 1 mL portions of water, elute with 3 mL MeOH. Evaporate eluate under nitrogen, add 200 jxL mobile phase, vortex, inject a 25 |xL aliquot. Urine. Dilute 100:1 (ratio may vary depending on concentration) with water, inject a 50 |xL aliquot.

HPLCVARIABLES

Column: 150 X 4.6 5 |xm Supelcosil LC-18-DB Mobile phase: MeOH:THF:buffer 16:4:80 (Buffer was 1 g sodium 1-heptanesulfonate + 15 mL triethylamine in 1 L water with the pH adjusted to 2.3 with concentrated phosphoric acid.) Column temperature: 30 Flow rate: 1.4 Injection volume: 25-50 Detector: UV 265
CHROMATOGRAM Retention time: 6

Internal standard: cephalexin (9.2) Limit of quantitation: 500 ng/mL

OTHER SUBSTANCES

Extracted: hydroxyloracarbef, loracarbef Noninterfering: acetaminophen, caffeine

KEYWORDS

plasma; serum; SPE; pharmacokinetics

REFERENCE
Kovach, P.M.; Lantz, R. J.; Brier, G. High-performance liquid chromatographic determination of loracarbef, a potential metabolite, cefaclor and cephalexin in human plasma, serum and urine. J.Chromatogr., 1991, 567, 129-139

SAMPLE

Matrix: blood, urine Sample preparation: 1 mL Plasma + 1 mL 6% trichloroacetic acid, mix, centrifuge at 4000 rpm for 10 min, inject an aliquot of the supernatant. Inject an aliquot of urine directly.
HPLCVARIABLES

Guard column: 10 X 4 7 i m m Lichrosorb RP 18 Column: 250 X 4 7 |xm Lichrosorb RP 18 Mobile phase: MeCN: 25 mM pH 7 phosphate buffer 10:90 Flow rate: 1 Injection volume: 10 Detector: F ex 385 em 485 following post-column reaction. The column effluent mixed with 200 |xg/mL fluorescamine in MeCN pumped at 0.25 mL/min and the mixture flowed through a 4.5 m X 0.25 mm ID coil of PTFE tubing to the detector.;UV 260
CHROMATOGRAM Retention time: 14

Limit of detection: 1.3 ng/mL (F); 1.6 ng/mL (UV)

OTHER SUBSTANCES

Also analyzed: cefroxadine, cephalexin, cephradine Noninterfering: amidopyrin, aspirin, barbital, caffeine, cefmenoxime, cefotaxime, ceftizoxime, ceftriaxone, cetazidime, diazepam, dibekacin, gentamycin, nycin, lidocaine, netilmicin, tetracaine, theophylline, tobramycin
KEYWORDS

post-column reaction; plasma; F detection may be less susceptible to interferences

REFERENCE
Blanchine, M.D.; Fabre, H.; Mandrou, B. Fluorescamine post-column derivatization for the HPLC determination of cephalosporins in plasma and urine. J.Liq.Chromatogr., 1988, 11, 2993-3010

SAMPLE Matrix: bulk Sample preparation: Dissolve in water at a concentration of 5 mg/mL, inject a 20 u,L aliquot. HPLCVARIABLES Column: 250 x 4.6 5 ujn YMC-ODS (YMC) Mobile phase: Gradient. A was 50 mM sodium dihydrogen phosphate adjusted to pH 4.0 with phosphoric acid. B was MeCN: 50 mM sodium dihydrogen phosphate adjusted to pH 4.0 with phosphoric acid 45:55. A:B from 95:5 to 75:25 over 30 min, to 0:100 over 15 min, maintain at 0:100 for 5 min, return to 95:5 and equilibrate for 14 min. Flow rate: 1 Injection volume: 20 Detector: UV 220 CHROMATOGRAM Retention time: 28 OTHER SUBSTANCES Simultaneous: impurities, excipients Also analyzed: cephalexin REFERENCE
Olsen, B.A.; Baertschi, S.W.; Riggin, R.M. Multidimensional evaluation of impurity profiles for generic cephalexin and cefaclor antibiotics. J.Chromatogr., 1993, 648, 165-173

SAMPLE Matrix: bulk, formulations Sample preparation: Dissolve in water, inject a 20 jxL aliquot. HPLCVARIABLES Column: 300 X 3.9 10 jxm ixBondapak C18 Mobile phase: MeOH: water: acetic acid 30:70:0.1 Flow rate: 1 Injection volume: 20 Detector: UV 254 CHROMATOGRAM Retention time: 7.5 Limit of quantitation: 2 (ig/mL OTHER SUBSTANCES Simultaneous: impurities, cefadroxil, cefamandole, cefamandole nafate, cefazolin, cefoperazone, cefotaxime, cefoxitin, ceftizoxime, cephalexin, cephalothin, cephapirin, cephradine REFERENCE
Ting, S. Reverse-phase liquid chromatographic analysis of cephalosporins. J.Assoc.Off.Anal.Chem., 1988, 71, 1123-1130

SAMPLE Matrix: solutions HPLC VARIABLES Column: 300 X 3.9 10 jim jxBondapak phenyl Mobile phase: MeOH: 10 mM phosphate buffer 27:73, pH 3.6 Column temperature: 27 Flow rate: 1 Detector: UV 254 CHROMATOGRAM Retention time: 6.7 OTHER SUBSTANCES Simultaneous: amoxicillin, ampicillin, cephalexin, cephradine REFERENCE
Huang, H.-S.; Wu, J.-R.; Chen, M.-L. Reversed-phase high-performance liquid chromatography of amphoteric P-lactam antibiotics: effects of columns, ion-pairing reagents and mobile phase pH on their retention times. J.Chromatogr., 1991, 564, 195-203

SAMPLE Matrix: solutions Sample preparation: Inject 100 jxL onto column A with mobile phase A, after 3 min backflush the contents of column A onto column B with mobile phase B, elute column B with mobile phase B, monitor the effluent from column B. HPLCVARIABLES Column: A 30 X 0.3 5 \xm ODS C18 (Nomura); B 150 X 0.3 5 \xm ODS C18 (Nomura) Mobile phase: A 10 mM ammonium acetate adjusted to pH 5 with acetic acid; B MeOH: water: acetic acid 40:60:0.5 Flow rate: A 0.1; B 0.004 Injection volume: 100 Detector: UV 262 CHROMATOGRAM Retention time: 6.71 Limit of detection: 10 ng/mL OTHER SUBSTANCES Simultaneous: cefaloridine, cefazolin, ceftizoxime KEYWORDS microbore; column-switching REFERENCE
Moore, CM.; Sato, K.; Katsumata, Y. High-performance liquid chromatographic determination of cephalosporin antibiotics using 0.3 mm LD. columns. J.Chromatogr., 1991, 539, 215-220

ANNOTATED BIBLIOGRAPHY

Muranushi, N.; Horie, K.; Masuda, K.; Hirano, K. Characteristics of ceftibuten uptake into Caco-2 cells. Pharm.Res., 1994, 11, 1761-1765 [also cefadroxil, cefazolin, cephalexin, cephradine, cyclacillin, latamoxef]

Lorenz, L.J.; Bashore, RN.; Olsen, B.A. Determination of process-related impurities and degradation products in cefaclor by high-performance liquid chromatography. J.Chromatogr.Sci., 1992, 30, 2 1 1 216 determination, [simultaneous impurities, degradation products; bulk; gradient; column temp 25] Nahata, M.C.; Jackson, D.S. Liquid chromatographic method for the determination of cefadroxil in its suspension and in serum. J.Liq.Chromatogr., 1990, 13, 1651-1656 [simultaneous cefadroxil; suspensions; cefaclor is IS] Nahata, M.C. Determination of cefaclor by high-performance liquid chromatography. J.Chromatogr., 1982, 228, 429-433 Rotschafer, J.C.; Crossley, K.B.; Lesar, T.S.; Zaske, D.; Miller, K. Cefaclor pharmacokinetic parameters: serum concentrations determined by a new high-performance liquid chromatographic technique. Antimicrob.Agents Chemother., 1982, 21, 170-172 Ullmann, U. High-pressure liquid chromatography and microbiological assay in the determination of serum levels using cefradine and cefaclor. Zentralbl.Bakteriol.fA]., 1980, 248, 414-421