Erythromycin

Molecular formula: C37H67NO13 Molecular weight: 733.9 CAS Registry No.: 114-07-8, 41342-53-4 (ethylsuccinate), 96128-89-1 (acistrate), 3521-62-8 (estolate), 304-63-2 (gluheptonate), 23067-13-2 (gluheptonate), 3847-29-8 (lactobionate), 134-36-1 (propionate), 643-22-1 (stearate), 84252-03-9 (stinoprate)

SAMPLE Matrix: blood Sample preparation: 200 jxL Plasma or whole blood + 10 jxL 10 |xg/mL oleandomycin + 20 |JLL saturated sodium carbonate + 1 mL diethyl ether, mix vigorously for 30 s, centrifuge at 6000 g for 2 min. Remove 750 |xL ether, evaporate to dryness under a stream of nitrogen at room temperature for 10 min, reconstitute residue in 50 |JLL mobile phase, inject 20 |xL aliquot. HPLCVARIABLES Guard column: 10 X 4.6 5 jmrn Asahi ODP-50G Column: 150 X 4.6 5 jxm Asahipak octadecyl polymer Mobile phase: MeCN: 50 mM pH 10.5 KH2PO4 37:63 Flow rate: 1 Injection volume: 20 Detector: E, Shimadzu L-ECD-6A, glassy carbon electrode +0.72 V versus an Ag/AgCl reference electrode CHROMATOGRAM Retention time: 12.1 Internal standard: oleandomycin (5.7) Limit of detection: 100 ng/mL OTHER SUBSTANCES Noninterfering: clenbuterol, diltiazem, dipyridamole, ketotifen, methacholine, orciprenaline, theophylline KEYWORDS plasma; whole blood REFERENCE
Kato, Y.; Yokoyama, T.; Shimokawa, M.; Kudo, K.; Kabe, J.; Mohri, K. Determination of erythromycin in human plasma and whole blood by high-performance liquid chromatography. J.Liq.Chromatogr., 1993, 16, 661-680

SAMPLE Matrix: blood Sample preparation: 1 mL Plasma + 20 |xL of 100 |xg/mL oleandomycin phosphate in ethanol + 60 |xL saturated potassium carbonate (final pH 10), mix briefly, add 5 mL tbutyl methyl ether, shake in a reciprocating shaker for 15 min, centrifuge at 800 g for 5 min. Remove 4 mL of the upper ether layer and evaporate it to dryness under a stream of nitrogen at room temperature. Wash down tube with 200 JJLL t-butyl methyl ether. Evaporate to dryness again, take up residue in 125 JJLL mobile phase, centrifuge 30 s, inject aliquot.

HPLCVARIABLES Guard column: Alltech Direct Connect packed with ^Bondapak C18 Column: 250 X 4.6 5 ixm Ultrasphere C18 Mobile phase: MeCN: MeOH: buffer 42:10:48, final pH adjusted to 6.30-6.35 (Buffer was 100 mM sodium acetate adjusted to pH 5.0 with 100 mM acetic acid.) Flow rate: 1.20 Injection volume: 20 Detector: E5 ESA model 5100A, guard cell +0.95 V, detector 1 +0.65 V, detector 2 +0.85 V CHROMATOGRAM Retention time: 6 Internal standard: oleandomycin phosphate (4) Limit of detection: 250 ng/mL OTHER SUBSTANCES Extracted: anhydroerythromyein, 2'-aeetylerythromycin KEYWORDS plasma; mobile phase recirculated REFERENCE
Laakso, S.; Scheinin, M.; Anttila, M. Determination of erythromycin base and 2'-acetylerythromycin in human plasma using high-performance liquid chromatography with electrochemical detection. J.Chromatogr., 1990, 526, 475-486

SAMPLE Matrix: blood Sample preparation: 1 mL Plasma + 400 [xL 0.1 M NaOH to adjust pH to 10, mix 30 s, add 5 mL methyl t-butyl ether, vortex 1 min, centrifuge at 3000 rpm for 5 min, evaporate organic layer to dryness under a stream of nitrogen at 40, dissolve residue in 150 |JLL mobile phase, vortex 2 min, inject 10 |xL aliquot. HPLCVARIABLES Column: 150 X 4 10 fxm Techopak T-15 C18 Mobile phase: MeCN:MeOH:THF:buffer 86:3:3:8 (Buffer was 75 mM sodium acetate adjusted to pH 4.1 with glacial acetic acid.) Flow rate: 1.5 Injection volume: 10 Detector: E, ESA Model 5100A with ESA model 5020 guard cell, analytical cell + 0.70 V (I) +0.85 V (II), guard cell +0.90 V, 0.5 |xm carbon filters CHROMATOGRAM Retention time: 10 Limit of quantitation: 50 ng/mL KEYWORDS plasma REFERENCE
Kokkonen, P.; Haataja, H.; Valttila, S. Determination of 2'-acetylerythromycin and erythromycin in plasma by HPLC using manual and robotic sample preparation. Chromatographia, 1987, 24, 680682

SAMPLE Matrix: blood, gastric juice Sample preparation: Centrifuge plasma or gastric juice at 1200 g for 5 min. 500 |xL Plasma or gastric juice + 500 |JLL pH 11 phosphate buffer (ionic strength 1=1.0) + 50 |xL 100 |xM oleandomycin in MeCN + 5 mL hexane: 2-butanol 80:20, shake for 15 min, centrifuge at 1200 g for 5 min, freeze in dry ice/acetone. Remove the organic layer and evaporate it to dryness under a stream of nitrogen, reconstitute the residue in 300 \xh mobile phase, vortex three times for 1 min, inject a 40 fxL aliquot. HPLC VARIABLES Guard column: 15 X 3.2 7 jxm Brownlee CN Column: 100 X 4.6 Hypersil C18 BDS base-deactivated Mobile phase: MeCN:2.1 mM NaH2PO4:27.1 mM Na2HPO4 40:30:30 Column temperature: 65 Flow rate: 1.2 Injection volume: 40 Detector: E, ESA Coulochem Model 5100A, Model 5020 guard cell before injector +1.0 V, model 5011 dual electrode analytical cell, screen electrode (detector 1) +0.65 V, sample electrode (detector 2) +0.85 V, ESA carbon filters before guard and analytical cells CHROMATOGRAM Retention time: 10.5 Internal standard: oleandomycin (5) Limit of quantitation: 20 nM (plasma); 100 nM (gastric juice) KEYWORDS plasma; rugged; pharmacokinetics REFERENCE
Toreson, H.; Eriksson, B.M. Determination of erythromycin in gastric juice and blood plasma by liquid chromatography and electrochemical detection. J.Chromatogr.B, 1995, 673, 81-89

SAMPLE Matrix: blood, saliva, urine Sample preparation: Plasma. 2 mL Plasma + 20 |xL 750 |Jig/mL roxithromycin in MeCN + 5 mL diethyl ether, shake vigorously for 3 min, centrifuge at 900 g at 4 for 5 min. Remove upper layer and evaporate it to dryness under a stream of nitrogen at 45. Reconstitute residue with 100 JJLL MeCN, vortex 5 s, inject 40 |xL aliquot. Urine. 1.5 mL Urine + 100 |xL 750 |xg/mL roxithromycin in saturated K2HPO4 + 4 mL diethyl ether, shake vigorously for 3 min, centrifuge at 900 g at 4 for 5 min. Remove upper layer and evaporate it to dryness under a stream of nitrogen at 45. Reconstitute residue with 100 jxL MeCN, vortex 5 s, inject 40 |xL aliquot. Saliva. 1.5 mL Saliva + 100 jxL 750 jxg/mL roxithromycin in saturated K2HPO4 + 4 mL diethyl ether, shake vigorously for 3 min, centrifuge at 900 g at 4 for 15 min. Remove upper layer and evaporate it to dryness under a stream of nitrogen at 45. Reconstitute residue with 100 jxL MeCN, vortex 5 s,
inject 40 JJLL aliquot.

HPLC VARIABLES Column: Nova-Pak C18 Mobile phase: MeCN:MeOH:56 mM sodium acetate buffer 50:4:56, final pH adjusted to 7.0 with glacial acetic acid Flow rate: 1.1 Injection volume: 40 Detector: E, Waters M460, +0.9 V versus Ag/AgCl reference electrode

CHROMATOGRAM

Retention time: 6.0 (erythromycin base), 7.1 (erythromycin B), 34.5 (erythromycin estolate), 35.5 (erythromycin ethylsuccinate) Internal standard: roxithromycin (14.7) Limit of detection: 12.5 ng/mL
OTHER SUBSTANCES

Simultaneous: 4"-acetylerythromycin, 6-O-methylerythromycin

KEYWORDS

plasma
REFERENCE
Croteau, D.; Vallee, F.; Bergeron, M.G.; LeBeI, M. High-performance liquid chromatographic assay of erythromycin and its esters using electrochemical detection. J.Chromatogr., 1987, 419, 205-212

SAMPLE

Matrix: blood, urine Sample preparation: Urine. Centrifuge urine at 2500 g for 5 min, inject a 100 uX aliquot. Whole blood. 200 |xL Whole blood + 100 |xL 10% EDTA H - 50 |xL 20 |xg/mL josamycin in MeOH: water 50:50, centrifuge to separate plasma. 200 JXL Plasma + 20 |xL saturated potassium carbonate + 1 mL MTBE, mix, centrifuge. Remove 800 |xL of the MTBE layer and evaporate it to dryness, reconstitute the residue in 200 |xL MeOH-.water 50:50, inject a 100 |xL aliquot.
HPLCVARIABLES

Column: 250 X 4.6 8 |xm PLRP-S 1000 A (Polymer Labs) Mobile phase: MeCN: t-butanol: 200 mM pH 9.0 phosphate buffer-.water 3:19:5:73 Column temperature: 70 Flow rate: 1.5 Injection volume: 100 Detector: F ex 365 em 450 following post-column extraction. The column effluent mixed with reagent pumped at 0.7 mL/min and this mixture flowed through a 1.5 m X 0.5 mm ID stainless steel coil. The effluent from the coil mixed with chloroform pumped at 1.5 mL/min and this mixture flowed through a 1.5 m X 0.5 mm ID stainless steel coil to a sandwich-type phase separator with a 40 (xL groove volume (Vrije Universiteit, Amsterdam). Part of the organic layer was separated and flowed through the detector at 0.5 mL/min. (Reagent was 5 jxM sodium 9,10-dimethoxyanthracene-2-sulfonate in 100 mM citric acid.)
CHROMATOGRAM

Retention time: 11 Internal standard: josamycin (28) Limit of detection: 12.5 ng/mL (plasma); 50 ng/mL (urine) OTHER SUBSTANCES Extracted: metabolites Simultaneous: midecamycin, troleandomycin
KEYWORDS

plasma; whole blood; post-column extraction

REFERENCE
Khan, K.; Paesen, J.; Roets, E.; Hoogmartens, J. Analysis of erythromycin A and its metabolites in biological samples by liquid chromatography with post-column ion-pair extraction. J.Liq. Chromatogr., 1994, 17, 4195-4213

SAMPLE Matrix: blood, urine Sample preparation: Dilute urine 1:2 with isotonic NaCl. 200 JJLL Plasma or diluted urine + 100 |JLL water + 600 |JLL pH 9 phosphate buffer + 3 mL dichloromethane, shake for 10 min, centrifuge at 2000 g for 5 min. Remove 2.5 mL of the organic layer and evaporate it to dryness under a stream of nitrogen, reconstitute the residue in 50 |xL MeOH, vortex for 10 s, inject a 15 \xL aliquot. HPLCVARIABLES Column: 300 X 3.9 10 ^m ixBondapak C18 Mobile phase: MeCN:MeOH: 83 mM ammonium acetate 55:22:23, pH adjusted to 7.5 with acetic acid Flow rate: 1 Injection volume: 15 Detector: E, ESA Coulochem Model 5100A, Model 5020 guard cell 1.0 V (before injector), Model 5010 dual-electrode cell, screen electrode El + 0.7 V, sample electrode E2 +0.9 V, 0.5 |xm ESA carbon filters placed before guard and analytical cells CHROMATOGRAM Retention time: 7.0 Internal standard: erythromycin OTHER SUBSTANCES Extracted: roxithromycin Simultaneous: amitriptyline, clomipramine, disopyramide, erythromycin estolate, erythromycin ethylsuccinate, erythromycin stearate, imipramine, josamycin, lidocaine, spiramycin KEYWORDS plasma; erythromycin is IS REFERENCE
Demotes-Mainaird, RM.; Vingon, GA.; Jarry, C.H.; Albin, H.C. Micro-method for the determination of roxithromycin in human plasma and urine by high-performance liquid chromatography using electrochemical detection. J.Chromatogr., 1989, 490, 115-123

SAMPLE Matrix: blood, urine Sample preparation: Prewash a 1 mL C18 Bondelut C18 SPE cartridge with 3 mL MeCN and 3 mL water. 1 mL Serum or urine + 0.25 mL (0.50 mL for urine) 6-12 |Jig/mL oleandomycin phosphate in water, add 1 mL MeCN, vortex 1 min, centrifuge at 1600 g for 5 min, add to 8 mL water, load onto the SPE cartridge, wash with 5 mL water, wash with 5 mL MeCN.water 1:1, suck dry, elute with two 0.5 mL aliquots of MeCN:50 mM pH 6.30 phosphate buffer. Dry under vacuum in a rotary vacuum centrifuge, reconstitute in 20 |JLL water, vortex 1 min, add 25 |JLL MeCN, vortex 1 min, centrifuge at 1600 g for 1 min, inject 3-5 JJIL aliquot of upper layer. HPLC VARIABLES Guard column: Waters Guard-Pak with Anatech 40-60 |xm glass beads Column: 150 X 3.9 Novapak C18 Mobile phase: MeCN: 50 mM pH 6.30 phosphate buffer 30:70 Column temperature: 35 Flow rate: 1 Injection volume: 3-5 Detector: E, Metrohm 656 with a glassy carbon electrode, 1.15 V versus Ag/AgCl reference electrode; also UV at 200 nm with LOD 250-1000 ng/mL (J.Pharm.Sci. 1985, 74, 11261128)

CHROMATOGRAM

Retention time: 6.3 Internal standard: oleandomycin phosphate (4.4) Limit of detection: 100 ng/mL OTHER SUBSTANCES Simultaneous: anhydroerythromycin
KEYWORDS

serum; SPE; stability-indicating

REFERENCE
Stubbs, C; Haigh, J.M.; Kanfer, I. A stability-indicating liquid chromatographic method for the analysis of erythromycin in stored biological fluids using amperometric detection. J.Liq.Chromatogr., 1987, 10, 2547-2557

SAMPLE

Matrix: formulations
HPLCVARIABLES

Column: 250 X 4.6 5 jim Bakerbond C18 Mobile phase: MeCN: MeOH: 200 mM ammonium acetate: water 45:10:10:25, pH 6.25 1
FIo W rate: 1 Detector: UV 215

CHROMATOGRAM

Retention time: 7.46 (erythromycin lactobionate)

KEYWORDS

injections; saline; water; stability-indicating

REFERENCE
Stiles, M.L.; Allen, L.V., Jr.; Prince, S. J. Stability of various antibiotics kept in an insulated pouch during administration via portable infusion pump. Am.J.Health-Syst.Pharm., 1995, 52, 7074

SAMPLE

Matrix: formulations Sample preparation: Weigh out material corresponding to ca. 250 mg erythromycin ethylsuccinate, add 10 mL acetone, sonicate 5 min, centrifuge at 2500 g for 5 min, dilute a 6 mL aliquot of supernatant to 10 mL with 200 mM pH 6.5 tetrabutylammonium hydrogen sulfate:200 mM pH 6.5 phosphate buffer:water 12.5:7.5:80.
HPLC VARIABLES

Column: 250 X 4.6 10 |xm RSiI LL C18 (RSL-Bio-Rad) Mobile phase: MeCN: 200 mM pH 6.5 tetrabutylammonium hydrogen sulfate (adjust pH with NaOH):200 mM pH 6.5 phosphate buffer:water 42.5:5:5:47.5 Column temperature: 35 Flow rate: 1.5 Injection volume: 20 Detector: UV 215
CHROMATOGRAM

Retention time: 24 (erythromycin A ethylsuccinate), 8 (erythromycin A)

KEYWORDS powders; tablets REFERENCE

Cachet, T.; Lannoo, P.; Paesen, J.; Janssen, G.; Hoogmartens, J. Determination of erythromycin ethyl succinate by liquid chromatography. J.Chromatogr, 1992, 600, 99-108

SAMPLE Matrix: solutions HPLCVARIABLES Column: 250 X 4.6 Zorbax C8 Mobile phase: MeCN: 200 mM pH 6.5 tetramethylammonium phosphate: 200 mM pH 6.5 ammonium phosphate: water 35:20:5:40 Column temperature: 35 Flow rate: 1.5 Injection volume: 20 Detector: UV 215 CHROMATOGRAM Retention time: 12 KEYWORDS better results with aged columns REFERENCE
Cachet, T.; Quintens, I.; Roets, E.; Hoogmartens, J. Improved separation of erythromycin on aged reversed-phase columns. J.Liq.Chromatogr., 1989, 12, 2171-2201

ANNOTATED BIBLIOGRAPHY

Zierfels, G.; Petz, M. [Fluorimetric determination of erythromycin residues in foods of animal origin after derivatization with FMOC and HPLC separation]. Z.Lebensm.Unters.Forsch., 1994, 198, 307312 Janecek, M.; Quilliam, M.A.; Bailey, M.R.; North, D.H. Determination of erythromycin A by liquid chromatography and electrochemical detection, with application to salmon tissue. J.Chromatogr, 1993, 619, 63-69 [electrochemical detection; fish; tissue; SPE; column temp 40; LOD 100 ng/g] Paesen, J.; Calam, D.H.; Miller, J.H.McB.; Raiola, G.; Rozanski, A.; Silver, B.; Hoogmartens, J. Collaborative study of the analysis of erythromycin by liquid chromatography on wide-pore poly(styrenedivinylbenzene). J.Liq.Chromatogr, 1993,16, 1529-1544 [column temp 70; simultaneous impurities] Cachet, T.; Quintens, L; Paesen, J.; Roets, E.; Hoogmartens, J. Improved separation of erythromycin on aged reversed-phase columns. II. J.Liq.Chromatogr, 1991, 14, 1203-1218 Paesen, J.; Roets, E.; Hoogmartens, J. Liquid chromatography of erythromycin A and related substances on poly(styrene-divinylbenzene). Chromatographia, 1991, 32, 162-166 Stubbs, C; Kanfer, I. A stability-indicating high-performance liquid chromatographic assay of erythromycin estolate in pharmaceutical dosage forms. Int.J.Pharm., 1990, 63, 113-119 Araman, A.; Temiz, D.; Guven, K.C. Stability studies of erythromycin in simulated gastric medium studied by high-performance liquid chromatography. Ada Pharm.Turc, 1988, 30, 37-42 Croteau, D.; Bergeron, M.G.; LeBeI, M. Pharmacokinetic advantages of erythromycin estolate over ethylsuccinate as determined by high-pressure liquid chromatography. Antimicrob.Agents Chemother, 1988, 32, 561-565 Grgurinovich, N.; Matthews, A. Analysis of erythromycin and roxithromycin in plasma or serum by high-performance liquid chromatography using electrochemical detection. J.Chromatogr, 1988, 433, 298-304

Haataja, H.; Kokkonen, P. Determination of 2'-acetylerythromycin and erythromycin in human tonsil tissue by HPLC with coulometric detection. J.Antimicrob.Chemother., 1988, 21, 67-72 Stubbs, C; Kanfer, I. High-performance liquid chromatography of erythromycin propionyl ester and erythromycin base in biological fluids. J.Chromatogr., 1988, 427, 93-101 [extracted erythromycin, erythromycin propionate; oleandomycin (IS); electrochemical detection; column temp 35; serum; urine; SPE; pharmacokinetics; LOQ 250 ng/mL] Cachet, T.; Kibwage, LO.; Roets, E.; Hoogmartens, J.; Vanderhaeghe, H. Optimization of the separation of erythromycin and related substances by high-performance liquid chromatography. J.Chromatogr., 1987, 409, 91-100 [column temp 35; simultaneous impurities, erythromycin A, erythromycin B, erythromycin C] Geria, T.; Hong, W.H.; Daly, R.E. Improved high-performance liquid chromatographic assay of erythromycin in pharmaceutical solid dosage forms. J.Chromatogr., 1987, 396, 191-198 Nilsson, L.G.; Walldorf, B.; Paulsen, O. Determination of erythromycin in human plasma, using column liquid chromatography with a polymeric packing material, alkaline mobile phase and amperometric detection. J.Chromatogr., 1987, 423, 189-197 Kibwage, 1.0.; Roets, E.; Hoogmartens, J.; Vanderhaeghe, H. Separation of erythromycin and related substances by high-performance liquid chromatography on poly(styrene-divinylbenzene) packing materials. J.Chromatogr., 1985, 330, 275-286 Stubbs, C; Haigh, J.M.; Kanfer, I. Determination of erythromycin in serum and urine by high-performance liquid chromatography with ultraviolet detection. J.Pharm.ScL, 1985, 74, 1126-1128 Duthu, G.S. Assay of erythromycin from human serum by high performance liquid chromatography with electrochemical detection. J.Liq.Chromatogr., 1984, 7, 1023-1032 [serum; electrochemical detection; also josamycin, oleandomycin, tylosin] Chen, M.L.; Chiou, W.L. Analysis of erythromycin in biological fluids by high-performance liquid chromatography with electrochemical detection. J.Chromatogr, 1983, 278, 91-100 Tsuji, K.; Kane, M.P. Improved high-pressure liquid chromatographic method for the analysis of erythromycin in solid dosage forms. J.Pharm.ScL, 1982, 71, 1160-1164